MOLECULAR TAXONOMY OF CERTAIN PLASMID HARBOURING ENTEROBACTERIA

DISSERTATION SUBMITTED.IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

Muittv of ^dtlo^oplip IN

Microbiology (Ag.)

BY

ABDUb MAblK

A G R I C U L T U R E C E N T R E / D E P A R T M E N T O F B I O C H E M I S T R Y

F A C U L T Y O F L I F E SCIENCES A L I G A R H M U S L I M U N I V E R S I T Y

A L I G A R H ( INDIA)

1991

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t\cc Nei

DS1719

DEPARTMENT OF BIOCHEMISTRY Ai i nARH MUSLIM UNlvrr?'-.! r v AUIGARH-202002 u, n. rwDiA

TfiLHIX ! 3642.10 c r A IN I'll rJK o r | - . - n 7 l l

CERTIFICATE

This i s to cer t i fy that the r e s e a r c h work

embodied in th i s d i s se r t a t ion ent i t led "MOLECULAR

TAXONOMY OF CERTAIN PLASMID HARBOURING

ENTEROBACTERIA" i s an or ig inal work, unless o the rwi se

s t a t ed , c a r r i ed out by Mr. ABDUL MALIK under my

superv is ion and i s su i t ab l e for submission for the award

of M.Ph i l , degree in Microbiology ( A g . ) .

Dated 7 ' ' ^ Jti-rv^-, n 1 ' ( MASOOD AHMAD )

DEDICATED

TO

MY

PARENTS

ACKNOWLEDGEMENT S

I f i n d words i n a d e q u a t e t o e x p r e s s my d e e p s e n s e of g r a t i t u d e and huinble r e g a r d s t o my e s t e e m e d t e a c h e r and s u p e r v i s o r . D r . Masood Ahmad, R e a d e r , Depa r tmen t of B i o ­c h e m i s t r y , \ander whose g u i d a n c e , keen i n t e r e s t , c o n s t a n t s u p e r v i s i o n and u n c e a s i n g e n c o u r a g e m e n t , I h a v e b e e n a b l e t o c a r r 7 o u t t h i s s t u d y . My a p p r e c i a t i o n f o r h i s s y m p a t h e ­t i c g u i d a n c e and c o n s t r u c t i v e c r i t i c i s m canno t ' ^e^q i ressed i n few w o r d s . He h a s b e e n c o n s t a n t s o u r c e of i n s p i r a t i o n t o me. Whatever m e r i t s t h i s d i s s e r t a t i o n p o s s e s s l i e w i t h h i m .

I am h i g h l y i n d e b t e d t o P r o f . S. I s r a r H u s s a i n , C o o r d i n a t o r A g r i c u l t u r e C e n t r e and P r o f . K h a l i d Mahmood f o r t h e i r v a l u a b l e s u g g e s t i o n s and c o o p e r a t i o n .

I am g r a t e f u l t o P r o f . M. S a l e e m u d d i n , Cha i rman , D e p a r t m e n t of B i o c h e m i s t r y , F a c u l t y of L i f e ) S c i e n c e s f o r h e l p f u l s u g g e s t i o n s and p r o v i d i n g n e c e s s a r y f a c i l i ­t i e s t o c a r r y o u t t h i s w o r k .

I am h i g h l y t h a n k f u l t o P r o f .AfM. S i d d i q i f o r f r u i t f u l a d v i c e and encou ragemen t t h r o u g h o u t t h i s s t u d y .

My s i n c e r e t h a n k s a r e due t o P r o f . S«M- H a d i , D r ( s ) A.N.K. Y u s u f i , M r s . B . Bano, M r s . N. Bano , F a s i h Ahmad, R iaz Mahmood, Qaiyyvim H u s s a i n and M i s s I . Naseem f o r t h e i r v a l u a b l e s u g g e s t i o n s .

A t o k e n of deep a p p r e c i a t i o n t o D r . J a v e d M u s a r r a t f o r h i s c o n s t r u c t i v e s u g g e s t i o n s and k i n d h e l p t h r o u g h o u t t h i s s t u d y .

I am d e l i g h t e d t o r e c o r d my deep a p p r e c i a t i o n and s i n c e r e t h a n k s t o Mis s Zehra Rehana f o r h e r a m i c a b l e h e l p and c o o p e r a t i o n d u r i n g t h i s w o r k .

I am a l s o t h a n k f u l t o M i s s S. I s l a m and Mr . S.A, Q a d r i f o r t h e i r h e l p f u l c o o p e r a t i o n .

My s i n c e r e t h a n k s a r e a l s o due t o my f r i e n d s and l a b c o l l e a g u e s , S h a h i d , R a s h i d , A s h g a r , S a i d , A d i l , Fahiro, Qasim, J a l a l , I b r a h i m and G o p a l , Mis s ( s ) R. Z a i d i , M.Mir2ay., R .D. G u p t a , T . Z e h r a , A i s h a , Veena , M r s ( s ) Afshan Naheed , A. Z a i d i , Pabeha and F a r a h .

M i n i s t r y of Env i ronmen t and F o r e s t s , Government of I n d i a , New D e l h i a r e g r a t e f u l l y acknowledged f o r f i n a n c i a l a s s i s t a n c e .

UL MALIK)

OONTENTS

C e r t i f i c a t e

Ac)aiowledgeiT»ent

L i s t o f a b b r e v i a t i o n s

L i s t of t a b l e s

L i s t of i l l u s t r a t i o n s

P r e f a c e ,

I n t r o d u c t i o n

M a t e r i a l and Methods

R e s u l t s

D i s c u s s i o n

B i b l i o g r a p h y

P a g e

V-

1.

36 .

5 3-

8 8 -

10 4-

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i i - i i i

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- 1 0 3

- 1 1 7

LIST OF ABBREVIATIONS

A d iT ip ic l l l l n

Ak a m i k a c i n

B b a c i t r a c i n

C c h l o r a n p h e n i c o l

CPU c o l o n y fofming u n i t s

Ct c h l o r o t e t r a c y c l i n e

Cx c l o x a c i l l i n

E e r y t h r o m y c i n

F r f u r a z o l i d o n e

FS f e c a l s t r e p t o c o c c i

G g e n t a m y c i n

h h o u r

K kanamyc in

MIC minimum i n h i b i t o r y c o n c e n t r a t i o n s

min m i n u t e s

ug micrograms

jul microxiters

MPN most probable number

Na nalidixic acid

N neomycin

P penicillin

Pb polymyxin B

R rifanpicin

S streptomycin

Sec seconds

T tetracycline

TBC total bacterial counts

TC total conforms

TEC total enterobacterial counts

ii

LIST OF TABLES

Table Page

1 Properties determined by bacterial plasmids. 1^-17

2 Taxonomic Relationship of the Entero- 29 bacteriaceae

3 Location of different sampling sites ^^

4 Seasonal variation in bacteriological para- 5 4-55 meters in the sewage water.

5 Seasonal variation in bacteriological para- 5 6-57 meters in the soil.

6 Locational variation and average bacterio- 59 logical parameters in the domestic sewage water.

Locational variation and average bacterio­logical parameters in the industrial sewage water.

60

8 L o c a t i o n a l v a r i a t i o n and average b a c t e r i o l o - - ^2 g i c a l pa r ame te r s of s o i l i n t h e v i c i n i t y of domest ic sewage w a t e r .

9 L o c a t i o n a l v a r i a t i o n and average b a c t e r i o l o - ^4 g i c a l pa rame te r s of s o i l i n t h e v i c i n i t y of i n d u s t r i a l a r e a .

10 A n t i b i o t i c r e s i s t a n c e p a t t e r n of E . o o l i ^5 s t r a i n s i s o l a t e d from sewage w a t e r .

11 A n t i b i o t i c r e s i s t a n c e p a t t e r n of E . c o l i 57 s t r a i n s i s o l a t e d from s o i l . ""

12 P e r c e n t r e s i s t a n c e a g a i n s t i n d i v i d u a l a n t i - 68 b a c t e r i a l agent moni tored i n t h e sewage w a t e r .

13 P e r c e n t r e s i s t a n c e a g a i n s t i n d i v i d u a l a n t i - 69 b a c t e r i a l agent moni tored in t h e s o i l . ,

14 I n c i d e n c e of metal r e s i s t a n c e and t h e i r MIC 7 i v a l u e in E . c o l i i s o l a t e d from sewage w a t e r .

15 . I n c i d e n c e of metal r e s i s t a n c e and t h e i r MIC 7 2 v a l u e in E . c o l i i s o l a t e d from s o i l .

C o n t d , , ,

iii

16. Incidence of metal resistance and their KIC 73 value in Enterobacter aeroqenes isolated from sewage water,

17. Antibiotic resistance pattern of 18 Enterobacter 75 aeroqenes strains isolated from sewage water.

18. Transforming-ability of AB1157 with the six test 79 plasmids.

19. Conjugative potential of multiply resistant 8 2 E.coli isolates with recipient AB1157 strain,

E.coli strains used in this study 44

i v

LIST OF ILLUSTRATIONS

F i g u r e P a g e

1 Aga rose g e l e l e c t r o p h o r e s i s p a t t e r n s of _ , p l a s m i d DNA i s o l a t e d from 5 E . c o l l Si and Sd i s o l a t e s .

2 Agarose g e l e l e c : ± r o p h o r e s i s p a t t e r n s o f 77 p l a s m i d DNA i s o l a t e d from 8 E . c o l i sewage w a t e r i s o l a t e s .

3 P l a s m i d c u r i n g and s u r v i v a l o f p l a s m i d 80 h a r b o u r i n g E . c o l i S i^ and Wd, s t r a i n s by t r e a t m e n t w i t h e t n i d i u m b r o m i d e .

Agarose g e l e l e c t r o p h o r e s i s of t h e p l a s m i d s p e c i e s i s o l a t e d from E . c o l i i s o l a t e s from w a t e r .

83

M o l e c u l a r s i z e Vs m o b i l i t y p l o t of t h e 85 t e s t s p e c i e s and t h e s t a n d a r d DNA.

Agarose g e l e l e c t r o p h o r e s i s of m u l t i p l e 8 6 p l a s m i d s p e c i e s i s o l a t e d from E . c o l i (Wig, Wd^, Wd^) s t r a i n s .

M o l e c u l a r s i z e Vs m o b i l i t y p l o t of t h e 97 t e s t ( m u l t i p l e s p e c i e s o f t h e R - p l a s m i d s i s o l a t e d from Wic# Wd^ and Wd s t r a i n s ) and s t a n d a r d DNA. " ^

P R E F A C E

The gram negative, non sporeforming bacilli make up a

large group of bacteria that include intestinal commensals/

such as Escherichia and Proteus, enteric pathogens, such as

Salmonella and Shigella and some strains of Escherichia and

Klebsiella as respiratory and urinary tract pathogens

Yersinia including the plague bacillus, and a variety of

saprophytes and plant pathogens (Freeman, 1985),

Application of sewage sludge on agricultural lands for

enhancing productivity has been a common practice in our

country for many years. Nowadays a serious attention is

being paid to the heavy metal contents of sewage sludge

before its land application, because of the tendency of up­

take of toxic metals and metalloids like Hg, Cd, Cu, Co, Cr,

Ag, Mo, Ni, Pb, Se, Te, and Zn by food crops and plants

(Webber, 1972; Wood, 1975; Lagerwerf e_t al_., 1976; Bhowal

et al,., 1987) .

Metal resistance in bacteria is frequently coded by

genes located on plasmids and transposons and is often

transferable intergenerically, interspecially and also from

in situ microflora to indigenous microflora (Datta and

Hedges, 1972; Smith, 1978; Bale et al_., 1988). Moreover the

metal resistance is often associated with resistance to

single or multiple drugs also (Kondo et a_l., 1974; Allen

et al., 1977; Foster, 1983) .

vi

Aiigarh city is famous for lock manufacturing facto­

ries. Hundreds of small and large scale factories are

supposed to spill tremendous amount of heavy metals into

the sewage and in the form of industrial effluents (Ajmal

fit al., 1980) .

The concentration of metal pollutants in the environ­

ment is usually low excepting in specific areas which are

polluted by various hospital and industrial wastes. However

unpolluted environment may also harbour metal resistant

organisms or organism that readily adapt to high concentra­

tion of metals. The incidence of plasmid bearing strain is

more in polluted site than in the unpolluted zone (Hada and

Sizemore, 1981) .

It is important to know the existence of plasmid

encoded metal resistances, their physiological and bioche­

mical characteristics, the mechanism of resistance and the

limiting factors in natural ecosystem so as to evolve a

"super bug" through recombinant DNA technology. It would

also be a fascinating task to search for new microbes that

would withstand natural milieu containing the metal pollu­

tants. Such microbes could be readily used to clean up

waste waters and soils containing toxic metals.

The constant exposure of the body flora of man and

animals to antibiotics has led to the selection and wide

dissemination in human population of antibiotic resi-stant

v i i

s t r a i n s of bac te r i a , R-factors which car ry a n t i b i o t i c and

metal r e s i s t ance can also be t ransmiss ible have become common

in the non-pathogenic E ,co l i of the alimentary t r a c t of man

and domestic animals. Many R"*" organisms e s p e c i a l l y those of

human and animal or ig in are expected to en te r sewage and

from t h i s , obviously to s o i l s (Smith, 1970; Bhowal e^ a l . ,

1987), E ,co l i with t ransmiss ib le r e s i s t ance to some broad

spectrum a n t i b i o t i c i s considered to be p o t e n t i a l l y very

dangerous because i t s r e s i s t a n c e might be t ransmit ted to

other pathogenic bac t e r i a l s t r a i n s , thus rendering the t r e a t ­

ment of in fec t ious diseases very d i f f i c u l t . Hence th i s sewage

water i s not onxy a source of infect ion but can also promote

the spread of aBbibiot ic r e s i s t a n c e .

In view of the c l i n i c a l and ecological s ignif icance

of the work as well as the urgency of the task and magnitude

of the problem coupled with the meagre work car r ied out in

t h i s d i r e c t i o n , attertpts have been made to study the i n c i ­

dence of R-piasmids harbouring s t r a i n of en terobac ter ia in

sewage water and soi l of the Aligarh c i t y . Another dimension

of the problem which i s equa l ly ra ther sometimes more iitpor-

tan t happens to be the taxonomy of the plasmid harbouring

organisms. Our study would also provide an appropriate index

for t he i r c l a s s i f i c a t i o n .

INTRODUCTION

General Properties of Enterobacteriaceae

The aerobic and facultatively anaerobic bacterial

flora of the large intestines of man and animals are corrpo-

sed of non-spore forming, nonacid fast, gram negative baci­

lli. They exhibit general morphological and biochemical

similarities and are grouped together in the large and

coirplex family Enterobacteriaceae (Ananthanarayan and

Paniker, 1986) . This family includes several genera like

Escherichia and Proteus, inhabiting in the intestines as

commensals. Salmonella and Shigella as enteric pathogens.

Yersinia as plague bacillus and a variety of other sapro­

phytes and plant pathogens (freeman, i985;?&'''»""*» ?£ oJ*/* " .

Since the enteric bacilli are closely related in

morphology, structure, biochemistry and antigenic make up,

some of their common properties are as follows :

Morphology i

Enteric bacteria are aerobic, facultatively anaerobic,

gram negative, nonsporulating bacilli. Capsules are formed

by a few, like Klebsiella and some strains of Enterobacter

and Serratia. Loose slime is frequently produced by Erwinia.

Most of the enteric bacilli are motile and when motility

occurs it is by means of peritrichous flagella.Shigella,

Klebsiella and Yersinia are typically nonmotile. Indivi­

dual cells vary some what in size, but typically they are

in range of 1.0 to 1.5 x 2.0 to 6.0 um. They occur singly

or in pairs (Braude, 1983) . The variation in size and

morphology is encountered, even within a species. In

some group, e.g. Proteus, there is evidence of filaments,

Spheroplast and other aberrant forms. Yersinia tend to be

some what coccoid or oval in shape and are smaller than

other members. Fimbriae are produced by many enterobacteria,

being found principally in Escherichia, Klebsiella, Proteus,

Salmonella and Shigella (Edwards and Ewing, 197 2) .

In colonial morphology, the enteric bacteria are all

very much alike, and it is not possible to make valid distin­

ction by these characters. On ordinary bacteriological media,

they tend to be smooth, convex with entire margins, moist

and shiny and are usually gray in colour. Rough colonial

forms are frequently seen. These are often dry, friable

and not easily emulsifiable in saline. The presence of cap­

sules or loose siime is usually reflected in dome shaped

colonies of mucoid and viscous consistency. Some strains

particularly of Escherichia and Proteus, are haemolytic on

blood agar. Although pigment production is rarely seen in

most genera, Serratia produce a red pigment under certain

growth conditions, while many Erwlnia colonies are cream

to yellow in colour (Martin and Washington, 1980) ,

Antigenic structure ;

Among the Enterobacterlaceae the designation of genus,

and in many cases species is based primarily on biochemical

parameters. Further differentiation to specific rank in

some instances and to serogroups/ serotypes, and varieties

is dependent upon serological reaction that detect surface

antigens. These antigens fall into three major classes i

somatic 0 antigen, flagellar H antigens, and capsular or

envelope K antigens.

0 antigens j The somatic 0 antigens are the heat stable

lipopolysaccharide common to all smooth, gram negative bac­

teria. They are present in outer membrane and are responsible

for the endotoxic activity allied with gram negative bacteria.

The antigenic specificity of these 0 antigens resides in the

0 specific side chains of the lipopolysaccharide and is

dependent upon the Kinds of sugar residues and their arran­

gement in the side chain (Simmons, 1971; OrsKov, 1977) ,

H antigens : The flagella of motile bacteria contain serolo­

gically specific proteins (flagellins) which unlike the 0

antigens, are heat labile and are destroyed by ethanol.

When these motile bacteria are reacted with specific anti-

flagellar antibodies, microscopic observation reveals a

loose association of many cells with tangling and inter­

twining of their flagella.

K antigens » A third class of antigens, the K antigens,

appear in Enterobacteriaceae as capsule or envelope anti­

gens which overlie the surface 0 antigens. Because of this.

the K antigens block agglutination by 0 specific antibody.

Antigens of this class are generally polysaccharides and

their reactivity is usually altered by heating, so that

treatment in this way yields strains agglutinable with 0

antiserum. The K antigens terminology varies some what in

different genera. In few Salrtonella that posses K antigens*

it is designated the Vi antigen. The K antigens of Bscheri-.

chia are generally polysaccharide, but in few cases, they

are coirposed of protein and occur as fimbriae on the cell

surface (Orskov, 1977) ,

Enterobacterial common antigens t In 1962, Kunin and his

colleagues described a haptenic substance of wide distri­

bution among enterobacteria that is now called the entero­

bacterial common antigen (EGA) or Kunin antigen. The hapten

occurs in two aggregative forms in the outer membrane. In

a few bacteria, the hapten is linked to the lipopolysaccha-

ride (LPS) and is immunogenic in the majority of enteric

bacteria, however it is in a free form and is not capable

of eliciting antibody response. The LPS-linked EGA is

relatively rare but is found in some R-mutants of Escherichia

coli and Shigella (Makela and Mayer, 1976; Mannel and

Mayer, 1978) ,

Flmbrial antigens : Most members of the Enterobacteriaceae

produce fimbriae or pili. The common pili are numerous on

the bacterial surface whereas the F or Sex pili are limited

in number 1-4 per cell and differ morphologically from

common pili (Rinno et al,, 1980),

Pyotein antigens t In common with other gram negative

bacteria, the Enterobacteriaceae contain several major

proteins in the outer membrane. These are antigenic and

contribute to the antigenic mosaic of enterobacteria.

There is considerable serological cross reactivity between

major outer membrane proteins from related strains e.g.

Escherichia coli/ and at least one of these may be common

to all Enterobacteriaceae (Hofstra and Dankert, 1979).

Besides major outer membrane proteins, Enterobac­

teriaceae also appear to possess a common lipoprotein

antigen (Griffiths et al,,, 1977),

Toxins I

In addit ion to the endotoxic a c t i v i t y associated

with the l ipopolysaccharide of the outer membrane, and

common to a l l wild s t r a i n s of en te robac te r i a , several

other toxins may be produced.

Those en te robac te r ia t h a t exh ib i t haeitolytic a c t i ­

v i t y such as enteropathic s t r a i n s of E ,co l i and some Proteus ,

usua l ly produce e x t r a c e l l u l a r haemolytic toxins t ha t may

have other cytotoxic p rope r t i e s as we l l . The diarrheagenic

enterotoxin produced by some s t r a i n s of E .co l i are of

considerable s ignif icance since these are responsible for

the secret ion of f l u id s and e l e c t r o l y t e s in to the lumen of

the snai l i n t e s t i n e . Several Shigellae especia l ly Shigella

dysentr iae type 1, produce substances with cytotoxic and

entero toxic a c t i v i t i e s . Enterotoxins are also occasional ly

encountered in ce r ta in Salmonella (Griesman, 1969; Richards

and Doughlas, 1978; Bergan and Olsvik, 1980; Brown, 1982).

Sal ient Features of Various Species In t h e Family Entero-

bac te r iaceae

Escherichia c o l i and i t s c h a r a c t e r i s t i c fea tures :

Escherichia co l i for t he f i r s t t ime was described

by Buchner (1885) and t h e genus was named a f t e r Theoder

Escherich (1888) who made a de ta i l ed study of t h i s micro­

organism. E.col i i s the predominant f acu l t a t i ve anaerobic

species in the la rge i n t e s t i n e and thus the species name

represents t h i s c h a r a c t e r i s t i c f ea tu re . According to the

Sergey 's manual/ the genus Escherichia has been grouped

under the family of Enterobacteriaceae (Bergey's manual,

1984), I t i s a gram negat ive s t r a igh t rod, measuring ( l -3 )x

(0.4-0.7) micron arranged singly or in p a i r s (Alcamo, 1987).

Cul tural c h a r a c t e r i s t i c s and colonial mozTpholoqy ; When

grown in l i qu id media, E .co l i produces a well dispersed

t u r b i d i t y in the temperature range of 10-46°C, but 37^C

i s t h e optimum temperature . Most E.col i s t r a i n s have f l a -

ge l l a and are mo t i l e . I t forms a var ie ty of colonies on

solid media e.g. large, thick, moist, smooth, greyish,

white or colourless, opaque or partially transluscent.

Smooth (S) type strains form shining convex colonies but

when repeatedly subcuitured they become rough (R) type and

form lustureless, granular colonies. The S —> R variation

is associated with loss of surface antigen and, usually

of virulence. Encapsulated variants produce mucoid colonies,

particularly when incubated at low teinperature and when

grown in media containing limited amount of nitrogen and

phosphorus and a high concentration of carbohydrate. Typi­

cal E.coli colonies are easily recognised by their character­

istic appearance on certain differential media. This bacter­

ium is largely a lactose fermenter and forms bright pink

colonies on MacConkey's medium. The colonies on the eosin-

methylene blue and the endo agar display a metallic sheen

characteristic.^-haemolysin is also produced on blood agar

in certain cases (Davis et al,, 1989),

Biochemical reactions t E.coli usually ferments carbohydrates

with the production of acid and gas, A few strains are how­

ever, anaerogenic producing acid without gas. Other subs­

tances with which different biotypes of E.coli react differe­

ntly include raffinose, rhamnose, sucrose, xylose, arginine,

glutamic acid and ornithine. Gram's staining and IMViC

reactions are commonly used for the identification of

enteric bacilli, and their identification is of prime

8

inportance in cont ro l l ing i n t e s t i n a l in fec t ions by preventing

contaminations of food and water suppl ies , E .col i d isplays

indol p o s i t i v e , methyl red p o s i t i v e , Voges-prauskaur nega­

t i v e and c i t r a t e u t i l i z a t i o n negative reac t ion i . e . IMViC++ ,

E .co l i i s also d is t inguished from other coliforms by i t s

a b i l i t y to form gas from lac tose in t e s t system incubated

a t 44 C (Cappucino and Sherman, 1987).

Host p a r a s i t e r e l a t i o n s h i p t E .col i i s a commensal t h a t

th r ives bes t in the human i n t e s t i n e . However, some micro­

organisms have the a b i l i t y to change g e n e t i c a l l y and become

v i r u l e n t . E .col i which was long considered an av i ru lent

commensal to humans, some times becomes oppor tunis t ic and

a t t a i n s pathogenic charac ter because ce r t a in toxin-producing

s t r a i n s have been i so l a t ed in the outbreaks of human d ia ­

rrhoea and ur inary t r a c t infect ions (Middiebrook and

Dorland, 1984) .

Salmonella t

The Salmonella are gram negative b a c i l l i whose

c e l l u l a r morphology c lose ly resembles and i s i nd i s t ingu i sh ­

able from other en te robac te r i a . All members except S ,

e n t e r i t i d i s serotypes pullorum and gal l inarum are a c t i v e l y

motile by pe r i t r i chous f l a g e l l a . No capsules are d i sce rn ib le

by l i g h t microscopy, although a polysaccharide surface

antigen designated as Vi, occurs in most s . typhi and a few

other Salmonella (Nolan, 1981) ,

The bac te r i a of "this group have simple n u t r i t i o n a l

requirements, growing readi ly on the usual nu t r i en t media.

Adequate growth obta ins in syn the t ic media containing

ammonium s a l t s as a ni t rogen source and simple carbon

sources such as glucose, pyruvate or l a c t a t e . A great

majority of s t r a ins do not requi re vitamins or amino ac ids ,

however some s t r a i n s of S.typhi require added t ryptophan.

They are f acu l t a t i ve anaerobes, growing equally well under

e i t h e r aerobic or anaerobic condi t ions . On the bas i s of

biochemical reac t ions , t h ree species of Salmonella are

d i s t ingu i shed . They are Salmonella cho le ra - su i s , S.typhi

and S . e n t e r i t i d i s (Harvey and P r i ce , 1979; Martin and

Washington, 1980) .

Salmonellosis in animals : Salmonellae infect a wide v a r i e t y

of animals and t h e i r importance as animal r e se rvo i r s of

human infec t ion i s well known. Food producing animals, i nc lu ­

ding swine, c a t t l e and fowl, may be na tu ra l ly infec ted . Man

cont rac t s Salmonellosis by consumption of infected f lesh

of such animals (Blaser, 1980) .

The Salmonellae responsible for animal in fec t ions

include S.cholera-suis and several serotypes of S . e n t e r i t i d i s .

Salmonella typhi in contras t t o most o ther Salmonella, i s

almost completely nonpathogenic for lower animals, but a

d isease closely resembling typhoid fever i s produced- in

Chimpanzees by ora l inoculat ion (Gaines, 1968) .

10

Salmonella enterltldls is generally responsible for

infections in rats and mice, and these animals become heal­

thy carriers of the bacilli (Kochan et al_., 1978).

Birds are quite commonly infected with Salmonellae,

Epidemics due to typhimurium serotype cause great destruc­

tion among canaries and other songbirds. Two barnyard

diseases of great importance are due to specific types,

the bacillary white diarrhoea of chicks is caused by sero­

type pullorum and fowl typhoid is caused by gallinarum

(Pelczar e^ al_., 1986). It is well known that cold blooded

animals may be infected with Salmonella. Ordinarily such

reservoirs of infection do not relate to human disease,

but a number of human infection have been acquired from

turtles and other aquarium pets (Chiodini and Sundberg,

1981), As a part of this problem fresh water aquarium

snails may also harbour these microorganisms (Bartlett and

Trust, 1976) .

Salmonella infections of man : Salmonellosis in humans is

almost always acquired by the ingestion of the microorganisms,

usually in contaminated food, milk or water. There are two

kinds of salmonellosis, one an acute gastroenteritis cha­

racterised by vomiting and diarrhoea in which a small propo­

rtions of patients usually the young children, become

septicaemic. The other is typhoid also called enteric fever.

11

a disease in which bacteremia and tissue invasion is noted

in the initial stages and synptoms are more general in

nature (Cohen and Gangarosa, 1978; Blaser and Newman, 198 2)

Enterobacter and Serratla :

The Enterobacter are found as commensals in the

intestine and are frequently isolated from soil and water.

They are only rarely encountered as infectious agents in

the community but may be responsible for a variety of

hospital associated or nosocomial infections (John et al.,

1982) . The predominant species responsible are E.cloacae

and E.aerogenes.

Serratia are, for the most part, free living sapro­

phytes but they are indeed capable of causing serious

infections under some circumstances and are regarded as

opportunistic pathogens. They have been iirplicated in infe­

ctions of the respiratory tract, UTI, meninges, and endo­

cardium (Yu# 1979). The most familiar of the Serratia are

the red pigmented varieties of S»marcescens«

Proteus, Providentia and Morqanella ;

The tribe Proteae is made up of three genera :

Proteus, Providentia and Morqanella. These were until

recently classified as Proteus and are thus related to

one another as well as to other enteric bacilli. Except

12

for some of the Providentla, these microorganisms are found

with some frequency in the intestinal tract of normal humans.

They are common inhabitants in soil and water that contain

decaying organic matter of animal origin and are usually

found in sewage. The phenomenon of swarming is typical of

Proteus and is consequence of the production of elongated

cells with excessive numbers of flagella (Williams and

Schwarzhoff, 1978). These microorganisms are regarded as

opportunistic pathogens and most infections are hospital

associated (Chow/ 1979). Among them urinary tract infect­

ions are the predominant type and these are most often due

to Proteus mirabilis (Turck and Stamm, 1981),

Klebsiella »

The genus Klebsiella consists of nonmotile, capsu-

lated rods that grow well on ordinary media, forming large

dome shaped, mucoid colonies of varying degrees of sticki­

ness, Klebsiellae are widely distributed in nature, occu­

rring both as commensals in intestines and as saprophytes

in soil and water. They have been classified into three

species based on biochemical reactions and into over 80

serotypes based on their capsular (K) antigens, K.pneumoniae

is the second most populous member of the aerobic bacterial

flora of the human intestines. It has become very impor­

tant cause of nosocomial infections, even replacing E,coli

in some centres (Martin and Washington, 1980; Baurnefied

et al_,, 1981) ,

13

Shigella ;

Shigella are gram negative, nonspore forming rods

related to other enteric bacteria. Some of them resemble

anaerogenic Escherichia and the typhoid bacillus in that

they ferment carbohydrates with the production of acid

but without gas. None of the dysentery bacilli are motile,

hence they do not contain flagellar antigens (Simmons, 1971)

Shigella are aerobic and facultatively anaerobic,

and their optimum temperature for growth is 37°C, Their

nutritive requirements are not conplex, since they can

grow upon ordinary nutrients media containing peptone and

beef extract. Colonies on MacConkey agar are colourless

due to the absence of lactose fermentation (Martin and

Washington, 1980) ,

Erwinia i

Erwinia is the irrportant phytopathogenic genus

belonging to the family Enterobacteriaceae and characteri­

sed by motile rods which normally do not require organic

N conpounds for growth. They produce acid with or without

visible gas from a variety of sugars. They invade tissues

of living plants and produce dry necroses, galls, wilt

and soft rots. In the latter cases the enzyme protopecti-

nase destroys the middle lamellar substances of the cells

(Singh, 1986) .

14

Soft rot of vegetables including potatoes caused

by different species of soft rot coliform bacteria is a

common storage and transit disease. In potatoes, these

bacteria not only cause soft rot of tubers but also black

leg and wilt of plants in the field. Several species of

Erwinia which cause plant diseases are E.carotovora«

E.atroseptica, E.aeroideae and E.phytophthora. Black leg

and soft rots of potatoes are caused by E.phytophthora

(Stapp, 1961).

Plasmids and their Characteristics Features

Plasmids are covalently closed circular extra-

chromosomal DNAs capable of autonomous replication. These

are ubiquitous among the bacterial genera and are also

found in some eucaryotic organisms (Davis and Rownd, 1972;

Meynell, 1973/ Lewin, 1977/ Broda, 1979).

Plasmids were discovered soon after the discovery

of bacterial conjugation in early 19501s by J, Lederberg

(Lederberg, 195 2) in family Enterobacteriaceae when it

became clear that there were two genetically determined

'mating* types and that genetic information was physically

transferred from a 'male' or donor type to a 'female' or

recipient type presumably involving the transfer of F

(Fertility) factor, Lederberg demonstrated that this

character for maleness was present in an extrachromosomal

15

genetic elements and in 195 2 he coined the term plasmid

to refer to all such extrachromosomal genetic system,

Plasmid encode a number of specialised function

that are generally nonessential for their bacterial host.

However, these plasmid encoded functions provide their

host cells with versatility and adaptability for growth

and survival under a variety of conditions, Plasmids and

transposable elements that they often contain may also

have played an integral role in the evolution of bacterial

species by promoting the distribution and exchange of

genetic information (Arber et al_, / 1985),

Plasmid mediated functions include genetic tran­

sfer, bacteriocin production and their resistance, anti­

biotic resistance and their production, resistance to

heavy metals and DNA damaging agents, metabolism of carbo­

hydrates and hydrocarbons, toxin and hemolysin production,

virulence and colonisation factors, tumorigenicity and

nitrogen fixation in plants. Recently, plasmids have be­

come the indispensable tools of modern molecular bio­

logical research (Womble and Rownd, 1988) , A detailed

list of properties determined by bacterial plasmids is

given in table I.

Classification of Plasmids

Identification and classification of plasmids are

16

Table - I i Properties determined by bacterial plasmids

1. Resistance properties ;

(a) Resistance to antibiotics - Aminoglycosides ( e . g . streptomycin, gentamycin,

amikacin - as a resu±t of enzymic modification by N-acetyla t ion. 0-nucxeotidylat lon or 0-phosphory-la t ion) ,

- Chloranphenicol (as a r e s u l t of enzymic modifica­t ion by 0 - a c e t y l a t i o n ) .

- Fusidic acid - Furans - p-Lactam antibiotics (e.g. benzyl penicillin, arrpi-

clllin, carbenicillin (as a result of enzymic clea­vage of the lactam ring).

- Sulphenamides, trimethoprim (as a result of alter­native drug-resistant target enzymes) .

- Tetracyclines (as a result of altered transport system)

- Erythromycin (as a result of altered ribosome bind­ing site,

(b) Resistance to heavy-metal cations - Mercury and organomercurials (as a result of reduc­

tases and hydrolases) - Nickel, cobalt, lead, cadmiumum, bismuth, antimony,

zinc, silver, thallium

(c) Resistance to anions - Arsenate, arsenite, tellurite, borate, chromate

(d) Other resistances - Radiation (e.g. u.v.. X-rays) - Phage and bacteriocin resistance (e.g. Inc P2 plas­

mids) - Plasmid - specified restriction, modification sys­

tems (e.g. Inc N plasmids)

2. Metabolic properties

- Ant ib io t ic and bac te r ioc in production ( e .g . by Streptomvces, Escher ichia , Pseudomonas, Baci l lus , Streptococcus)

- Metabolism of s inp le carbohydrates ( e . g . l ac tose , sucrose, raffinose)

- Metabolism of coirplex carbon compounds (e .g . octane and other n-alkanes, p or m-xylenes, p or m-toluenes, cartphor, nicoline)

- Metabolism of p r o t e i n s (e .g . case in , gelat in) - Metabolism of opines (by Ti^ Aqrobacterium) - Nitrogen f ixation (by Rhizobivim)

17

- S"-Endotoxin by sporulating B. thurlnqlensls - Other properties (e.g. citrate utilization, H2S production, leucine biosynthesis)

3. Properties contributing to pathogenicity or sygribiosis

- Antibiotic resistance and bacteriocin production - Toxin production (e.g. enterotoxins of Escherichia coli and Staphylococcus aureus, exfoliative toxin by S.aureus, haemolysins by Escherichia, Staphylo­coccus and Streptococcus)

- Colonization antigens of E.coli (e.g. K88, K89, CFAI, CFAII) "•

- Tumorigenicity (by Ti Aqrobacterium) - Host specificity (of Aqrobacterium and Rhizobium) - Nodulation (by Rhizobium)

4. Conluqal properties

- Sex pili and associated sensitivity to pilus-specific phages

- Surface exclusion - Response to and inhibition of pheromones (in Streptococcus)

- Fertility inhibition

5, Replication-maintenance properties

- Sensitivity to curing agents -, Inconpatibility - Host range - Copy number - Tenperature-sensitive replication

6, Other properties

- Gas vacuole formation in Halobacterium - Interference in sporulation of Bacillus pumilis - Sensitivity to bacteriocins (in Aqrobacterium and Streptococcus)

- Translucent-opaque colony change in Mycobacterium - Regulation of melanin production in Streptomyces

Adopted from "Methods in Microbiology" (1984), Bennejt, P.M. and Grinsted, J. (eds.). Academic Press.

18

especially inportant in medicine because genes for clini­

cal traits such as drug resistance and virulence factors

are frequently present on plasmids (Couturier £i 5 .«

1988), Three major classes of plasmids have been charac­

terised most extensively, which include conjugative (F)

plasmids, bacteriocinogenic plasmids and drug resistance

(R) plasmids. The F plasmid is the classic fertility

factor that is capable of its own transfer, the transfer

of other plasmids and the transfer of the host bacterial

chromosome during bacterial conjugation, Col-plasmids and

R-plasmids in contrast mediate the production of colicins

and confer drug-resistance to their bacterial host cell

respectively (Womble and Rownd, 1988).

Some authors have classified R-plasmids on the

basis of (a) their transfer by conjugation (b) inconpati-

bility, and (c) curing or elimination (Novick, 1969;

Couturier e_t aJ,., 1988) .

R-Pla?mid3 and their Salient Features

The best known plasmids from the stand point of

the human medicine are those that specify antibiotic resis­

tance (Eiwell and Shiply, 1980) , R-plasmids in bacteria

have resistance markers, either for one antibiotic (Thom­

son and Biigeri, 1982; Hirsh et al ,, 1989) or for more

than one antibiotics (Hardy and Haifeli, 1982; Amyes, 1989

19

Hirsh et al,., 1989). R-plastnid mediated antibiotics

resistance in enteric bacteria also referred to as mul­

tiple antibiotic resistance or infectious antibiotic

resistance is known ever since its discovery in Japan

in 1959 (Akiba, 1959; Feary et aJ,,, 1972; Womble and

Rownd, 1988). The R-plasmids are most common in bacteria

from clinical and veterinary sources where exposure to

antibiotics is likely to be high (Smith, 1977; 0 Brien

et al,, 1986), Many R-organisms especially from human

origin, would be expected ultimately to enter the sewage

(Smith, 1970). Many authors have reported their occurre­

nce at low frequency In bacteria from water and other

sources (Sizemore and Colwell, 1977; Talboot et al.,19e0).

Besides antibiotic resistance, R-plasmids can often

confer certain additional properties on their host cell

and can be accordingly characterised. Some of the proper­

ties include resistance to heavy metals and their salts

like arsenate, arsenite, nickel and cobalt ions (Smith,

1967; Dabbes and Sole, 1988), Plasmids also confer resis­

tance to UV radiations in Streptococcus, Pseudomonas and

Enterobacteriaceae (Jacob et al ,, 1977; Clewell, 1981) ,

In addition to their resistance properties some other

properties of R-plasmids Include : fertility inhibition

to a male bacterium, propagation of specific phages,

phage restriction, colicin production, colicin resistance.

20

plasniid transfer, plasmid inconpatibility, plasmid insta­

bility and plasmid curing (Siccardi, 1966; Meynell et al.*

1968; Bannister and Qlover, 1968j Novakova, et al.., 1969|

Khatoon et a_l.. # 1972; Stanisich, 1984; couturier et al.,

1988; Womble and Rownd, 1988; Hirsh et al.., 1989; Jewell

and Collins-Thoitpson, 1989) .

Plasmid encoded resistance »

(i) Metal resistance t Heavy metal resistance in bacteria

is frequently coded by genes located on plasmids and trans-

posons and is often transferable integenerically, inter-

specially and also from iri situ microflora to indigenous

microflora (Datta and Hedges, 197 2; Smith, 1978; Bale

et £l., 1988). The metal resistance is often associated

with resistance to single or multiple drugs (Hall, 1970;

Kondo et al ., 1974; Fostor, 198 3), phenolic compounds

(Pickup et al,., 198 3) and pesticides (Don and Parriberton,

1981) .

The concentration of metal pollutants in the envi­

ronment is usually low excepting in specific areas which

are polluted by various hospital and industrial wastes.

However, unpolluted environments may also harbour metal

resistant organism or organisms that readily adapt to high

concentration of metals. The incidence of plasmid bearing

strains is more in polluted site than in unpolluted region

(Hada and Sizemore, 1981) .

21

Resistance to toxic metals such as mercury, arsenic

and cadmium is frequently plasmld encoded in both gram

positive and gram negative bacteria (Summers and Silver,

1978; Tynecka et al,, 198l7 Mobiey and Rosen, 198 2).

Trevors et al.. (1985) have reported four basic mechanisms

by which plasmids encode resistance against metals and

these are -

i) inactivation of the metal

ii) alteration of the site of inhibition

iii) impermeability of the metal

iv) metal by pass mechanism

Plasmld determined mercury and orqano-mercury resistance ;

Mercury resistance determinants are common, among the

naturally occurring and clinically isolated bacterial

populations and are frequently found on plasmids (Trevor's

et al., 1985; Summers, 1986; Foster, 1987; Silver and

Misra, 1988) .

The earliest observation of the presence of bacteria

with strong resistance towards mercury and organomercurials

came from Japan where resistance bacteria were isolated

from organomercurlal polluted soil (Vaczi e^ al.., 1962;

Tonomura and Kanzaki, 1969) and from the united kingdom

among the penicillinase producing clinical isolates of

S. aureus (Richmond and John, 1964; Dyke et al,., 1970) and

subsequently, it was described in Escherichia coli (Smith,

1967) .

22

Many of the antibiotic resistance plasmids in both

gram negative and gram positive bacteria have the genes

2+ to determine resistance to Hg (Novick and Roth, 1968;

Schottel et al_., 1974; Summers and Jacoby, 1978) . In the

Hammersmith hospital London, in a collection of over 800

plasmids transferred into a common host, E.coli K12 from

a wide range of host species including, Proteus, Provi-

dentia. Salmonella, Serratia, Shigella and Klebsiella,

the frequency of genetic determinants of mercury resis­

tance was about 25% (Schottel et ad., 1974) .

Plasmid determined cadmium resistance : Genes resljstant

to cadmium are located on both plasmids and chromosomes

(NovicK and Roth, 1968; TynecKa et al,., 1981; Surowitz

1984), In S.aureus, cadmium resistance is attributed to

reduced uptake of Cd via Mn system (Chopra, 1971;

Chopra, 1975). Recently Silver and Kisra (i988) have

suggested six or more cadmium resistance systems in bac­

teria. The first two systems involved are Cad A and Cad B

of S.aureus plasmids that also confer resistance to seve­

ral other heavy metals (Perry and Silver, 198 2) .

Plasmid determined arsenic resistance j Role of plasmids

in resistance to arsenate has been studied by Silver

et al. (1981), though it has been known for years in S_,

aureus (Novick and Roth, 1968) and Escherichia coli (Hed­

ges and Baumberg, 1973). The plasmids involved are pl524

23

and R773. Arsenite resistance is associated with anti­

biotic (Smith, 1978; Summers et ad,, 1978) and mercury

resistance (Nakahara et al.., 1977), Silver et al_. (i98l)

have reported that piasmid mediated arsenate, arsenite

and antimony (III) resistances are coded for by an indu­

cible operon like system in both , aureus and E.coli.

Mobley et al . (i983) have demonstrated that in IncF

plasmid R773 of E.coli, arsenate, arsenite and antimonate

resistance are carried by a29Kb EcoRI fragment, Beliiveau

et al, (1987) suggested that three plasmids encoded pro­

teins Ars A,B and C are involved in the resistance of

E.CO1i to arsenate. Arsenite is not oxidised to the less

toxic arsenate by the plasmids of E.coli or S,aureus

(Bellieveau e_t al., 1987) ,

A 69Kb conjugative plasmid pDG 10l was found in

Corynebacterium flaccumfaciens sub spp, oortii which codes

for resistance to arsenate, arsenite and antimony (Hende-

rick et al,, 1984) ,

Other plasmid determined metal resistance t Tellurite and

tellurate resistances have been detected from plasmid

mediated determinants (Summers and Jacoby, 1911 i Taylor

and Summers, 1979). Jobling and Ritchie (1987, 1988) have

recently cloned the tellurium determinants from a large

Alcaliqenes plasmid into E.coli, where it was expressed

from 3,55 Kb DNA fragment.

24

Resistance to cobalt and nickel has been reported

in 12 clinical isolates of Enterobacter spp, harbouring

R-plasmids (Smith, 1967) .

Some Staphylococcus aureus plasmids determine resis­

tance to inorganic lead conpound (Novick and Roth, 1968),

Copper resistance is associated with an antibiotic

resistance plasmid in E.coli (Ishihara et al_,, 19 78).

(ii) Drug resistance : The increasing use of antimicrobial

agents has provided a strong selective force favouring the

survival of those bacterial strains that have acquired

resistance to such agents either by mutation or by inheri­

ting R-plasmids from unknown sources (Davis and Smith,1978),

Resistance plasmids are found in wide variety of

bacterial genera including both gram positive and gram

negative organism, Plasmids may be endowed with the capa­

city of self transmission (Conjugative) or non transmi­

ssible (non conjugative) .

Various biochemical mechanisms have been proposed

for resistance to different antibiotics (Davis and Maas,

195 2; Davis and Smith, 1978) . These resistance mechanisms

include (a) alteration of the target site in the bacterial

cell, (b) interference with drug transport, (c) enzymatic

detoxification of antibiotics, and (d) by-pass mechanism.

In addition to these mechanism there are some unknown drug

resistance mechanisms also (Davis and Smith, 1978).

25

Some Inpor tan t Charac te r i s t i c s for Studies on the

R-plasmld Harbouring Cells

The a b i l i t y of a proper ty to be t ransfer red from

one bacterium to another through conjugation and/or t r a n s ­

formation provides a good presunptlve evidence of plasmid

involvement, p a r t i c u l a r l y i f the frequency of t ransfer i s

high. Transfer experiments t ha t are conducted by sirrply

mixing cu l tu res of the t e s t bacterium with a su i t ab le r e c i ­

p i en t s t r a i n can give r i s e to progenyby conjugation and

transformation.

(1) Con luqation »

Conjugation i s the highly spec i f i c process whereby

DNA is t ransfer red from donor to r e c i p i e n t bac ter ia by

a mechanism involving c e l l to c e l l contac t (Lederbeirg, 195 2) .

Conjugation has been detected in many members of the Entero-

bac ter laceae (Jacob e_t al. , , 1977) and in other gram negative

and gram var iable b a c t e r i a .

Almost a l l types of R-plasmlds of more than 5x10

daltons are conjugative in nature . R-plasmids are composed

of two g e n e t i c a l l y and phys ica l ly d i s t ingu i shab le compo­

nents J (1) a r e s i s t ance t ransfer factor (RTF) tha t har­

bours the genes for se l f t r a n s m i s s l b i l i t y ( tra) and auto­

nomous r e p l i c a t i o n ( rep) , and (11) a r e s i s t ance determinant

(R-determinant) , The R-determinants harbours the majority

of r e s i s t ance genes (Clowes, 1972; Davis and Rownd, 1972) ,

26

The plasmid transfer by conjugation is possible

both within and outside the boundaries of the genus (Marniur

et al,,, 1963; Datta and Hedges, 1972; Datta, 1975). E.coli

is also reported to transfer its R-plasmids to various

pathogenic as well as non pathogenic bacteria (Baron and

Faikow, 1961; Oleson and Gonzalez, 1974; Jeanclaude et al.,

1988) .

(ii) Transformation j

This is the process by which DNA from one cell (the

donor) is taken up by another (the recipient) directly

from the surrounding medium (Cohen e_t al.., 197 2) , There

are many genera of bacteria in which transformation has

been successfully demonstrated e.g. all genera in Entero-

bacteriaceae. Bacillus. Haemophilus (Smith e_t £1./ 1981) ,

Staphylococcus (Lacey, 1975) and Streptococcus (Clewell,

1981) , Some genera of bacteria are able to undergo physio­

logical transformation, but E.coli is not conpetent to

physiological transformation (Saunders et al_,, 1984).

However the E.coli can be made susceptible to take up for­

eign DNA under artificial conditions like treatment with

transition metals (Ca"*"*", Rb"*"*") , with intermittent heat

shock etc. (Lederberg and Cohen, 1974; Kushner, 1978).

(ili) Plasmid instability and curing ;

One of the ways to ascertain whether or not a

particular character is associated with plasmid, the

27

el iminat ion (curing) of plasmids provides an appropriate

experimental b a s i s . The c r i t e r i o n of curing can be used

both in conjugative and non-conjugative plasmids{Khatoon

and Ali-Mohammad, 1986). I t i s expected, therefore , t h a t

in any growing population of plasmid harbouring bac t e r i a ,

p lasmid- less segregants w i l l occasional ly be produced as

the e r ro r in the process of plasmid r e p l i c a t i o n or pajfti-

t ioning to daughter b a c t e r i a . Thus t h i s loss of plasmid,

or curing can occur e i t h e r spontaneously (Novick, 1969)

or under the influence of the physical ( s t ad l e r and Adel-

berg, 197 2) and chemical agents (Novick, 1969; Stanis ich

1984) . Some of these curing agents can mutate DNA (Will-

e t t e s , 1967) , These agents are ind iv idua l ly e f fec t ive

only aga ins t some plasmids, and t h e i r e f f ec t on newly

discovered plasmid cannot be predicted (Mitsuhashi e t a l . ,

1961; S tan is ich , 1984).

The genera l ly used curing agents are acr idine

dyes (Hirota, 1956; HirOta and l i j lman, 1957) , ethldium

bromide (Bounchaud e t al, . , 1969; Jones and Sneath, 1970;

Hardman e t al,, , 1986; Khatoon and Ali-Mohammad, 1986;

El-Syed e^ SJL»» 1988), many mutagens (Wi l l e t t e s , 1967;

Chakrabarty, 1972), some a n t i b i o t i c s (Ikeda e t al^., 1967;

Yoshikawa and Sevag, 1967; Hahn and Ciak, 1971; Lacey,

1975; McHugh and Swartz, 1977; Molnar e^ a_l,, 1978;

Selan e_t al_,, 1988), chemicals l ike sodium dodecyl s u l -

28

phate (Tomoeda et ed., 1968; Inuzaka et a^., 1969;

Tomoeda et ad., 1970), metal ions like Ni, Co (Hirota,

1956) and also some physical agents like heat (May e_t al.,

1964; Stadler and Adelberg, 1972; Jayaratne et al,, 1987) .

Classification of Bacteria and their Taxonomic Studies

The classification of organisms or their taxonomy

has two purposes : one is to group the organisms of same

kind, the descriptive classification and the second pur­

pose is to provide a natural phylogenetic classification.

In the descriptive classification, organisms have been

grouped on the basis of information collected about a given

kind, that can be pooled and conpared. This type of class­

ification is based on the characters of the organisms

which a taxonomist can enploy (Mandel, 1969) ,

Bacteria have been classified by microscopic exa­

mination on the basis of shape, size and staining charac­

teristics. Bacteria also possess certain characteristic

structures such as the capsules, flagella, spores, etc.

whose presence or absence is used as a tool in identifi­

cation. Morphological, cultural and colony characteristics

provide useful information about the different organisms

but these can not be used as the sole criteria for class­

ification. Further, biochemical, serological and specific

tests are often required to be performed before bacteria

are cleariy identified (Pelczar et al.., 1986).

29

Tabic 2 J 'lnx<tn<)inic Rela t ionships of (he F.u(cr<»l)ac(criacc'ac

Tribe

llsi.hcrichic.ic

ildvk,ini!>icllc.ic

S.ilnioncllc.ic

KlehMcllciic

I'rotccac

Ycn>m)C3c

[lr^^lnlC.lC

Genus

l.schfnchia

.Shij^'fllii

I'llwtirdsielta . .

r Salmonella

Arizona

r

Ciirobacter

Klebsiella ..

Enierobacter.

Hafnia.

Serrano

Proteus

Provideniia.

[_ Morganella.

Yfrsinia

Erwiniu

I'ectinohacienitm

Species

/. col,

S dvseniernie S flexnen S hovdii S ionnei

E tarda

S. cholerasut^ S. typhi S. enteniidi-s

A. /iias/iuvMi

C frcundn C divcrsus C anialonaiiciis

i' •>i\c,iii\i)n\ae h ozacnae K rhinoscleromaiis K oxyioca

E aerogenes E cloacae E. agfitomeraiis E. saKdsakii

[_ E. gergoviae

H alvei

S. marcescens S liquifaciem S. rubtdaca

P. mirabdis P. vulgaris

P. reltgeri P. sluartii P. alcaliquifaciens

M. morganii

Y. pesiis Y pieudoiubcrculoiis Y. enterocolitica y. ifiwrmedia Y frcdenksenii Y. ruckeri

Adapted from ihc projiDs.ils in Brenner,_c/ d/_. , ( 1 9 7 7 )

30

A c l a s s i f i c a t i o n system was given by Kaufman and

his co l labora tors in 1940s. In th i s system E,col i was

subdivided on the bas is of antigen l i k e 0, K and H (Vahlne,

1945; Kaufman, 1975) , In 1976, an analys is of OjH se ro ­

types and biotypes was used for c lass i fy ing E.col i causing

diarrhoeal diseases (OrsXov e t al_., 1976) , Numerous taxo-

nomists continue to use serotyping and biotyping for

c lass i fy ing bac te r ia , not s o l e l y because of t r a d i t i o n ,

but these r e s u l t s also allow ready pigeon holing of E ,co l i

i s o l a t e s and often c o r r e l a t e well with disease s p e c i f i c i t y .

A successful taxonomic analys is of E .co l i population

s t ruc tu re s would include quan t i t a t i ve est imates of the

re la tedness between b a c t e r i a l groups. This might yield

valuable ins ights into microbial ecology and pathogenic i ty

(Achtman and Pluschke, 1986),

Many c l a s s i f i c a t i o n methods have been used in E .co l i

taxonomic s t u d i e s . Among these , s tarch gel e l ec t rophore t i c

analysis of cytoplasmic isoenzymes, a c l a s s i c a l technique

in the taxonomy of eucaryot ic organisms has been ex tens ive­

ly used. Other methods based on r e s i s t ance to co l i c ins

and other toxic chemical, DNA-DNA hybr id iza t ion or seque­

nce analys is of rRNA or p ro te ins have a lso been employed

in taxonomic s tudies (Achtman and Pluschke, 1986) ,

Moreover, SDS-PAGE CMP (Outer Membrane Protein)

p a t t e r n s and OiK serotypes have also been used to c l a s s i f y

31

E.coll from epldendologlcal sources into various groups

(Paahkanen e^ al,., 1979; Jaan and Jaan, 1980).

Isoenzyme analysis (Ochman and Salander, 1984) ,

OMP analysis and serotyping collectively defined E.coli

into 15 different clones (Achtman and Pluschke, 1986).

Whereas, it was typed into 160 0 groups, by Orskov and

Orskov, (1984). The 0 serotyping is based on the reacti­

vity of the somatic 0 sugar chains of LPS (lipopolysaccha-

ride) with hyperimmune rabbit antigens.

Restriction of phages is a property associated with

sorrte plasmids including the R-plasmids (Siccardi, 1966) .

Plasmids also interfere with phage propagation (Duckworth

et al., 1981). These two properties of phages with their

bacterial hosts have been exploited for the grouping of

the bacteria like E.coli (Meynell et al., 1968).

Phage typing of certain bacterial strains is some­

times difficult and confusing for examples S_, aureus

strains are not typable by bacteriophages of international

typing set. Supplementary bacteriophages therefore have

been isolated to distinguish these non typable strains of

S. aureus (Vickery et al.., 1983; Richardson et al_,, 1988) ,

Antibiotic sensitivity patterns or antibiograms

have been used for the classification and grouping of

bacterial strains by a number of workers. This typing

32

method is faster and more cost effective than biochemical

analysis and bacteriophage typing (Gillespie et al.,

1990). E.coli isolated from porcine fecal origin, patients

of urinary tract infection and patients from Egypt have

been divided into various groups on the basis of their

multiple antibiotic resistance pattern or antibiograms

(Ohmae et al.., 1980; Elkhovly, 1981; Gees et al., 1982).

Molecular Genetic Approach to Bacterial Taxonomy :

The approaches like serotyping, biotyping, phage

typing and numerical taxonomy described for the classifi­

cation have ordered bacteria rather tentatively, in terms

of phenotypic traits. The development of molecular gene­

tics, however has now revolutionised taxonomy. This taxo­

nomy is based on macromolecular sequence homology (Brenner

and Falkow, 1971) .

The sinplest comparison is the base conposition of

DNA, This can be estimated easily from melting tenperature

or buyont density. The base corrposition is essentially the

same in all vertebrates about 40 moles per cent guanine,

cytosine [G-^-C) and 60 per cent adenine, thymine, (A+T) .

But the base conposition among bacteria vary remarkably

from about 30-70 moles per cent (Marmur et al_., 1963).

DNA sequence homology (DNA-DNA homology) can be

measured quantitatively in terms of the ability of the

33

DNA strand from two different sources to form molecular

hybrids iji vitro. Among bacteria, DNA-DNA hybridisation

is useful only within closely related groups because it

quickly vanishes in the broad range of variation between

more distant organisms, Ribosomal RNA hybridisation to

DNA is useful for estimating more distant kinship among

bacteria. These processes have been employed in descrip­

tive classification of bacterial strains (Brenner and

Falkow, 1971; Schiffer and Stackebrandt, 1983).

DNA-DNA hybridisation of R-plasmid harbouring

airpicillin resistant gram negative bacterial strains was

used for their characterisation and classification. All

of the anpicillin resistant gram negative strains demons­

trated sequence homology (Foek-Ruediger and Rainer, 1982).

Since the antibiotic resistance in bacteria is

usually conferred by plasmids. Plasmid profiles have

therefore, been used for identification and corrparison

of bacterial strains in addition to antibiograms and phage

typing, Eiectrophoretic patterns of plasmid were used for

bacterial identification. 7 2 E.coli strains conferring

resistance to multiple antibiotics have been shown to

contain single type of plasmid by their eiectrophoretic

patterns (Ohmae et al ., 1980). Similarly 5-6 plasmids

ranging from 30-70 Mdal, have been isolated from 30 stra­

ins of bacteria (Vakulenko et ad,, 1980) .

34

Antibiograms, plasmid profiles and phage typing

have been jointly used for the classification of E.coli,

Salmonella typhimurium and Staphylococcus aureus strains

from clinical origin (Frost et al., 1982; Martinez et

al., 1987; Gillespie et al,., 1990).

Restriction endonuclease fragmentation of DNA or

plasmids and molecuxar size differences have also been

used for taxonomic studies of various bacterial spp.

(Achtman and Plusckhe, 1986).

Hardman et_ al.. (1986) have used restriction frag­

mentation and molecular size difference of plasmids for

the characterisation and classification of four Pseudomonas

and two Alcaliqenes species. Shinji and Yokiko (1988) have

classified E.coli strains by corrparing their plasmid pro­

files and the restriction fragments of these plasmids.

Burnie et al, (1989) have used EcoRI restriction enzyme

fragmentation patterns and immunoblotting to differentiate

and classify methicillin resistant isolates of Staphylo­

coccus aureus.

Objectives of the M.Phil work :

As we have already defined our problem in preface,

the present literature survey enlightened us to initiate

the work on the following lines j

1. Estimation and Enumeration of bacterial densities viz.

35

TBC, TEC, TC and FS in sewage and soil in the neigh­

bourhood of Industrial area of Aligarh,

2. Correlation between the bacterial population from domes­

tic sewage water and industrial effluents as well as

from soils of the same area,

3. Prevalence of antibiotic resistance if any, in entero­

bacterial population of sewage and soil origin.

4. Prevalence of metal resistance in enterobacterial popu­

lation derived from soil and sewage water.

5. Incidence of multiple antibiotic and metal resistance,

if any, in the enterobacterial population.

6. Evaluation of correlation between the drug and metal

resistant enterobacterial population in domestic sewage

water and industrial effluents as well as that between

soils and sewage.

7. Studies on the involvement of plasmids in mediating

such resistances and their possible transfer by con­

jugation and transformation.

8. Confirmation of the role of plasmids in displaying the

test resistance characteristics, exploiting the pheno­

menon of curing of harboured cells under certain

conditions,

9. Isolation and characterisation of R-plasmids from the

above mentioned isolates by agarose gel electrophoresis,

10. Determination of molecular size using known plasmid

markers and DNA fragments.

MATERIAL AND METHODS

36

MATERIALS

MEDIA

Azlde dextrose broth (pH 7.2)

Peptone i 15 g/1

Beet extract i4.5 g/1

Dextrose t7,5 g/1

Sodium chloride jO,2 g/1

Sodium azide »0.2 g/1

EMB agar (pH 7. 2)

Peptone t 10.0 g /1

Lactose : 5.0 g /1

Sucrose ; 5.0 g /1

Potass ium phospha te t 2.0 g /1

Agar X 13.5 g / 1

Eosin Y J 0.4 g / 1

Methylene Blue » 0 , 0 6 5 g / l

MacConkey's agar (pH 7.1)

p a n c r e a t i c d i g e s t of g e l a t i n t 17.0 g / 1

P a n c r e a t i c d i g e s t of ca se in j 1.5 g / 1

Tissue i 1.5 g / 1

Bi le s a l t s i 1.5 g /1

Sodium c h l o r i d e : 5.0 g / 1

Agar 1 15.0 g / 1

Neutra l red » 30.pmg/l

Crys t a l v i o l e t ; l.Omg/1

37

MacConkey's broth (Double Strength) (pH 7.4)

Peptone . 40.0 g/1

Lactose : 2O.O g/1

Bile s a l t s ; 10.0 g/1

Sodium chlor ide • 10.0 g/1

Neutral red ; 0 . i 5 g / l

MacConkey's borth (Single strength) (pH 7.4)

Peptone , 2O.O g/1

Lactose , 10.0 g / i

Bile s a l t s ; 5,0 g/1

Sodium chlor ide • 5.0 g/1

Neutral red » 0.075g/l

Nutr ient agar (pH 7.4)

Peptone , 5.0 g/1

Sodium ch lor ide $ 5,0 g/1

Beef ex t r ac t • 1.5 g / i

Yeast e x t r a c t • 1,5 g / i

Agar » 15.0 g/1

Nutr ient broth (pH 7.4)

Peptone j 5.0 g/1

Sodium chlor ide ; 5,0 g/1

Beef e x t r a c t . 1,5 g / i

Yeast e x t r a c t j I .5 g / i

38

SIM agar (pH 7.3)

Peptone

Beef extract

Ferrous ammonium sulfate

Sodium thiosulfate

Agar

Simmons citrate agar (pH 6.9)

Ammonium hydrogen phosphate

Dipotassium phosphate

Sodium chloride

Sodium citrate

Magnessium sulfate

Agar

Bromo thymol blue

Soft or Top agar

Nutrient broth

Agar powder

Triple sugar iron (pH 7.4)

Peptone

Tryptone

Yeast extract

Beef ex t r ac t

La'ctose

Sacchasose

30.0 g/1

3.0 g/1

0.2 g/1

0.2 g/1

3.0 g/1

: 1.0 g/1

: 1.0 g/1

J 5.0 g/1

; 2.0 g/1

i 0.2 g/1

. 15.0 g/1

: 0.08g/l

: 13.0 g/1

: 7.0 g/1

t 10.0 g/1

» 10.0 g/1

* 3.0 g/1

» 3.0 g/1

s 10.0 g/1

: 10.0 g/1

39

Dextrose i 1,0 g/1

Ferrous sulphate : 0.2 g/1

Sodium chloride j 5,0 g/1

Sodium thiosulphate j 0,3 g/1

Phenol red : 0,024g/l

Agar ; 12.0 g/1

Trypticase soy broth (pH 7. 2)

Trypticase : 15.0 g/1

Phytene x 50.0 g/1

Sodium chloride t 5,0 g/1

REAGENTS AND BUFFERS i

Alkaline SDS

SDS ; 1.0 %

NaOH I 0,2 N

Ammonium acetate Solution

Ammonium acetate i 0,1 K

Barrits reagent

Soln. A ; Alpha-naphthol : 5,0 gm

Ethanol (absolute) : 5.0 ml

Soln. B : Potassium hydroxide : 40 gm

Calcium chloride solution

Calcium chloride : O.l M

40

C r y s t a l v i o l e t

C r y s t a l v i o l e t ( 8 5 % d y e c o n t e n t ) ; 1,0 gm

D i s t i l l e d w a t e r : lOO ml

D e c o l o r l z e r

E t h a n o l (95%) ; 250 mi

A c e t o n e ; 250 ml

E l e c t r o p h o r e s i s b u f f e r ( T r l s - a c e t a t e , pH 8 , 0 )

T r l s J 9 . 7 gm

EDTA : 0 . 7 gm

A c e t i c a c i d ( g l a c i a l ) j 2 . 2 8 ml

D i s t i l l e d w a t e r j 197 ml

G r a m ' s I o d i n e

I o d i n e c r y s t a l s . 1,0 gm

P o t a s s i u m I o d i d e j 2 . 0 gm

D i s t i l l e d w a t e r , 30O ml

K o v a c ' s r e a g e n t

p - D l m e t h y l a m i n o b e n z a l d e h y d e ; 5 , 0 gm

Amyl a l c o h o l : 7 5 . 0 ml

HCl ( c o n c e n t r a t e d ) j 2 5 , 0 ml

L y s i s b u f f e r

T r i s - c l (pH 8 , 0 ) i 2 5 . 0 mM

EDTA i 10 mM

G l u c o s e . 50 mM

L y s o z y m e , 2 i r g /ml

41

Marker dye

Glycerol

Bromophenol blue

EDTA J

Methyl red s o l u t i o n

Methyl red ;

Ethyl a l coho l (95y«) :

Reagent A

TE buffer c o n t a i n i n g

5% dow co rn ing ant ifoam RD emuls ion .

Reagent B

1 M NaOH saturated at 20°C

with SDS. Distilled water ;

Safranin

Safranin 0

(25% solution in 95% ethyl alcohol);

Distilled water ;

Sodium acetate solution

Sodium acetate ;

CHEMICALS

The following chemicals were used

; 50.0 %

I 0.05 %

J 40 mM

; 0.1 gm

: 300 ml

200 ml

10 ml

100 ml

3.0 M

Chemicals Source

Acetic acid BLH, India

42

Agarose

Agar powder

Anpicillin

Ammonium acetate

Antifoam RD emulsion

Azide dextrose broth

Butanol

Calcium chloride

Chlorai tphenicol

Dipotassium phosphate

Ethylene d i a m i n e t e t r a a c e t a t e

Eosin methylene b lue agar

Glucose

Glycerol

Kanamycin

Lambda DNA

Lysozyme

KacConkey's agar

MacConkey's b r o t h

Kagnessium s u l p h a t e

N u t r i e n t agar

N u t r i e n t b r o t h

Potass ium a c e t a t e

Potass ium dihydrogen

Sigma, USA

Hi-Media, I n d i a

Ranbaxy, I n d i a

Hi-Media, I n d i a

Hopkins and Williams,

England

HI-Media, India

Sisco Laboratories, India

Sisco Laboratories, India

Ranbaxy, Indid

BDH, India

sd Fine Chemicals , Ind ia

Hi-Media, India

Hi-Media, India

Hi-Media, India

Ranbaxy, India

Sigma, USA •

Sisco L a b o r a t o r i e s , Ind ia

Hi-Media, I n d i a

Hi-Media, I n d i a

E. Merck, I n d i a

Hi-Media, I n d i a

Hi-Media. I n d i a

Ci ty Chemicals Corpn. , USA

BDH, I n d i a

43

ortho phosphate

Sodium acetate

Sodium citrate

Sodium chloride

Sodium hydroxide

Sodium thiosulphate

Simmons citrate agar

SIM agar

Sucrose

Streptomycin

Tetracycline

Tris HCl

Triple sugar i ron agar

Trypticase soy broth

BDH, India

Loba Chemicals, India

BDH, India

Sisco Laboratories, India

sd Fine Chemicals, India

Hi-Media, India

Difco, USA

Qualigens fine Chemicals,

India

IDPL, India

Pfizer Ltd., India

Sigma, USA

Hi-Media, India

Difco, USA

Note } The chemicals which have not been included in this

list were of analytical grade.

44

Following E .co l l s t r a in s have been used in t h i s study

S t ra in Relevant genet ic d e s i g n a t i o n marke r s

Source

AB1157

EcRp

SWi,

SWd.

swd

S S i

SSd.

SSd.

t h i - 1 , a r g E ~ 3 , t h r - 1 , l e u B 6 , p roA2 , h i s G 4 , l acYl ,F- , S^-, X

r T r r r r A^,B , C t ^ E ' - ^ P b ,R S ^ , S z ^ , H g ^ , C d ^ , 2 n ^ , P b ^ / l a c A ^ , B ^ , C t ^ , E ^ , F r ^ , K ^ R ^ , s r , S z ^ , H g ^ , C d ^ , Z n ^ , P b ^ , l a c

A ^ , A k ^ , B ^ , C t ^ , E ^ , F r ^ , N ^ R ^ , S z ^ , H g ^ , C d ^ , E n ^ , P b ^ , l a c

A ^ , B ^ , C ^ , C t ^ , E ^ , F r ^ , S z ^ H g r , c d ^ , Z n ^ , P b ^ . l a c

A ^ , B ^ , C t ^ , E ^ , F r ^ , S ^ , S z ^ H g ^ , C d ^ , Z n ^ , P b ^ , l a c

A ^ , B ^ , C t ^ , E ^ , F r ^ , K ^ , N ^ ,

l a c

Srivastava, B. S,

Srivastava, B.S.

Isolated from industrial effluents

Isolated from domestic sewage

Isolated from domestic sewage

Isolated from soils of industrial area

Isolated from soils in the proxi­mity of domestic sewage drains.

Isolated from soils in the proxi­mity of domestic sewage drains.

(r = resistant, s = sensitive)

45

Collection of Sanples

Water Sairples j

Water sanples were collected routinely from the

different drains of the Aiigarh city including industrial

estate (Table III), The sampling sites were severely

affected by human and industrial activities. Water sanp­

les were collected in 250 ml sterilized glass bottles and

transferred to the laboratory immediately for initial

processing. The elapsed time between the collection of

sanple and initial processing did not exceed 2 h.

Soil samples :

The soil sanples were collected in sterilized

container with the help of sterilized scapula. The sar-ple

were taken at a depth of 6 inch from the upper surface of

soil (Table lH) .

Isolation and Identification of Enterobacteria from Water

and Soil

A variety of media were enployed. The isolation of

E.coli and Enterobacter aeroqenes was done according to

the standard methods described in APHA (1985). 10 ml ali-

quots was taken in double strength MacConkeys broth and

incubated at 37°C for 24 h. Furthermore the sanples indi­

cating acid and gas production was spread on eosin methyl­

ene blue (EMB) agar plates and incubated at 37°C for 24 h.

Some colonies were small showing green metallic sheen and

46

Table III

S.No. Test sairples Various sources of the sarrples collec­ted for the isolation of E.coli and Enterobacter aeroqenes

Water Samples

1. SW Muddy and dirty water from the drain near V.M, Hall crossing,

2. SW Sewage water from the Jamalpur drain, 2 Aiigarh.

3. SW Sewage water from the Quwarsi drain, ^ Aiigarh.

4. SW. Blackish sewage water near Herkut 4 Udyog Industrial area, Aiigarh

5. SW Brownish dirty water near ITI, Indus­trial area, Aiigarh

6, SW Stagnant water from the Aiigarh drain,

near GT Road, Aiigarh.

Soil Sarrples

7. SS Soil from agricultural field near

V.M. Hall crossing, Aiigarh 8, SS Soil from agricultural field near 2 Jamalpur drain, Aiigarh.

9. SS Soil from agricultural field near • Quwarsi drain, Aiigarh.

10. SS Soil, near Herkut Udyog, Industrial area, Aiigarh.

11. SS Agricultural field soil near ITI, Industrial area, Aiigarh.

12. SSg Soil from agricultural field near GT Road in the vicinity of Aiigarh drain Aiigarh.

47

others were large pinkish mucoid colonies. The colonies were

picked and grown individually in nutrient broth at 37°c for

12 to 15 h (Martin and Washington, 1980).

100 gm of soil in a beaker were taken and suspended

in an equal amount of normal saline solution. The soil sanples

were homogenised in a waring blender for about 5 to 10 min,

and then 0.5 ml of the supernatent to culture media were

plated and incubated at 37 C for 12 to 16 h, the suspected

colonies were picked up and subcultured on different selec­

tive media for the isolation of pure cultures of entero-

bacteria e.g. E.coli and Enterobacter aeroqenes (Martin and

Washington, 1980) .

MPN count from soil was done according to the method

of Topping (l9 38) .

Gram's Staining »

The gram's staining of all E.coli and Enterobacter

aerogenes was done according to the standard procedure of

Cappucino and Sherman (1987).

Biochemical Reactions ;

The isolated E.coli and Enterobacter aeroqenes were

finally identified on the basis of their biochemical proper­

ties and enzymatic reactions m the presence of specific

substrates. The IMViC series of reactions were performed for

E.coli and Enterobacter aeroqenes according to the method of

Cappucino and Sherman (i987) ,

48

Determination of Minimum inhibitory Concentrations (MIC5)

of Heavy Metals

This was done by plate dilution method. The metals

of, Hg, Cd, Pb and Zn were used as HgCl , CdCl , Pb(CH-COO)

and ZnCl , varying in concentrations from 3.25 ug/ml to

6400 jug/ml and were supplemented in nutrient agar which were

then spot inoculated with overnight broth culture of different

test strains of E.coli and Enterobacter aeroqenes. The

plates were incubated at 37°C for 24 h. The sensitivity

and resistance criteria were established in accordance with

the data of Joly e^ al_, (1976), Austin e^ al.. (1977), Marques

et al . (1979) and Ahmad and Yadava (i988) .

Antibiotic Sensitivity test

All the isolates of E.coll and Enterobacter aeroqenes

were tested for sensitivity to antimicrobial agents by

means of disc diffusion method enploying multidiscs (Bauer

et al., 1966; Coleman et .., 1985), This test was carried

out by mixing 0,3 ml of fresh exponential culture of the

test strains with 3.0 ml of molten top agar (held at 45°C) .

This mixture was layered over a freshly prepared nutrient

agar plate and allowed to set for about half an hour. The

antibiotic discs were placed on the agar layer using steri­

lised forceps and the plates were Incubated overnight at

37°C. The zone of growth inhibition around the antibiotic

discs were measured and the results recorded. The following

49

antibiotic discs (all from HI Media) were used. Concen­

tration of the antibiotics used was in micrograms per disc,

except where otherwise is stated. The symbols and concen­

trations of respective antibiotics are given in the paren­

thesis I Amikacin (Ak 30) , Anpicillin (A 10), Bacitracin

(B 10 units), Chloranphenicol (C 30), Chlorotetracycline

(Ct 30) , Erythromycin (E 15) , Furazolidone (Fr 15) , Genta-

mycin (G 10) , Kanamycin (K 30) , Nalidixic acid (Na 30) ,

Neomycin (N 30), Peniciilin-G (P 10 units), Polymyxin B (Pb

300) , Rif anpicin (R 5) / Streptomycin (S 10) , Sulphadiazine

(Sz 30) , Tetracycline (T 30) , These antibiotics were selected

on the basis of their common use by the humans and domestic

animals.

Isolation of Plasmids

The isolation of plasmid from satisfactory cleared

lysate from any bacterial species is frequently a combina­

tion of skill, luck and patience, especially if large plasmids

are to be isolated. Plasmids were routinely isolated from

E.coli isolates of soil and water. The mlniprep method was

followed essentially as described by Birnboim and Doly (l979),

Determination of Solecuia Weight

The plasmid size estimates of the isolated plasmid

were obtained by conparing their relative mobilities on

agarose gel with standard known molecular weight plasmid

50

and XDNA fragments. The standard curve was drawn by p l o t t i n g

r e l a t i v e mobil i ty Vs log of the molecular weight of the s t an ­

dard markers. The molecular weight of the unknown plasmids

was determined by p lo t t i ng the r e l a t i v e mobil i ty on the s t an ­

dard curve (Ohman, 1988) .

Agarose Gel Electrophoresis

The i so la ted plasmids were charac te r i sed by agarose

gel e lec t rophores i s according to the standard procedure of

Maniatis e t a l . (1982). About 30 to 40 Jul f rac t ion along

with g lycerol dye mixture (50% glycerol , 0.25% bromophenol

blue) were applied on the s l o t s . Horizontal s lab gel e l e c t r o ­

phoresis was car r ied out using 1% agarose. The gel was p r e -

electrophoresed a t 40 mA for 20 nriin and the normal run was

performed a t 20 mA in e lec t rophores i s buffer (0.004 M T r i s -

ace ta te , 0.00 2 M EDTA, pH 8.0) for 3 to 5 h. The DNA bands

were s ta ined with ethidium broniide and f luorescent p r o f i l e

was photographed by uv i l luminat ion through photodyne UV 300

t r ans i l l umina to r .

Plasmid Transfer

Conjugation ;

Standard method of conjugation was followed enploying

the donor, t e s t E .col i s t r a i n s and the r e c i p i e n t AB1157 (Ohman,

1988). An overnight cu l tu re of r ec ip ien t and donor E .co l i

s t r a in s were added to fresh nu t r i en t broth and incubated

a t 37 C for 30 mln and 60 mln respec t ive ly . For cont ro l , the

51

parental culture alone was added to fresh nutrient broth

and incubated at 37°C. These cultures were serially diluted

and spread on plain and antibiotic supplemented plates.

The selection was done on the basis of appearance

of plasmidial markers along with the original markers of

the parent recipient strain. The AB1157 strain is lac mutant

and also lacking antibiotic resistance markers except for

streptomycin resistance. The selection of transconjugants

was therefore based on appearance of the donor antibiotic

resistance plus non pink colonies on the MacConkey agar

plates.

Transformation t

The transfer of R-plasmid to recipient E,coli

AB1157 cells was also studied through transformation follow­

ing the method of Lederberg and Cohen (1974), The overnight

grown AB1157 cells were made coirpetent by suspending in 0,1 M

MgCl for 30 min on ice and then 0,1 M CaCl under similar

conditions. To 1,0 ml cortpetent cells/ 2,0 ugm of plasmid

DNA, was added and left on ice for 30 min. Heat shock was

given to these cells at 42 C for 3 min. These cells were

then allowed to grow in nutrient broth for 2 h at 37°C,

The cells were spread on the antibiotic supplemented plates

after suitable dilutions. The plates were incubated at 37°c

for 16 h.

52

Plasmld Curing j

The curing of R-plasmids was performed by the method

described by Hirota (i960) and Hardman et al. (1986). Over­

night cultures of E.coli were grown in the nutrient broth

in the presence of curing agent (ethidium bromide) at concen­

tration ranging from 0.5 to 100 wgn/ml. A control tube lacking

the curing agent was also included. All the tubes were incu­

bated overnight at 37 C. Contents of the tube were then

plated, after making required dilutions, on antibiotic supple­

mented and plain nutrient agar plates. The plates were incu­

bated at 37 c overnight and the number of colonies appeared

on the plates were then- recorded. The number of colonies

on antibiotic supplemented plates was subtracted trom that

on the plain plate.

The number of cured cells = number of colonies on

the plain plate at a specific concentration - number of

colonies on the antibiotic supplemented plate at the same

concentration.

RESULTS

53

Water samples for bacteriological analysis were

collected from six different locations. The results of

analysis in various seasons are shown in table 4. The

maximum TBC was observed during spring season in SW2

test samples while the lowest TBC was recorded in SW,

test samples during the spring season again. Total entero­

bacterial count was highest (3.66x10 CFU/lOO ml) during

summer in SW^ test samples while the lowest enterobac­

terial count (0.78x10^ CFU/lOO ml) was observed in SW^

test samples during winter season. Maximum number of

coliform (i.e. 1.6x10^ MPN/100 ml) was present at 3^^

location in all the seasons while the minimum counts (i.e.

0.24x10 MPN/lOO ml) was observed in SW^ test samples. In

terms of Fecal streptococci the SW and SW- locations

were maximally polluted.

Soil samples for bacteriological analysis were also

collected from agricultural fields in the proximity of

drains. The results of analysis in various seasons are

shown in table 5. A remarkably higher TBC compared with

sewage samples was invariably observed throughout the

period of study in all the locations. The total entero­

bacterial counts were highest in SS. location during

spring season while it was lowest in SS. location during

winter season. As compared with sewage samples, the soil

sampling sites displayed relatively lower counts to the

54

TABLE 4 : S e a s o n a l v a r i a t i o n i n b a c t e r i o l o g i c a l p a r a m e t e r s i n t h e sewage w a t e r

P a r a m e t e r s S p r i n g Summer W i n t e r Mean sf % CV** 90 90 9 0 - 9 1

T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml

T o t a l e n t e r o ­b a c t e r i a l c o u n t s x l 0 3 CFU/100 ml

T o t a l c o l i f o r m x l 0 3 MPN/100 ml

F e c a l s t r e p t o ­c o c c i x l 0 3 MPN/lOOml

64800 72800 62400 66667 4446 6 .6

3080 3260 1360 2567 856 33 .4

1600 1600 900 1366 330 2 4 . 1

900 1600 500 1000 454 4 5 . 5

T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml

83200 69800 43800 65600 16356 24.9

Total entero­bacterial counts xl03 CFU/lOO ml

2540 3660 2280 2827 599 21.1

Total coliform 1600 xl03 MPN/100 ml

Fecal streptococci 900 xl03 MPN/100 ml

1600

1600

1600 1600 0 0

500 1000 454 45.4

Total bacterial counts xl03 CFU/lOO ml

55800 58400 46800 53667 4970 9.3

Total entero­bacterial counts xlO^ CFU/lOO ml

2180 2280 1580 2013 309 15.3

Total coliform xl03 MPN/100 ml

300 500 240 347 111 32.0

Contd,...

55

Fecal streptococci 130 220 130 160 42.4 26.5 xl03 MPN/lOG ml

SW. 4_

Total bacterial 12100 36200 17800 22033 10284 46.6 counts xl 03 CFU/lOO ml

Total entero- 980 1900 780 1220 488 39.9 bacterial coxints xl03 cFU/lOO ml

Total coliform 240 xlO^ MPN/lOO ml

Fecal streptococci 70 xl03 HPN/lOO ml

Total bacterial 31800 41800 39000 37800 4320 11.4 counts xl03

300

130

240

30

260

76.6

28

41

10.8

53.6

CFU/lOO ml

T o t a l e n t e r o ­b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml

1360 2260 1120 1580 491 3 1 . 0

T o t a l c o l i f o r m 300 300 220 2 7 3 . 3 38 13 .7 x l 0 3 MPN/100 ml

F e c a l s t r e p t o c o c c i 240 280 50 190 100 5 2 . 8 x l 0 3 MPN/lOO ml

SW, 6

T o t a l b a c t e r i a l 32600 42600 20800 32U00 8910 2 7 . 8 c o u n t s x l 0 3 CFU/lOO ml

T o t a l e n t e r o - ^^^^ 2100 176.0 1900 145 7 . 6 b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml

T o t a l c o l i f o r m x l 0 3 MPN/lOO ml

F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOO ml

* Standard d e v i a t i o n

** Percent c o e f f i c i e n t of v a r i a t i o n

500

220

500

300

70

110

357

210

203

78

56.8

' 37.0

56

TABLE 5 ; S e a s o n a l v a r i a t i o n i n b a c t e r i o l o g i c a l p a r a m e t e r s i n t h e s o i l

P a r a m e t e r s S p r i n g 90

Summer 90

W i n t e r 9 0 - 9 1 Mean SD %cv

SS.

T o t a l b a c t e r i a l c o u n t s x l 03 CFU/lOO ml

T o t a l e n t e r o ­b a c t e r i a l c o u n t s x l 0 3 CFli/100 ml

T o t a l c o l i f o r m xlO^ MPN/lOO ml

1024000 8X8000 712000 851333 129536 15 .2

1960

24

2420

13

1860 2080

7 . 0 1 4 . 6

244 11 .7

7 .0 4 8 . 2

F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOO ml

24 13 7 . 0 1 4 . 6 7 . 0 4 8 . 2

T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml

1064000 962000 886000 970667 72926 7 .5

T o t a l e n t e r o ­b a c t e r i a l counts x l 0 3 CFU/lOO ml

3320 3080 2460 2953 362 12 .2

T o t a l c o l i f o r m x l 0 3 MPN/lOO ml

50 160 13 74 62.4 84.0

Fecal streptococci xl03 MPN/lOO ml

SS.

2.3 5 . 1 2 . 0 3 9 . 6

T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml

698000 558 000 47600 577333 91656 15 .8

T o t a l e n t e r o ­b a c t e r i a l counts x l 0 3 CPU/100 ml

2280 2240 2180 2233 41 1.8

C o n t d . . .

57

T o t a l c o n f o r m x l03 MPN/100 ml

Fecal s t r e p t o c o c c i x l03 MPN/100 ml

SS,

28 30 13 23.6 7.5 32.2

3.4 4.1 0.6 15.7

Total bacterial counts xl03 CFU/100 ml

Total entero­bacterial counts xl03 CFU/100 ml

Total coliform xl03 MPN/100 ml

Fecal streptococci xl03 MPN/100 ml

848000 624000 596000 689333 112775 16.4

1890

13

7

i860

7

2.3

1460 1737

4.1

19 6 11.2

2.8 31.4

2.0 50.4

Total bacterial counts xl 03 CFU/100 ml

Total entero­bacterial counts Xl03 CFU/100 ml

Total coliform xl03 MPN/100 ml

Fecal streptococci xl03 MPN/100 ml

622000

244 0

742000

2680

340000 568000 168499 29.6

11

13

1520 2213

8.3

2.3 7.4

500 22.5

2.5 29.9

4.3 58.9

Total bacterial 728000 counts xl03 CFU/100 ml

Total entero- 2860 bacterial counts xl03 CFU/100 ml

Total coliform 11 xl03 MPN/100 ml

Fecal streptococci 7 xl03 MPN/100 ml

732000

3020

376000 612000 166885 27.2

22

11

2420 2767

4 12.3

7.6

254 9.2

7.4 60.0

2.5 32.0

58

t o t a l coliform in a l l the seasons. Fecal s t reptococci count

in the so i l sample was also invar iably low ra ther lowest in

a l l the t e s t samples comparing with other b a c t e r i a l counts .

Table 6 shows the loca t iona l va r i a t ion and average

bac t e r io log i ca l parameters in domestic sewage water . The

average TBC was highest in the spring season (67.9x10 CFU/

100 ml) while t o t a l en te robac te r i a l count was highest during

svimmer (3.1x10 CFU/100 ml) . The minimum TBC and TEC were 6 6

recorded in winter season i . e . 51x10 and 1.7x10 CFU/100 ml

r e spec t ive ly . As far as the en te robac te r i a l populat ion in

t h e t e s t sanples was concerned, i t was maximxjm (4.5%) in

summer and lowest (3.4%) in winter season. The proportion

of t o t a l conforms among the t o t a l b a c t e r i a l (TBC) was nearly

same (1.7%) in a l l the seasons. However the t o t a l coliforms

in the en te robac te r i a l population was maximim in winter-

seasons (52.4%) and was minimum in the summer (40.2%). The

average fecal s t reotpcocci were found t o be highest (1.1x10

MPN/lOO ml) during summer and lowest (0,3x10 MPN/100 ml)

in winter seasons.

Table 7 p resen ts the loca t iona l va r i a t i on and

average bac t e r io log ica l parameters in i n d u s t r i a l e f f l u e n t s .

The average TBC was highest in summer season (40.2x10^ CFU/

100 ml) and TEC was also maximum (2.1x10^ CFU/100 ml) in

t he same season again. As far as the r e l a t i v e proport ion

59

TABLE 6 : L o c a t i o n a l v a r i a t i o n a n d a v e r a g e b a c t e r i o l o g i c a l ~ p a r a m e t e r s i n t h e d o m e s t i c s e w a g e w a t e r

E a r a m e t e r SW. sw. SW. Mean SD

T o t a l b a c t e r i a l c o T i n t s X 10^ CFU/lOO m l

T o t a l e n t e r o ­b a c t e r i a l c o u n t s x l 0 3 CFU/iOO m l

T o t a l c o l i f o r m x l 0 3 MPN/100 ml

SPRING 9'0

6 4 8 0 0 8 3 2 0 0 5 5 8 0 0 6 7 9 3 3

3080 2540 ( 4 . 7 ) ( 3 . 0 5 )

1600^ 1600^ ( 2 . 4 ) ^ , ( 1 . 9 )

2180 ( 3 . 9 )

2 6 0 0 ( 3 . 8 )

F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOO ml

( 5 1 . 9 )

9 0 0

* * ( 6 2 . 9 )

9 0 0

• *

SUMMER 9 0

300 1 1 6 6 . 6 , ( 0 . 5 ) ^ ^ ( l - 7 ) _

( 1 3 . 8 ) * * ( 4 4 . 8 )

130 64 3

11403

613

363

1 6 . 7

3 6 9 . 8 1 5 . 2

5 2 . 5

5 6 . 4

T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/IOO m l

T o t a l e n t e r o ­b a c t e r i a l c o u n t s x l 0 3 CFU/lOO m l

T o t a l c o l i f o r m x l 0 3 MPN/lOO ml

7 2 8 0 0 69800 5 8 4 0 0 6 7 0 0 0

3 2 6 0 3660 ( 4 . 4 ) ( 5 . 2 )

1 6 0 0 ^ 1 6 0 0 ^ / 2 . 1 ) ( 2 . 2 h ( 0 . 8 ) ; ( 1 . 8 ) " ( 4 9 . 0 ) ( 4 3 . 7 ) ( 2 1 . 9 ) ( 4 0 . 2 )

2280 ( 3 . 9 )

500 .

3 0 6 6 ( 4 . 5 )

1233 ,

F e c a l s t r e p t o c o c c i 1600 x l 0 3 MPN/lOO m l

1600 220

T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml

T o t a l e n t e r o ­b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml

T o t a l c o l i f o r m x l 0 3 MPN/lOO m l

F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOO m l

WINDIER 1 9 9 0 - 9 1

6 2 4 0 0 4 3 8 0 0 4 6 8 0 0

1360 ( 2 . 1 )

9 0 0 (1 .4):

2280 ( 5 . 2 )

1600

1580 ( 3 . 3 )

240 ,

1140

5 1 0 0 0

1 7 4 0 ( 3 . 4 )

9 1 3

( 6 6 . 2 ) * * ( 7 0 . 2 ) * * ( 3 . 6 ) ^ ^ ( 0 . 5 ) _ ( 1 . 7 )

500 500

( 1 5 . 2 )

130

* * ( 5 2 . 4 )

376

* *

6 2 0 3

580

5 1 8

65 0

8 1 5 3

392

555

174

9 . 2

1 8 . 9

42.U

5 7 . 0

15 .9

22.5

6 0 . 8

4 6 . 3

60

TABLE 7 : Locational variation and average bacteriological parameters in the industrial sewage water

P a r a m e t e r s

T o t a l b a c t e r i a l c o u n t s X 103 C F U / 1 0 0 m l

T o t a l e n t e r o ­b a c t e r i a l c o x i n t s x l 0 3 CFU/100 m l

T o t a l c o l i f o r m x l 0 3 MPN/100 m l

4 ^ 5

SPRING 90

1 2 1 0 0

9 8 0 ( 8 . 0 )

31800

1360 ( 4 . 2 )

2 4 0 ^ 300 .

( 2 4 . 4 ) ( 2 2 . 0 )

F e c a l s t r e p t o c o c c i 70 x l 0 3 MPN/lOO m l

T o t a l b a c t e r i a l c o i i n t s x l 0 3 CFU/100 ml

T o t a l e n t e r o ­b a c t e r i a l c o u n t s x l 0 3 CFU/100 ml

T o t a l c o l i f o r m

3 6 2 0 0

1900 ( 5 . 2 )

3 0 0 ^

240

SUMMER 90

4 1 8 0 0

2260 ( 5 . 4 )

, 300^

^^^6

3 2 6 0 0

1840 ( 5 . 6 )

5 0 0

( 2 7 . i f 220

4 2 6 0 0

2 1 0 0 ( 4 . 9 )

, 5°?*

Mean

2 5 5 0 0

1 3 9 3 . 3 ( 5 . 4 )

3 4 6 . 6 ^

( 2 4 . 8 )

1 7 6 . 6

4Q200

2087 ( 5 . 2 )

3 6 6 . 6 ^

SD

9 4 8 0 .

3 5 1 ,

1 1 1 ,

7 5 .

284 2

147

94

.8

, 8

,15

,8

%CV

3 7 . 1

2 5 . 2

3 2 . 0

4 2 . 9

7 . 0

- 7'°

2 5 . 7 xl03 MPN/lOO ml (0.8).. (0.7).. (1.2)_ (0.9)^^

( 1 5 . 7 ) ( 1 3 . 2 ) ( 2 3 . 8 ) ( 1 7 . 6 )

F e c a l s t r e p t o c o c c i 130 280 3 0 0 237 76 3 2 . 0 x l 0 3 MBN/100 m l

T o t a l b a c t e r i a l 17800 c o u n t s x 103 CFU/100 ml

T o t a l e n t e r o - 7 8 0 b a c t e r i a l coxonts ( 4 . 3 ) x l 0 3 CFU/100 ml

T o t a l c o l i f o r m 240 x l 0 3 MPN/lOO m l ( 1 - 3 ) ^ ^ ^ 0 . 5 ) " ^ ^ ( 0 . 3 ) " ( 0 . 7 ) '

( 3 0 . 7 ) * * ( 1 9 . 6 ) * ( 3 . 9 ) * * ( 1 4 . 4 ) * "

F e c a l s t r e p t o c o c c i 30 5 0 n o 63 3 3 . 9 5 3 . 7 x l 0 3 MPN/lOO m l

WINTER

39800

1120 ( 2 . 8 )

, 2 2 0 ^

9 0 - 9 1

2 0 8 0 0

1760 ( 8 . 4 )

,. 19*

2 6 1 3 3

1220 ( 4 i 6 )

, IZ?*

9 7 4 1

4 0 6 . 3

7 5 . 8

3 7 . 2

3 3 . 3

4 2 . 9

61

of en te robac te r i a l covint with respect t o TBC was concerned,

t he TEC was highest in the t e s t b a c t e r i a l populat ions

during spring (5.4%) and lowest in winter (4.6%). The

maximtun proportion of t o t a l colifoxm in the t o t a l bac­

t e r i a l population was observed in spring (1.3%) while

t h e minimum was recorded in the winter (0.7%). The avera­

ge proport ion of TC with respect to en te robac te r ia l count

was a l so maximum in spring (24.8%) and minimum in winter

again (14.4%). Fecal s t reptococci showed the maximum count

in summer (0.23x10 MPN/lOO ml) and lowest count was reco­

rded in winter (0.063x10^ MPN/lOO ml) .

Table 8 gives the loca t iona l va r i a t ion and average

bac t e r io log i ca l parameters of s o i l in a g r i c u l t u r a l f i e ld s

in t h e proximity of domestic sewage d r a i n s . The average TBC

was maximum in spring (9 28.6x10 CFU/lOO ml) and minimum

in winter season (691.3x10 CFU/lOO ml) . As far as the

r e l a t i v e proport ion of en te robac te r ia in the t o t a l b a c t e r i a l

populat ion was concerned, i t was approximately same in a l l

seasons (0.3%). Total colifoxms in the b a c t e r i a l populat ions

(TBC) were maximum in suimier (0.0008%) and a minimxan during

winter (0.0001%). The r e l a t i v e proportion of t o t a l c o l i -

form out of en t e robac te r i a l counts was again maximum during

summer season (2.6%). Average fecal s t reptococci was maximum

during spring (0.01x10 MPN/lOO ml) but i t was minimiim

(0.004x10^ MPN/lOO ml) during winter season.

62

TABLE 8 : L o c a t i o n a l v a r i a t i o n and a v e r a g e b a c t e r i o l o g i c a l p a r a m e t e r s of s o i l xxi t h e v i c i n i t y of d o m e s t i c sewage w a t e r

P a r a m e t e r s SS SS SS Mean SD %CV

SPRING 90

T o t a l b a c t e r i a l 1024000 1064000 698000 928666 163921 1 7 . 6 c o u n t s x l 0 3 CFU/lOO ml

T o t a l e n t e r o - 1960 33 20 b a c t e r i a l c o u n t s ( 0 . 2 ) ( 0 . 3 ) x l 0 3 CFU/lOO ml

T o t a l c o l i f o r m 2% 50^ x l 0 3 MPN/lOO ml ( . 0 0 0 2 ) ( . 0 0 0 4 )

( 1 . 2 ) * * ( 1 . 5 ) * *

F e c a l s t r e p t o c o c c i 24 7 x l 0 3 MPN/lOO ml

SUMMER 9 0

2280 ( 0 . 3 )

28 ( . 0 0 0 4 ^

( 1 . 2 )

5

25 20 ( 0 . 3 )

34 * ( . 0 0 0 3 ^ ^

( 1 . 3 )

12

5 8 0

1 1 . 4

8 . 5

2 3 . 0

3 3 . 6

7 1 . 0

T o t a l b a c t e r i a l 818000 962000 558000 779333 167183 21 .4 c o u n t s x l 0 3 CFU/lOO ml

T o t a l e n t e r o - 2420 3080 2240 2580 361 13 .9 b a c t e r i a l c o u n t s ( 0 . 3 ) ( 0 . 3 ) ( 0 . 4 ) ( 0 . 3 ) x l 0 3 CFU/lOO ml

T o t a l c o l i f o r m 13 ^ 1 6 0 ^ 30 6 7 . 6 ^ 66 9 7 . 0 x l 0 3 MPN/lOO ml ( .OOOlsf ( . 0 1 6 ) ^ ^ ( . 0 0 0 5 L ( . 0 0 0 8 )

( 0 . 5 ) ( 5 . 1 9 ) ( . 1 3 ) ( 2 . 6 ) *

F e c a l s t r e p t o c o c c i 13 6 4 7 . 6 3 . 8 5 0 . 3 x l 0 3 MPN/lOO ml

WINTER 9 0 - 9 1

T o t a l b a c t e r i a l 712000 886000 476000 691333 168018 24 .3 coxints x 103 CFU/lOO ml

T o t a l e n t e r o - 1860 2460 2180 2166 .6 245 1 1 . 3 b a c t e r i a l c o u n t s ( 0 . 3 ) ( 0 . 3 ) ( 0 . 5 ) ( 0 . 3 ) x l 0 3 CFU/lOO ml

T o t a l c o l i f o r m 7 ^ 13 ^ 13 ^ 11 ^ 2 . 8 25 .7 x l 0 3 MPN/lOO ml ( . 0 0 0 0 4 ) ( . 00014) ( . 0 0 0 2 7 ) ( .OOOll)

( 0 . 3 7 ) * * ( 0 . 5 2 ) * * ( 0 . 5 9 ) * * ( 0 . 5 ) * *

F e c a l s t r e p t o c o c c i 7 2 . 3 3.4 4 . 2 2 .0 4 7 . 4 x l 0 3 MPN/lOO ml

63

Table 9 shows the loca t iona l va r ia t ion and average

bac t e r io log ica l parameters of so i l from a g r i c u l t u r a l f i e l d s

in the v i c i n i t y of i ndus t r i a l a rea . The average TBC was

maximum in spring season (732.7x10^ CFU/lOO ml) while i t

was minimum during winter (437.3x10^ CFU/iOO ml) . As far

as the r e l a t i v e proport ion of en te robac te r i a l counts with

TBC was concerned, i t was maximum (0.4%) in winter and mini­

mum in spring season (0.3%). Total ooliform count with

respect t o t he TBC was lowest (0.0001%) in a l l the seasons

while the t o t a l coliform count with respect to en te robac te r i a l

population was maximum in summer (0.5%) and minimum in

winter (0.3%). In terms of fecal s t reptococci / t he average

FS count was nearly the same (0.008x10^ MPN/lOO ml) in

spring and summer seasons but r e l a t i v e l y lower in w in t e r

(0.003x10^ MPN/lOO m l ) .

A t o t a l 49 E.col l i s o l a t e s from sewage out of 5 3

were found t o be multiply r e s i s t a n t t o a n t i b i o t i c s . Among

the 15 commonly used a n t i b i o t i c s / d r u g s t e s t ed for s e n s i t i ­

v i t y , 9 d i f fe ren t mul t ip le a n t i b i o t i c s / d r u g s r e s i s t ance

p a t t e r n were observed. The number of a n t i b i o t i c s or drugs

against which res i s t ance was observed ranged from 3 t o 9 .

Nine d i f fe ren t E.col i i s o l a t e s were iden t i f i ed on the b a s i s

of antibiograms (Table lO). EigHt types of a n t i b i o t i c s /

drugs r e s i s t ance pa t t e rn s among the so i l i s o l a t e s were

64

TABLE 9 : L o c a t i o n a l v a r i a t i o n and a v e r a g e b a c t e r i o l o g i c a l p a r a m e t e r s of s o i l i n t h e v i c i n i t y of i n d u s t r i a l a r e a

P a r a m e t e r s SS^ SS^ SS^ Mean SD >iCV

SPRING 90

Total bacterial 848000 622000 728000 732667 92323 12.6 counts xl03 CFU/lOO ml

Total entero- 1890 2440 2860 2397 397 16.5 bacterial counts (0.2) (0.4) (0.4) (0.3) xl03 CFU/100 ml

Total ooliform 13 ^ 9 ^ 11 ^ H * 1'6 14.8 '^ "' " • 00015) (.000111 (.00015) (.00015:

(' x l 0 3 MPN/100 ml ( . 00015) ( . 00014) ( . 00015) ( . 0 0 0 1 5 )

( 0 . 7 8 ) * * ( 0 . 4 ) (0.41)^** ( 0 . 5 ) * *

F e c a l s t r e p t o c o c c i 7 7 7 7 0 0 x l 0 3 MPN/100 ml

SUM14ER 9 0

T o t a l b a c t e r i a l 624000 742000 732000 699333 53424 7 . 6 c o u n t s x l 0 3 CFU/lOO ml

T o t a l e n t e r o - 1860 2680 3020 2520 487 1 9 . 3 b a c t e r i a l c o u n t s ( 0 . 3 ) ( 0 . 4 ) ( 0 . 4 ) ( 0 . 4 ) x l 0 3 CFU/lOO ml

T o t a l c o l i f o r m 7 ^ 11 ^ 2 2 ^ 1 3 . 3 ^ 6 . 3 4 7 . 5 xlO 3 MPN/100 ml ( . O O O i n ( . 00014) ( . 0 0 0 3 ) ^ ( .OOOl) .

( 0 . 4 7 ) * * ( 0 . 4 ) * * ( 0 . 7 ) * * ( 0 . 5 )

F e c a l s t r e p t o c o c c i 2 . 3 13 11 8 . 7 4 . 6 5 2 . 9 x l 0 3 MPN/lOO ml

WINTER 9 0 - 9 1

Total bacterial 596000 340000 376000 437333 113153 25.8 counts xlo3 CFU/lOO ml

Total entero- 1460 1520 2420 1800 439 24.3 bacterial counts (0.2) (0.4) (0.6) (0.4) xl03 CFU/lOO ml

Total coliform xl03 MPN/lOO ml Total coliform 7 ^ 5^. 4^ 5 . 3 ^ 1.2 23.3

(.00011) (.00014) (.0001) (.0001) (0.5)** (0.3)** (0.2)** (0.3)**

Fecal s t reptococci 3 2.3 5 3.4 1.2 33.3 xl03 MPN/100 ml

* Total coliform counts expressed in terms of percentage of the t o t a l b a c t e r i a l count

** Total coliform expressed in terms of % of t o t a l en te robac te r i a l count.

65

TABLE 10 : A n t i b i o t i c r e s i s t a n c e p a t t e r n of E.co 1 i s t r a i n s i s o l a t e d from sewage w a t e r * ""

S .No. E . c o l i R e s i s t a n c e a g a i n s t No. of a n t i -a n t i b i o t i c s / d r u g s b i o t i c s

a g a i n s t w h i ­ch r e s i s ­t a n c e wag^ o b s e r v e d

Number of s t r a i n s abu-showing nda-r e s i s t a - n e e n e e

1 .

2 .

3 .

4 .

5 .

6 .

7 .

8 .

9 .

Wi.

Wi ,

Wi .

Wi .

Wi .

Wd.

Wd,

Wd,

Wd,

A, Ak, R 3

A,B,E ,Fr ,Na , .R 6

A , B , E , F r , P b / R , S z 7

A , A k , B , E , F r , P b , R , 8 Sz

A , B , C t , E , P b , R , S,Sz 8

A , A k , B , C , E , F r , P b , R 8

A , B ^ C t , E , F r / N a , R , 9 S/ Sz

A , B , C t , E , F r , K , R , S , 9 Sz

A , A k / B , C t , E , F r , N , R , 9 Sz

3

2

26

4

4

4

1

3

2

5 . 6

3 .7

4 9 . 0

7.5

7 .5

1.8

5 . 6

3 . 7

be T o t a l number of E . c o l i s t r a i n s found t o ^ s e n s i t i v e t o a l l a n t i b a c t e r i a l a g e n t s = 4 ( 5 . 6 2 % ) .

* T o t a l number of s t r a i n s employed f o r t h e s t u d y = 53

* * T o t a l number of a n t i b a c t e r i a l a g e n t s employed f o r t h e s t u d y = l 5

I n f a c t we had u s e d 17 a n t i b a c t e r i a l a g e n t s b u t t h e r e was no

e x c e p t i o n w i t h r e g a r d t o t h e r e s i s t a n c e a g a i n s t a m p i c i l l i n

and p e n i c i l l i n a s w e l l a s c h l o r o t e t r a c y c l i n e and t e t r a c y c l i n e .

T h e r e f o r e o p e r a t i o n a l l y we were work ing on 15 a g e n t s o n l y .

66

recorded (Table 11) . E ,col l i s o l a t e s of i n d u s t r i a l or ig in

(Wi ) were r e s i s t a n t to only 3 a n t i b i o t i c s whereas the

domestic sewage i s o l a t e s displayed res i s t ance t o 9 diff­

erent a n t i b i o t i c s simultaneously (Table 10) .

Table 12 and 13 show the percent r e s i s t ance agains t

the individual a n t i b i o t i c s . Out of 15 an t ib io t i c s /d rugs

t e s t e d , the s e n s i t i v i t y to gentamycin was invar iably obser­

ved in a l l the i s o l a t e s from water and soil whereas r e s i s ­

tance to ampici l l in and rifampicin was equally prevalent

(92.45%). Moreover, 86.7% of i s o l a t e s were r e s i s t a n t t o

both the Erythromycin and Baci t racin whereas 79.2% were

against furazolidone in t he sewage water samples. Further-

more, 95.5% E.co l i i s o l a t e s from so i l were at l e a s t r e s i s ­

t a n t to a m p i c i l l i n / p e n i c i l l i n and erythromycin whereas

88.8% were simultaneously r e s i s t a n t against furazolidone

and sulphadiazine (Table 13) .

A t o t a l of 30 E.col i i s o l a t e s from sewage were

t e s t e d from re s i s t ance against four metals eg. Hg, cd, Pb

and Zn. 66.6% of s t r a in s showed re s i s t ance t o Hg"*"*" of which

53.3% sanples were of i ndus t r i a l o r i g i n . Among the E.col i

s t r a i n s i so l a t ed from i n d u s t r i a l e f f luen t s , 13.3% showed

MIC of 200 ug/ml towards Hg"*""*" whereas 10% of the s t r a i n s

showing MIC at 12.5 ug/ml t o Hg" "*". 93.3% of E.col i i s o l a t e s

exhibi ted r e s i s t ance t o Pb" "*" and Zn"*" of which 66.6% and

67

TABLE 11 : Ant ib io t ic r e s i s t ance pa t te rn of E.col i s t r a i n s i so la t ed from soi l

S.No. E.col i s t r a i n s

Resistance against ant ib io t ic s/drug s

No. of a n t i - Number % b i o t i c s of s t r -abun-against a ins dance

which showing res i s t ance re s i s -was observed** tance

1.

2.

3 .

4 .

5.

6.

7 .

8 .

S i .

S i ,

S i .

S i ,

Sd.

Sd,

Sd.

Sd,

A /Ak ,E ,Pb ,R

A, A k , C , E , F r , Sz

A , B , E , F r , P b , R , Sz

A , B , C , C t , E , F r , Sz

A , B , C t , E , F r , S, Sz

A , B , C t , E , F r , K , N , R , Sz

A , B , C t , E , F r , N a , R , S, Sz

A , B , C t , E , F r , K , R , S, Sz

5

b

7

7

7

9

9

9

3

3

27

2

3

2

6 . 6

6 .6

6 0 . 0

4 . 4

6 .6

4 . 4

4 .4

2 . 2

Total number of E.col i s t r a i n s found t o ^ e n s i t i v e t o a l l a n t i b a c t e r i a l agents = 214,4%).

*Total number of s t r a i n s ertployed for the study = 45

**Total nxomber of a n t i b a c t e r i a l agents employed for the study = 15 .

68

TABLE 12 ; P e r c e n t r e s i s t a n c e aga ins t i n d i v i d u a l a n t i b a c t e r i a l agent moni tored in t h e sewage wa te r

T o t a l Number Number of i s o l a t e s r e s i s t a n t Pe rcen t (%) of E . c o l i t o t h e fo l lowing a n t i b i o t i c s / r e s i s t a n c e s t r a i n s i s o l a - drugs t e d from sewage wa te r

53 Ampic i l l i n -49 92.4

Amikacin - 13 24.5

B a c i t r a c i n - 46 86.7

Chloramphenicol - 4 7.5

C h l o r o t e t r a c y c l i n e - 1 0 18.8

Erythromycin - 46 86.7

Furazo l idone - 42 79.2

Gentamycin - 0 0

Kanamycin - 3 5.6

N a l i d i x i c acid - 3 5.6

Neanycin - 2 3.7

polymyxin B-41 77 .3

Rifampicin - 49 92.4

Streptomycin - 8 15.0

Su lphad iaz ine - 40 75.4

69

TABLE 13 : Percent r e s i s t ance against individual a n t i b a c t e r i a l agent monitored in the soi l

Total number Number of i s o l a t e s r e s i s t a n t Percent (%) of E .co l i s t r a - t o a n t i b i o t i c s / d r u g s res i s tance ins i s o l a t e d from so i l

45 A i n p i c i l l i n - 43

Amikacin - 6

B a c i t r a c i n - 37

C h l o r a m p h e n i c o l - 5

C J i l o r o t e t r a c y c l i n e - 1 0

E r y t h r o m y c i n - 43

F u r a z o l i d o n e - 40

Gentamycin - 0

Kanamycin - 3

N a l i d i x i c a c i d -2

Neomycin - 2

Po lymyxin B - 3 0

R i f a m p i c i n - 35

S t r e p t o m y c i n - 6

S u l p h a d i a z i n e - 40

9 5 . 5

13 .3

8 2 . 2

1 1 . 1

22 .2

9 5 . 5

8 8 . 8

0

6 . 6

4 . 4

4 . 4

6 6 . 6

7 7 . 7

1 3 . 3

8 8 . 8

70

7 3.3% were derived from i n d u s t r i a l or ig in r e spec t ive ly .

The highest MIC of 3200 ug/ml was observed in both cases .

89.9% of t he i s o l a t e s showed re s i s t ance t o Cd"*"*" of which

73.3% of the i s o l a t e s were from i n d u s t r i a l e f f l u e n t s . The

maximum MIC at a concentration of 3200 ug/ml was exhib i ­

t ed by these s t r a i n s t owards Cd"*""*" (Table 14).

Table 15 descr ibes the incidence of r e s i s t ance t o

var ious metals and t h e i r MIC value in the t e s t E .col i i s o ­

l a t e s frcxn so i l o r i g i n . 46.7% of the t e s t s t r a i n s showed

r e s i s t ance t o Hg"*""*" of which 26.6% of the s t r a i n s were i s o ­

l a t ed from i n d u s t r i a l a rea . The highest MIC of 100 ug/ml

t o Hg"*""*" was observed in 6.7% of t he s t r a i n s i s o l a t e d from

i n d u s t r i a l area whereas the minimum MIC t o Hg' "*" was 12.5

ug/ml. 100% of the i s o l a t e s exhibi ted res i s t ance t o Pb" "*",

Cd"*"'" and Zn"*"*" of which 5 3.3% Pb"*"*" r e s i s t a n t , 70% Cd'*"''

r e s i s t a n t and 63.3% 2n"*""*" r e s i s t a n t E.col i s t r a i n s had been

i s o l a t e d from the i n d u s t r i a l a r ea . The maximum MIC of 3200

ug/ml was observed for these meta ls , which were mostly of

i n d u s t r i a l i s o l a t e s . The minimum MIC of 800 ug/ml was

displayed for Pb"*""*" (Table 15).

Table 16 shows the MIC values in the 18 s t r a i n s

of Enterobacter aeroqenes against four heavy metals

i s o l a t e d from sewage. 61% of the s t r a i n s were r e s i s t a n t

t o mercury whereas 100% of the s t r a i n s showed re s i s t ance t o

TABLE 14 i i n c i d e n c e of meta l r e s i s t a n c e and t h e i r MIC^ v a l u e in E . c o l i i s o l a t e d from sewage water

71

Heavy Me ta l s

Minimal I n h i b i t o r y concen­t r a t i o n s (MICs) (iig/ml)

No. of s t r a i n s showing r e s p e c t i v e MIC v a l u e I

Domestic sewage

Indus­t r i a l e f f l ­u e n t s

Inc idence of a b s o l u t e r e ­s i s t a n c e aga­i n s t t h e r e s p e c t i v e me ta l i

I n d u s ­t r i a l e f f l u e n t s

T o t a l

Frequency

Mercury (Hg)

12.5 25 50

100

200

1 1 1 1 0

2 4 1 5 4

53.3% 66.6%

10.0%

16.7%

6.7%

20.0%

13 .3%

Lead (Pb)

800

1600

3200

3

3

2

0

9

11

66.6% 93.3%

10.0%

40.0%

44 .3%

Cadmium 1600 (Cd)

3200

11

11

73.3% 89.9%

4 3 .3%

4 6.6%

Zinc (Zn)

1600

3200 15

7 3 .3% 9 3 .3%

36.6%

5 6.6%

*Total number of isolates « 30

72

TABLE 15 t I nc idence of meta l r e s i s t a n c e and t h e i r MIC va lue in E . c o l l i s o l a t e d from s o i l

Heavy M e t a l s

Mercury (Hg)

Lead (Pb)

Cadmium (Cd)

Z inc (Zn)

Min ima l I n h i b i ­t o r y con-r c e n t r a t i on (MICa) u g / m l

12 .5

25

5 0

100

8 0 0

1600

3200

1600

3200

1600

Number o: showing . t i v e MIC

1 D o m e s t i c sewage a r e a

3

2

1

0

6

7

1

6

3

5

f s t r a i n s r e s p e c -v a l u e

I n d u s ­t r i a l a r e a

0

3

3

2

0

11

5

13

8

7

I n c i d e n c e of a b s o l u t e r e s i s t a n c e a g a i n s t t h e r e s p e c t i v e m e t a l ]

1 I n d u s - T o t a l t r i a l a r e a

26.6% 46.7%

53 .3% 100%

70% 100%

Frequency

10.0%

16.7%

13.3%

6.7%

20.0%

60.0%

20.0%

63 .3%

36.6%

40.0%

3200 63.3% 100%

11 60.0%

* T o t a l number of i s o l a t e s = 30

73

TABLE 16 : I n c i d e n c e of m e t a l r e s i s t a n c e and t h e i r MIC v a l u e i n E n t e r o b a c t e r a e r o q e n e s i s o l a t e d from sewage w a t e r

Heavy M e t a l s

Mercury (Hg)

Min ima l I n h i b i t o r y c o n c e n ­t r a t i o n s (MICs) u g / m l

12 .5

25

50

No. of s t r a i n s showing t h e i r MIC v a l u e

5

4

2

I n c i d e n c e of a b s o l u t e r e s i ­s t a n c e a g a i n s t t h e r e s p e c t i v e m e t a l

F requ­ency

6 1 . 1 %

27.7%

22.2%

1 1 . 1 %

Lead (Pb)

1600 11

100%

6 1 . 1 %

3200 38.8%

Cadmium (Cd)

1600

100%

33 .3%

3200 12 66.6%

Z i n c (Zn)

1600 13

100%

72.2%

3200 27.7%

* Total number of i s o l a t e s = 18

74

lead, cadmixjm and z inc . Highest MIC of 3200 ug/ml was

observed agains t Pb"*""*", Cd"*" and Zn'*"''" and minimum was at

1600 iig/ml ^Table 16) .

All the 18 i s o l a t e s of E.aeroqenes were a l so t e s t ed

for t h e i r r e s i s t ance against 15 a n t i b a c t e r i a l agents and

these were invar iably found to be multiply r e s i s t a n t . The

nxjmber of ant ib iot ics /dr^igs against which r e s i s t ance was

found ranged from 6 t o 8 a n t i b i o t i c s / d r u g s . Thus out of

18 E.aeroqenes i s o l a t e s , 5 subtypes were i den t i f i ed on the

b a s i s of antibiograms as shown in t a b l e 17.

Although the aforementioned parameters s trongly

suggested for the presence of plasmids, yet a d i r ec t

experiment was requi red . All the aforementioned E .co l i

and E,aeroqenes i s o l a t e s were found to harbour plasmids

based on the agarose gel e lec t rophores i s (data not shown).

Fig . 1 shows the gel e l ec t rophore t i c p r o f i l e s of plasmid

i so l a t ed from 5 d i s t i n c t E.col i i s o l a t e s from s o i l . Only

a very s l i gh t difference was observed in t h e i r r e l a t i v e

mobi l i ty . Fig . 2 shows the agarose gel e l ec t rophore t i c

p r o f i l e s of plasmids in the 8 E .col i s t r a i n s i so l a t ed

from the sewage water . Most of the s t r a i n s show mul t ip le

copies of plasmids in the sewage i s o l a t e s whereas s ingle

plasmid appeared in s o i l i s o l a t e s IFig . l ) .

75

TABLE 17 t A n t i b i o t i c r e s i s t a n c e p a t t e r n of 18 E n t e r o b a c t e r a e r o q e n e s s t r a i n s i s o l a t e d from sewage w a t e r

S .No. E , a e r o q e n e s R e s i s t a n c e a g a i n s t No. of Nxoraber of % s t r a i n =„+. v ^ ^.^ ^ ^ / H V . , , ^ „ a n t i b i o - s t r a i n s a b u -

a n t i b i o t i c s / u r u g s . . T_ j j ' ^ t i c s a g a - s h o w i n g ndan-

i n s t r e s i s t a - ce which n e e r e s i s t a ­n c e was o b s e r v e d *

A , B , E , R , S , S z

A , B , E , K , R , S z

A , B , E , F r , R , Sz

A,B , C t , E , F r , R , SZ

A,B , C t , E , F r , R , S, Sz

T o t a l number of E n t e r o b a c t e r a e r o q e n e s s t r a i n s foxond t o b<£

s e n s i t i v e t o a l l a n t i b a c t e r i a l a g e n t s = 2 ( 1 1 . 1 % ) .

* T o t a l niamber of a n t i b a c t e r i a l a g e n t s employed f o r t h e s t u d y

= 1 5 .

In fact we had used 17 antibacterial agents but there was

no exception with regard to the resistance against ampicillin

and penicillin as well as chlorotetracycline and tetracycline.

Therefore operationally we were working on 15 agents only.

1 .

2 .

3 .

4 ,

5 .

EA^

EA^

EA3

EA4

EA5

6

6

6

7

8

3

1

7

3

2

1 6 . 6 %

5 . 5 %

3 7 . 7 %

1 6 . 6 %

1 1 . 1 %

Fig. 1. Agarose gel e lec t rophores i s p a t t e r n s of

plasmid DNA i so l a t ed from 5 E.col l Si and Sd

i s o l a t e s ( lanes a to e ) .

*arrow ind ica tes t h e pos i t ion of the wel ls

( s t a r t i n g point) .

Fig, 2. Agarose gel electrophoresis pat terns of

plasmid DNA isolated from 8 E.coli sewage

water Isolates (lanes a to h ) .

*arrow indicates the position of the wells

(s tar t ing po in t ) .

a b c d e f g h

78

Transfonaation : To ascer ta in whether the drug re s i s t ance

in the aforementioned E«coli s t r a i n s Ci .e . Wi, Wd, Si and

Sd) was a plasmid mediated character , tne transformation

of E»coli AB115 7 with R-plasmids i so la t ed from these s t r a i n s

was performed. Tne transformation frequency of AB1157 with

these four R-plasmids i . e . Wx , Wd-, Wd., Si . as well as

two more plasmids i so l a t ed from soi l* Sd. and Sd^ i s shown

in Table 18. The transformation frequency of r ec ip i en t c e l l s

with R-plasmids Wi^/ Wd_, Wd., a l l derived from sewage was -3 -3 -3

ca lcu la ted t o be 1.7x10 , 3.83x10 and 2.96x10 •" respec­

t i v e l y , on the o ther hand, t he plasmids of so i l o r i g i n . S i . ,

Sd , Sd- displayed t h e transformation frequencies of 2.54x

10"-^, 2.46x10"'^ and 3.29x10-3 respec t ive ly (Table 18) .

Plasmid curing t Further confirmation in favour of t he

plasmid mediated res i s t ance charac ter was ca r r i ed out by

means of curing experiment. For t h i s purpose we made use

of two E.col i s t r a i n s i . e . Si . and Wd. los t the res i s tance

character on treatment with ethidium bromide. Fig . 3 shows

a curve of percent curing Vs ethidixom bromide concentra t ions .

Curing was observed at the minimum concentration of 0.5

ug/ml and increased with t h e increasing concentrat ion of

ethidixjm bromide. One E.col i s t r a i n s i so l a t ed from so i l

(S i . ) displayed 7.4% curing while other from water i s o l a t e

(Wd.) displayed 37.7% curing on treatment with 0.5 ug

79

TABLE 18 ; T r a n s f o r m i n g a b i l i t y of AB1157 w i t h t h e s i x t e s t p l a s m i d s

S.No. T e s t T o t a l n o . No. of t r a n s - T r a n s f o r m - M a r k e r s P l a s m i d s of c e l l s f o r m a n t s a t i o n t r a n s f e r -

f r e q u e n c y r e d

1 .

2 .

3 .

4 .

5 .

6 .

pWis

pWd3

pWd^

p S i 4

pSd^

pSd_

1.80x10"^ 3.07x10'^

LOSxlo"^ 4 . 1 4 x 1 0 ^

1.68x10"^ 4 .98x10^

1.27x10*^ 3 .77x10*

1.48x10*^ 3 .13x10*

l .OSxlo '^ 3 .46x10*

1.70x10

3.83x10"^

2.96x10"^

2.54x10"^

2.46xl0~^

3.29x10"^

A,Ct,Hg, Cd,Zn

A, Ct,Hg, Cd

A,Ct,N,R, Cd

Cd,Zn

A,Ct,Hg, Cd

Hg

Fig . 3 . Plasmid curing and survival of plasmid

harbouring £.co 1 i Si. and Wd. s t r a i n s

by treatment with ethidium bromide.

Symbols - curing ( so l id l i n e ) ; survival (broken l i n e ) ;

E .col i Si^' ( © ) ; g . co l i Wd ( O ) .

100

CT C

cr D o •«—>

c 0 u

a.

-vQO (i> - 1

o (V> ^ ^ ^ r 60-CO c -^ <

< 40-

2o|

60 80 100 Ethidium Bromide(ug/m()

81

ethidium bromide per ml of cu l tu re on l6 h of t rea tment .

A maximum curing of 92% was observed with maximum dose of

(80 Aig/ml) of ethidium bromide (Fig. 3 ) .

Lethal e f fec t s of curing agent : The curing agent ethidium

bromide was a lso observed t o be l e t h a l a t the concentrat ions

used (Fig . 3 ) . The survival was decreased by 3% in Si^

s t r a i n of E .col i on 0.5 aig/ml ethidium bromide treatment

and was only 7.0% at the concentrat ions of 100 ug/ml

(Fig . 3 ) . In E .co l i Wd s t r a in t he survival was decreased

by 7% at 0.5 ug/ml ethidium bromide treatment and was

reduced to a mere 4,6% at t he concentrat ions of 100 ug/ral

(Fig . 3 ) .

Conjugation : Table 19 gives the conjugative p o t e n t i a l

of the four t e s t E .col i s t r a i n s . E .col i Wi_ demonstrated

58.7% conjugation when mated for 60 min with r ec ip i en t

E .co l i AB115 7 s t r a i n . The E.col i Wd. and Wd exhibi ted

5 2.8 and 63.15 per cent conjugation respect ive ly under the

same experimental condi t ions . The conjugation was observed

t o be 62.9 per cent in another E .co l i s t r a i n . Si . which

was i so la t ed from s o i l .

Molecular weight of R-plasmids : F ig . 4 shows the r e l a t i v e

mobi l i ty of R-plasmids on agarose gel i so l a t ed from E.col i

Wi2# Wi-# Wi- and Wic s t r a i n s in lane d,e/,f and g respec­

t i v e l y . Other lanes carr ied t he marker DNAs of known

molecular weight.

8 2

TABLE 19 : Conjugative p o t e n t i a l of mult iply r e s i s t a n t E.col i i s o l a t e s with rec ip ien t AB1157 s t r a in

Donor Time Colonies on Colonies on Percent s t r a i n (min) p la in p l a t e s A+T+Cd conjugation

p l a t e

28.1%

58.7%

22.6%

5 2.8%

26.4%

63.1%.

31.3%

62.9%

SWi^

SWd3

SWd^

SSi^

30

60

30

60

30

60

30

60

194

228

146

176

121

152

204

232

54

134

33

93

32

96

64

146

Fig. 4 . Agarose ge l e lec t rophores i s of the plasmid

species i so l a t ed from E.cxjli i s o l a t e s from

water.

lane a - Plasmid pBR322

lane b - R-plasmid i so la ted from marker Ec Rp4.

lane c - I n t a c t A DNA

lane d - R-plasmid i so l a t ed from E.col i Wi2 s t r a i n . ""

lane e- R-plasmid i so l a t ed from E.col i Wi s t r a i n ~

lane f- R-plasmid i so la ted from E.col i Wi. s t r a i n .

lane g -R-plasmid i so la ted from E.col i Wic s t r a i n .

*arrow ind ica t e s t he pos i t ion of the wells ( s t a r t i n g p o i n t ) .

a b c d e f g

84

Fig. 5 gives the ca l ib ra t ion curve for the molecular

weight determination making use of the data from Fig . 4 .

The molecular weight of t he plasmids i so l a t ed from the four

aforementioned E.col i s t r a i n s ca lcula ted on the b a s i s of

t h i s c a l i b r a t i o n p lo t was found t o be 46 and 50 Kb (Kilo-

base p a i r s ) .

Fig. 6 shows the r e l a t i v e mobil i ty of mult iple

plasmids i so l a t ed from E,co1i Wi, and Wd s t r a i n s . The

s t r a i n s Wi appears to harbour two plasmids (lane c ) .

The molecular weight ranged from 17,5 t o 50 Kb (Fig. 7 ) .

The s t r a in v;d- a l so appear to harbour two d i s t i n c t plasmids

(lane d) and the molecular s ize of the two plasmids

ranged from 22.0 t o 51 Kb. In con t ras t , the s t r a i n s Wd.

seems t o harbour four plasmids (lane e) and the molecular

s ize of these plasmid DNA calcula ted t o be carying from

5.6 t o 52 Kb.

Fig . 5 . Molecular .size Vs mobil i ty p lo t of the t e s t

species ,4nd the standard DNA.

Symbols - R-pLasmids i s o l a t e d from Wi„ and Wi_ (O )

a ;^rox. s i ze i s 52 Kb.

asmid i so l a t ed from Wi and Wi (©)

approx. s ize i s 50 Kb.

- Standard DNA markers ( • )

60

50

N

I 30 o

20

IHO-I± i baJ -Bloa i abiiTTaXlq-/!

, a.'i £cf 3± aJTia . x o a t

10

0 10 20 30

Relative Mobility (mm)

W

Fig . 6. Agarose gel e lect rophoresis of mult iple

plasmid species i so la ted from E.col i (Wij-,

Wd^, Wd.) s t r a i n s .

lane a - Plasmid pBR3 2 2

lane b - R-plasmid i so la ted from -marker Ec%>4.

lane c - Plasmid i so la ted from E.col i Wij. s t r a i n .

lane d - Plasmid i so la ted from E. co 1 i Wd_ s t r a i n .

lane e - Plasmid i so la t ed from E . co l i Wd. s t r a i n . 4

lane f - Intact A DNA

*arrow indicates the position of the wells

(starting point).

Fig. 7. Molecular size Vs mobility plot of the t e s t

(multiple species of the R-plasmids isolated

froB Wi,

Symbols - Wij. p

- Wi^ p2

- " S f>l -^.Wd3 P2

- Wd^ p ^

' - Wd^ P3

- w d ^ p .

Wd and Wd strains) and standard DNAr*

0 ) approx. size 50 Kb

Q ) approx. size 17.5 Kb

L ) approx. size 51 Kb

A ) approx. size 22.0 Kb

n ) approx. size 5 2 Kb

01 ) approx. size 4 3.5 Kb

a ) approx. size 32.5 Kb

• ) approx. size 5.6 Kb

Standard DNA markers - ( • )

60

50

40

N

in 30

Z3 O

o

20

W

0' 10 20 30 Relative Mobility (mm)

40

DISCUSSION

88

Seasonal and loca t iona l va r i a t ions in TBC, TEC, TC

and FS in sewage water and so i l s are shown in t a b l e 4 - 9 .

The average t o t a l b a c t e r i a l covmts were found t o be retnark-

ably higher in domestic sewage water (679x10 CFU/100 ml)

as compared with t h e i n d u s t r i a l e f f luen t . Almost s imi la r

r e s u l t s were repor ted e a r l i e r by Biemond (1963). The t o t a l

en t e robac t e r i a l coxints and t o t a l coliforms were always

much l e s s than the t o t a l bac t e r i a l coionts. This i s q u i t e

obvious in nature because t o t a l b a c t e r i a l counts represent

a l l b a c t e r i a including TEC and TC groups of microorganisms.

The mean populat ions of t o t a l colifozm and feca l 4

s t rep tococc i in danes t i c sewage were 12.3x10 MPN/ml and 4

0.7x10 MPN/ml r e s p e c t i v e l y . The r e s u l t s were almost simi­l a r with the f indings of Al-Shahwani et a l . (1986) who

repor ted t o t a l coliform and fecal s t reptococci counts to 4 4

be 20.9x10 ce l l s /ml and 8.0x10 cel ls /ml in sewage water .

Bel l et_ al_. (1980) a lso reported nearly s imi la r d e n s i t i e s

of t he se b a c t e r i a l f l o ra in the sewage system.

Higher c e l l densi ty as well as a high propor t ion

of coliforms with respect to t o t a l b a c t e r i a l populat ion

was a l so observed in domestic sewage and in i n d u s t r i a l

e f f l uen t s as compared to s o i l s . Remarkably high t o t a l

coliforms and fecal coliform count in sewage compared

with s o i l a l so reported by several authors (Geldreich

e t a l . , 1964; Gordon, 1972; Cohen and Shuval, 1973).

89

In the present inves t iga t ion , t he average d e n s i t i e s

of t o t a l en te robac te r ia l counts, t o t a l coliforms and fecal

s t reptococci obviously including the t o t a l bac t e r i a l

counts were found t o be more in domestic sewage water

than the i ndus t r i a l e f f luen t s . This i s q u i t e ovbious in

na tura l system due to contamination of more animal and

human excreta t o domestic sewage whereas i n d u s t r i a l e f f lu ­

ents are expected t o contain t o x i c heavy metals which

would ra ther inh ib i t the growth and k i l l the bac te r i a at

high concentra t ions . Jana and Bhattacharya (1988) studied

the inhib i tory ef fect of various heavy metals l i ke Hg, Cd,

Pb, As, Cu and Cr on the growth of feca l E .col i and demo­

ns t r a t ed a gradual decl ine in i t s growth with the increase

of exposure time as well as concentrat ion of meta l s . More­

over, several authors have reported t h a t heavy metals

introduced into the water not only br ing about several

chemical changes and high degree of v a r i a t i o n in metal

concentration but a lso affect the e n t i r e ecosystem inc lu­

ding the bac t e r i a l population (Fostner and Wittman, 19 79;

Vinkour et al_., 1980; Moore and Ramamoorthy, 1984) .

I t i s a well deocumented fac t t ha t t he Aligarh c i ty

not only possesses the large scale lock manufacturing

i n d u s t r i e s but also provides s h e l t e r t o innumerable num­

ber of small scale lock manufacturing p l an t s re leas ing

t h e i r e f f luents to domestic d ra ins (Ajmal et al.»# 1980) .

90

In our system t h e average t o t a l b a c t e r i a l counts

which largely included t h e aerobes and facu l t a t ive anae­

robes was found to be higher during spring season in dome­

s t i c sewage water . Reddy e t al^. (1986) also reported a

s ign i f ican t ly higher count of t o t a l aerobic bac te r i a

during spring season in po l lu ted water .

The average t o t a l en t e robac t e r i a l count, t o t a l c o l i -

forms and fecal s treptococ 'ci were recorded to be higher

in our study during summer and spring season in sewage

water . I t i s cons is ten t t o the findings reported by seve­

r a l authors who obtained r e l a t i v e l y more en te robac te r i a l

populat ions during t h e warm season (Rudolf et a_l., 1950;

Sommers and Nelson, 197 6) . Such seasonal va r i a t ion has

been a t t r i bu t ed to c e r t a i n environmental fac tors l i k e

temperature, so la r r a d i a t i o n , seasonal v a r i a b i l i t y and

concentration of n u t r i e n t s .

The present study a lso c lear ly ind ica tes tha t sewage

system was not a good r e se rvo i r for the b a c t e r i a l f lora

(TBC) compared with s o i l both in terms of locat ion as well

as of season (Table 4-9) . Such a lower t o t a l b a c t e r i a l

counts may be due t o t h e presence of t ox i c metals and o ther

undesirable substances in the sewage samples which would

often be inh ib i to ry t o b a c t e r i a . This i s qu i te reasonable

because of the lack of p a r t i t i o n i n g between the i n d u s t r i a l

and domestic sewage.

91

Our contention gains fu r ther support from the

s imi la r data on TBC derived from s o i l s in the v i c i n i t y

of i n d u s t r i a l as well as domestic d ra ins thereby sugg­

es t ing a common type of n u t r i t i o n a l and physiological

s t a t u s of the two l o c a t i o n s .

Similar t o the f indings obtained with the sewage in

the seasonal v a r i a t i o n , we found a comparatively lower

t o t a l b a c t e r i a l counts in s o i l during winter season. This

may be largely due t o t he overflow of sewage carrying

the t o x i c metals and o ther undesirable substances during

the post monsoon season extending upto the win te r . Fur­

thermore the environmental fac to rs l i k e temperature, so la r

r ad ia t ion , seasonal v a r i a b i l i t y and concentration of nut ­

r i e n t s would also s i g n i f i c a n t l y cont r ibute to such v a r i a ­

t i on (Santo-Domingo et_ al . . , 1989).

Comparing the r e l a t i v e proport ion of en te robac te r i a l

count within the t o t a l b a c t e r i a l f lo ra , i t was qu i t e

obvious t ha t the so i l did not contain impressive q u a n t i ­

t i e s of enterobacter ia r a the r ce r ta in nonenterobacter ia l

f lora would be p r e sen t . This was t r u e not only in terms

of aforesaid r e l a t i v e proport ion but also in terms of

absolute number of e n t e r o b a c t e r i a l populat ion. Contrary

t o t h i s sewage systan ca r r i ed a lo t of en te robac te r ia

presumably due t o t h e feca l contamination.

92

Another s ign i f i can t obsejrvation t o be mentioned

here was regarding the seasonal va r i a t i on which was much

more pronounced in case of t o t a l b a c t e r i a l f lora but such

a systematic v a r i a t i o n was absent in the en te robac te r ia l

count. This pa t t e rn might r e f l e c t a sor t of p ro tec t ive

effect on the en te robac te r i a but not on other b a c t e r i a l

f lora brought about by ce r t a in mechanism of res i s tance

against the undesi rable substances l i k e metals and a n t i ­

b a c t e r i a l agen ts .

The r e l a t i v e propor t ion of t o t a l coliform within

the en te robac te r i a l populat ion appears t o be s i gn i f i can t ly

reduced in the s o i l sartples (0.00 2%) compared t o t h a t in

sewage (1.3%). This probably r e f l e c t not only the presence

of such organisms because of the la rge amount of intake

into the sewage system but a l so due to the capacity t o

withstand under t h e unfavourable condit ions resu l t ing

out of the tox ic metal contamination.

The r e l a t i v e propor t ion of t o t a l coliform with

respect to e n t e r o b a c t e r i a l count did not indicate t he

expected reduction in winter season in the sewage system

ra the r the opposite t r end was recorded. The aforementioned

and other parameters d i r ec t ed us t o in tens ively work

upon the following two l i n e s : -

i ) The s t a t u s of metal and a n t i b i o t i c res i s tance in po­

l l u t i o n ind ica to r coliform bacterium of fecal or ig in

93

i . e . E . c o l i .

i i ) The s t a tu s of t h e metal and a n t i b i o t i c res i s tance in

the enterobacterium of nonfecal o r ig in i . e . Enterobac-

t e r aeroqenes.

Almost a l l L .co l i and Enterobacter aeroqenes i s o ­

l a t e s from sewage water and s o i l s were found to be mult iply

r e s i s t a n t t o an t imic rob ia l agents (Table 10,11 and 17) .

The organisms were mostly r e s i s t a n t t o ampic i l l in , amika-

cin, b a c i t r a c i n , chloramphenicol, ch lo ro te t racyc l ine ,

erythromycin, furazol idone, kanamycin, na l id ix i c acid,

neomycin, polymyxin B, r i fampicin , streptomycin and su l -

phadiazine, con f l i c t ing data have been reported from v a r i ­

ous p a r t s of the world as f a r as the percentage of r e s i s ­

tance character i s concerned (Feary et_ al_,, 19 72; Niemi

et a l . , 1983; Sameer et_ a l , . , 1988).

A high l eve l of r e s i s t a n c e against p e n i c i l l i n

(9 2.45%) was observed in t h e sewage water i s o l a t e s and

86.6% of i s o l a t e s were r e s i s t a n t t o rifampicin and 81.1%

to polymyxin B. Shears e t al_. (1988) studied the prevalence

of res i s tance t o s ix commonly used ant imicrobial agents

in fecal coliform from chi ldren in Khartoum (Sud<S-"n) and

he found r e l a t i v e l y very high frequency of r e s i s t ance

against ampic i l l in (96%) and 705'o t o chloramphenicol.

94

Baya et^ al_. (1986) i s o l a t e d E.col i from sewage

e f f luen ts t h a t were r e s i s t a n t t o a combination of a n t i ­

b i o t i c s including kanamycin, chloramphenicol, and t e t r a ­

cyc l ine .

In the same year , Aljebouri and Meshhadoni examined

po l lu ted water samples from r i v e r T i g r i s m the v i c i n i t y

of raw sewage o u t f a l l for the incidence of a n t i b i o t i c

r e s i s t ance among E .co l i and found t h a t 70% or more of '

t hese organisms were r e s i s t a n t t o one or more a n t i b i o t i c s

(Aljebouri and Meshhadoni, 1986) . Vinayagamoorthy et_ a l .

(1986) found t r a n s f e r a b l e r e s i s t a n c e to ampic i l l in ,

chloramphenicol, t e t r a c y c l i n e and sulphamethoxazole, among

1207 c l i n i c a l i s o l a t e s . Further more, Chong (1986) i s o ­

l a t ed 25% s t r a i n s of en te robac te r ia from man in penin­

su la r , Malaysia of which 7 were found t o be Enterobacter

spp.and 5 were E . c o l i . They were a l l sens i t ive t o n a l i ­

d ix i c acid but r e s i s t a n t t o more than 3 other a n t i b i o t i c s .

The nvimber of r e s i s t a n c e charac ter ranged from 3 t o 19

a n t i b i o t i c s .

In our study, E.col i i s o l a t e s frcxn so i l s a lso

showed a renarkably high l eve l of r e s i s t ance t o ampici­

l l i n (95.5%). Frederickson (1988) a lso found t h a t E .col i

i s o l a t e s from s o i l s were r e s i s t a n t t o ampici l l in with

maximum frequency and a lso showed t h a t these i s o l a t e s

were mul t ip ly r e s i s t a n t to ant imicrobia l agents l i ke

95

ampic i l l in , p e n i c i l l i n , c a r b e n i c i l l i n , streptomycin,

kanainycin and t e t r a c y c l i n e .

The higher l e v e l of r e s i s t a n c e as conferred by

these E.col i i s o l a t e s agains t ampici l l in and some other

ant imicrobial agents (Table 12 and 13) seems t o r e f l e c t

wide spread chemotherapeutic use of these drugs in the

v i c i n i t y of t e s t r eg ion . The indiscr iminate use i s one of

t he major f ac to r s t h a t r e su l t ed in the energence of a n t i ­

b i o t i c r e s i s t a n t b a c t e r i a ( an i th , 1970; Levy, 1983; Al-

Doori et_ a l . , I9b6) .

The E .co l i and K.aeroqenes isola ted from sewage

water and s o i l s have been c l a s s i f i e d into many d i f fe ren t

an t ib io types on t h e b a s i s of antibiograms. The E.col i

i so l a t ed frcxn sewage have been c lass i f i ed in to 9 groups

and from s o i l s i n t o 8 group on the basis of antibiograms

obtained against d i f f e ren t a n t i b i o t i c classes (Table 10

and 11). The E.aeroqenes i s o l a t e s have been c l a s s i f i e d

in to 5 biotypes on t h e b a s i s of antibiograms (Table 17) .

Among the var ious methods of taxonomic s tud ies , the

bac te r i a have been commonly c l a s s i f i ed on the b a s i s of

antibiograms alongwith t h e i r plasmid prof i l es (Gi l lesp ie

et a l . , 1990). The E .co l i i so l a t ed from sewage samples

were multiply r e s i s t a n t t o t he antimicrobial agents

ranging from 3 to 9 . These findings are in agreement with

those of Bell et a]^. (1980) and Al-Doori et a l . (1986).

96

The p r e s e n t i n v e s t i g a t i o n i n d i c a t e d t h a t 66.6% of

E . c o l i s t r a i n s i s o l a t e d from sewage harboured r e s i s t a n c e

t o Hg" "*" whereas 93.3% i s o l a t e s were r e g i s t a n c e t o Pb"*""*"

and Zn++ both and 89.9% t o Cd"*" o n l y . Nakahara and Yone-

kura (1987) found t h e f requency of meta l r e s i s t a n c e in t h e

a q u a t i c E . c o l i i s o l a t e s t o be 49% fo r Hg"*""*", 74% for Cd"*"*"

and 84% for As"*"*"*".

In t h e p r e s e n t s t u d y , E . c o l i and E .aeroqenes

i s o l a t e d from sewage and s o i l were a l s o t e s t e d f o r t h e

r e s i s t a n c e a g a i n s t f o u r heavy m e t a l s i . e . Hg, pb , Cd and

Zn, Major i ty of E . c o l i i s o l a t e s from sewage and s o i l s sho­

wed MIC of more t h a n 25 ug/ml t o Hg"*""*". F u r t h e r , MIC v a l u e s

upto 3 200 iig/ml were r e c o r d e d f o r Cd"*"*", Pb"*"*" and Zn"*""*•

(Tgble 14-16) whxch were e x c e p t i o n a l l y high compared t o

p r e v i o u s s t u d i e s (Summers and S i l v e r , 1972; Austin et_ a l . ,

1977; Clark e t a l . . , 1977 ; Marques e t a l . , 1979; and

Hor i t su et. a l . , 1985) . Summers and S i l v e r (1972) and

Clark et. al. . (1977) r e p o r t e d t h e maxiravim i n h i b i t o r y

concen t ra t ion t o be on ly 10 ug/ml fo r Hg . Moreover,

H o r i t s u et. aul- (1986) r e p o r t e d t h e E . c o l i s t r a i n s r e s i s ­

t a n t t o as high c o n c e n t r a t i o n s of Cd"*"" , Pb"*"*" and Zn"*""

a s 1283 iig/ml, 650 jug/ml and 3 22 ug/ml r e s p e c t i v e l y . In

our study most of t h e t e s t b a c t e r i a d i s p l a y i n g t h e h i g h ­

e s t MIC had been i s o l a t e d from i n d u s t r i a l e f f l u e n t s . T h i s

may be due t o t h e s t r a t e g i c p o s i t i o n of t h e area of ou r

97

study namely t he o u t s k i r t s of Aligarh c i ty which houses

many lock manufacturing fac to r i e s and t h e i r e f f luents

would e s s e n t i a l l y contain q u i t e a large amount of these

meta l s .

According t o some authors , the emergence of

m e t a l l i c r e s i s t a n c e could have been due to the exposure

of these t o x i c metals t o human and animals (Allen et_ a l » ,

1977; Clark e t a l . , 1977; Dutta e t al^., 1980). However

such a p o s s i b i l i t y is not f ea s ib l e in our case because

t he to l e rance l i m i t s in human and animals for Hg' '*', Cd" "

Pb" "*" and Zn" "*" have been reported t o be 0,001 ug/ml, 0.005

ug/ml, 0,05 ug/ml and 5.0 ug/ml respec t ive ly . The l eve l s

exceeding the se value i s not expected t o be present in

animal and host (WHO, 1985).

On the contrary , we have found the en terobac ter ia

r e s i s t a n t t o 20 0 ug/ml for Hg"*" , 3 200 ug/ml for Cd"*"*",

3 200 ug/ml t o Pb" "*" and 3200 ug/ml for Zn" " (Table No. 14-

16). The incidence of highly m e t a l l i c r e s i s t a n t en tero­

bac t e r i a t he re fo re r e f l e c t such a high concentrat ions of

these metals i n t h e environment o ther than the hos t . The

only poss ib le explanation the re fore i s the presence of

high concentrat ion of t o x i c metals in our t e s t systems

i . e . sewage and s o i l .

This s u b s t a n t i a l l y supports our contention tha t

the coliform populat ion present in the sewage could be

98

r e l a t i v e l y more due t o both the reasons : -

i) The la rge in take of mul t ip le drug r e s i s t an t coliform

into t he sewage system,

i i ) The acqu i s i t i on of metal r e s i s t a n c e of these coliforms

within the sewage a f t e r the entry in to the system.

The f i r s t point c a r r i e s weight because the sewage

i s not ej<pected t o carry a s ign i f i can t amount of the drugs/

a n t i b i o t i c s t o acqui re mul t ip le r e s i s t ance unless t h e host

i s exposed t o a high concentrat ions of these cnemothera-

peu t ic agent for q u i t e a long t ime . tttJweVer t he second

point could be proved on the b a s i s of the presence of the

large q u a n t i t i e s of t o x i c metals (Ajmal et al_., 1980) which

would be l e t h a l t o t h e host and thus a acquis i t ion of metal

r e s i s t ance in b a c t e r i a ins ide the host i s most un l ike ly .

To a sce r t a in whether or not the res i s tance was

plasmid mediated, we fur ther extended our s tud ies in t h i s

d i r e c t i o n . 5 s t ra ins from 8 ant ib io types i so la ted from so i l

and 8 s t r a i n s from sewage were screened for plasmid i s o l a ­

t i o n and found t o harbour plasmids (Fig. 1 and 2) . These

r e s u l t s were almost s imi la r to t h a t of vakulenko and h i s

coworkers who found 30 out 32 a n t i b i o t i c r e s i s t a n t E.col i

s t r a i n s were carrying plasmids (Vakulenko et_ al^., 1980).

Hada and Sizemore (1981) suggested t h a t t h e i n c i ­

dence of p lasnid bear ing s t r a in s i s more in po l lu ted s i t e

99

than in the unpolluted zone. The incidence of high metal

r e s i s t a n t populat ion in E.col i and E.aeroqenes from sewage

and s o i l , suggest t h e p o s s i b i l i t y of increasing environ­

mental po l lu t ion t o these me ta l l i c s a l t s .

As f a r as t h e number of plasmids in the plasmid

harbouring s t r a in i s concerned most of t he s t r a i n s from

sewage were carrying mul t ip le copies of plasmids (Fig . 2

and 6)but so i l i s o l a t e s harboured s ingle plasmid. Baya

et a l . (1986) i s o l a t e d E.col l from sewage e f f luen ts r e s i s ­

tance to 9 d i f f e ren t a n t i b i o t i c s and these s t r a i n car r ied

mul t ip le bands of plasmid. Schuett (1988) also showed multi­

p l e bands of plasmid in t he s t r a i n i s o l a t e d from Sksshuts-

zyoen lake , whereas i s o l a t e s from brown water lake in south

Sweden showed t o harbour single plasmid of high molecular

weight O 3 0 Mdal) .

In t he present i nves t iga t ion , one of the s t r a in s

appears t o carry 4 types of plasmids ranging in molecular

weight from 5.6 t o 52 Kb whereas two other s t r a i n s ca r r ied

2 plasmids varying in molecular s ize from 17.5 t o 50Kb and

22.0 t o 51 Kb r e spec t ive ly .

Vinayagamoorthy et al^. (1986) i so la ted E.col i from

drinking water and demonstrated t h i s bacteriiom t o harbour

5 p lasn ids one of which having a molecular s ize of 2.4 Kb

ca r r i ed res i s t ance t o sulphonamide. He has also found the

100

E.col i from c l i n i c a l i s o l a t e s exh ib i t ing t r ans fe rab le

res i s tance to ampic i l l i n , t e t r a c y c l i n e , streptomycin,

chloramphenicol and tr imethoprim. These markers were

present in the s ing le plasmids of 60-120 Kb.

To provide evidence i f the r e s i s t a n c e markers were

ac tua l ly on the plasmid DNA, we ca r r i ed out the t r a n s ­

formation experiment employing plasmids i so la t ed from

t e s t E.ooli s t r a i n s . The expression of r e s i s t ance markers

in a l l the transformants suggested t h a t the a n t i b i o t i c

as well as metal r e s i s t a n c e was plasmid mediated. The

transformation frequency obtained with the rec ip ien t

s t r a in i . e . E.col i AB1157 (Table 18) was comparable t o

t h a t obtained by Bopp _et a_l. (1983) . He found t h a t E.cioli .

P.put lda and P. f luorescens s t r a i n s could be transformed

with P .putida derived RP. plasmid DNA at frequencies

ranging from l.SxlO" t o 3.5xlO" transformants per r e c i ­

p i e n t . All the t e s t E.col i s t r a i n s t r a n s f e r r e d 4 or more

res i s tance markers out of 7 t o 13 markers (Table 10 and

11) . Tantulivanchi et_ al_. (1981) had shown t h a t 9 out of

15 res is tance markers were t r a n s f e r a b l e in E .co l i •

The final proof in favour of t h e presence of

R-plasmids in t h e t e s t E .co l i s t r a i n s was provided

through the curing experiment. A s ign i f i can t f ract ion of

t e s t s t r a i n s was found t o lose t h e t e s t r es i s tance markers

101

in presence of curing agent i . e . ethidium bromide (Fig. 3 ) .

Our r e s u l t s of curing with ethidium bromide were even

b e t t e r than those obtained by El Syed and h i s coworkers

(1988) in case of c l i n i c a l i s o l a t e s of i^ .col i .

The t e s t E .co l i s t r a i n s were able t o t r a n s f e r some

of t h e i r r e s i s t ance markers t o standard E.col l r ec ip ien t

s t r a i n by conjugation CTable 1-9) . Moreover t h e percent

conjugation was much higher than those observed by Bell

et a l . (1980) in feca l coliform i so l a t ed from sewage e f f l ­

uen ts , but coincides with t he r e s u l t s obtained by Genthner

e t a l . (1988) in aqua t i c gram negative b a c t e r i a . This

t r a n s f e r of mul t ip le r e s i s t ance markers strongly suggests

t h a t t h i s property of a n t i b i o t i c as well as metal r e s i s ­

tance i s plasmid mediated (Summers, 1986).

Cavalcante et_ al. . (1988) i so l a t ed E.col l s t r a i n s

harbouring the conjugative plasmids from waste treatment

p lan t and demonstrated t h a t the i s o l a t e s were carrying

r e s i s t ance t o both an t imicrobia l agents and heavy meta ls .

Several workers have demonstrated the t r a n s f e r of

plasmids through conjugation in various systems including

the s o i l , water and o the r environmental condi t ions (Trevors

and Oddie, 1986> Trevors and Starodxib, 1987) .

We had performed our conjugation experiments in

l iqu id which might not be a b e t t e r environment as compared

t o so l id media. Since Genthner et a l , (1988) demonstrated

10 2

t h a t t h e c o n j u g a t i o n was much e f f i c i e n t on s o l i d media

r a t h e r t h a n i n l i q u i d i n c a s e o f a q u a t i c gram n e g a t i v e

o r g a n i s m s .

I n c o n c l u s i o n , we can s u g g e s t t h a t t h e s o i l and

sewage sys tem i n t h e v i c i n i t y of A l i g a r h c i t y i s h e a v i l y

p o l l u t e d w i t h s e v e r a l t y p e s o f t o x i c m e t a l s a s w e l l a s

t h e p o t e n t i a l l y h a z a r d o u s m i c r o b i a l f l o r a b e c a u s e of t h e i r

c a p a c i t y t o r e s i s t one o r t h e o t h e r w e l l known c h e m o t h e r a -

p e u t i c a g e n t s . The p r o b l e m t a k e s t h e s e r i o u s t u r n i n v i ew

of t h e r e s i s t a n c e t o m u l t i p l e a n t i b i o t i c s / d r u g s wh ich

would b e i n e f f e c t i v e even i f t h e y a r e g iven i n c o m b i n a t i o n ,

One s t e p a h e a d o f t h e a b o v e , we can e n v i s a g e o n l y t h e

a l a r m i n g s i t u a t i o n p r e v a i l i n g i n o u r system and s u r r o u n d ­

i n g s i n t h e l i g h t of t r a n s m i s s i b l e n a t u r e of R - p l a s m i d s .

T h i s s t u d y c a l l s f o r p a r t i c u l a r a t t e n t i o n t o w a r d s t h i s

p o t e n t i a l h a z a r d of i n c r e a s i n g i n c i d e n c e of t r a n s m i s s i b l e

m u l t i p l e d r u g r e s i s t a n c e . One s o o t h i n g a s p e c t of t h e

p r o b l e m however , i s t h e h i g h i n c i d e n c e of m e t a l r e s i s ­

t a n c e which wou ld somehow d e t o x i f y t h e e f f e c t of m e t a l l i c

p o l l u t a n t s and t h e s e b a c t e r i a can s e r v e a s n a t u r a l p u r i ­

f i e r t o t h e t o x i c i t y p r o d u c e d b y t h e t e s t m e t a l s i . e .

Hg"^" , Cd^''", Pb++ and Zn'^'^.

B e s i d e s t h e a p p l i e d a s p e c t of t h e p r o b l e m , one

can n o t even i g n o r e t h e s i g n i f i c a n c e of t h e m o l e c u l a r

103

taxonomy of plasmid han^ouring bac te r i a which would

provide a quick and r a t i o n a l approach t o c lass i fy then

on the b a s i s of antibiograms and plasmid p r o f i l e s . This

study i s one s tep forward towards t h i s end.

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