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DETECTION AND ANTIBIOTIC SENSITIVITY TESTING OF
BRUCELLA SPECIES
Apexa Suryawanshi and Dr. Rajashree Gandge*
1Livestock Development Officer, Nashik, Maharashtra.
2Associate Professor, Department of Microbiology, Bombay Veterinary College,
Parel, Mumbai- 400 012.
ABSTRACT
Infection with Brucella spp. continues to pose a human health risk
globally despite strides in eradicating the disease from domestic
animals. The timely and accurate diagnosis and treatment of human
brucellosis had been challenge for clinicians because of its non-
specific clinical features. Surveillance of disease or identifying
etiology is a key element for management of prevention and control
program. Therefore, the present study was designed to detect human
brucellosis in occupationally exposed humans to infected animals or
their products and to study antibiogram of Brucella spp. for detection of emergence of
resistance and to find out the suitable antibiotic for treatment of brucellosis. A total of 205
sera and 100 blood samples from animal handlers, farmers, milkers and practicing
veterinarians from different parts of Konkan and Western Maharashtra region were
processed. Overall seroprevalence of human brucellosis was found to be 16.58%, in a total of
205 sera samples tested by RBPT and STAT. Seroprevalence of brucellosis was found higher
in humans from Western Maharashtra region (23.52%) as compared to Konkan region
(9.09%). Only 4 Brucella isolates (6.6%) could be recovered from total 60 (22 seropositive +
38 seronegative) human whole blood samples attempted for isolation of Brucella spp.
employing biphasic media. Out of 4 Brucella spp. isolates recovered, 3 were identified as
Brucella abortus and 1 could not be identified upto its species level. Antibiotic susceptibility
test didn’t show development of drug resistance amongst Brucella spp. and isolates were
found sensitive to tetracycline, doxycycline and cephotaxim. PCR assays was carried out
using six different primers for detection of Brucella isolates at their genus and species level.
The PCR using BCSP31 (223 bp and 428 bp), DNAJ/BM (503 bp) and DNAJ/AB (388 bp)
*Corresponding Author
Dr. Rajashree Gandge
Livestock Development
Officer, Nashik, Maharashtra.
Article Received on
21 May 2017,
Revised on 13 June 2017,
Accepted on 2 July 2017
DOI: 10.20959/wjpps20177-9621
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
S SJIF Impact Factor 6.647
Volume 6, Issue 7, 1817-1829 Research Article ISSN 2278 – 4357
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Rajashree et al. World Journal of Pharmacy and Pharmaceutical Sciences
primers were proved useful in confirming Brucella organisms at genus level. Whereas,
IS711/AB PCR produced abortus specific (Biotype1, 2, 4) amplification (498 bp) confirming
the isolates as B. abortus. None of 4 isolates showed B. melitensis specific amplification in
IS711/BM PCR. Direct detection of brucellosis by DNAJ/BM PCR from 100 human blood
samples revealed amplification product of 503 bp in 48% humans. PCR assays were found to
be most sensitive followed by serological and cultural methods in detection of human
brucellosis. Although, previous reports show prevalence of Br. melitensis in human
brucellosis; in present study Br. abortus was found to be predominant species existed in
chronic or subclinical form of occupationally exposed human population.
KEYWORDS: Bacterial isolation, Brucella abortus, Brucella melitensis, Human brucellosis,
PCR, Serology.
INTRODUCTION
Infection with Brucella spp. continues to pose a human health risk globally despite strides in
eradicating the disease from domestic animals. Brucellosis is one of the important diseases of
animals causing high economic losses in developing countries[9]
and having zoonotic
importance. The global burden of human brucellosis remains enormous; it causes more than
500,000 infections per year worldwide.[29]
The interest in brucellosis has been increasing
because of the growing phenomena of international tourism and migration, in addition to the
potential use of Brucella as a biological weapon.[23]
Although brucellosis is an important
disease in India, as per literature search it seems that, not much detailed research on human
brucellosis is carried out and only few recent studies have addressed the prevalence and
importance of brucellosis as a human disease problem in India. The timely and accurate
diagnosis and treatment of human brucellosis had been challenge for clinicians because of its
non-specific clinical features, slow growth rate in blood cultures and the complexity of its
serodiagnosis. However, with recent development in prompt diagnosis and treatment with
appropriate antibiotics may greatly reduce the time a patient may be incapacitated. Moreover,
surveillance of disease or identifying etiology is a key element for management of prevention
and control program. Therefore, the present research is designed with reference to study
prevalence of human brucellosis as an important re-emerging disease in occupationally
exposed humans by different diagnostic techniques.
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MATERIALS AND METHODS
Reference bacterial strains and Human specimens
The reference bacterial strains Brucella abortus (544) and Brucella melitensis Rev 1 were
used in present investigation. A total of 305 samples (205 sera + 100 whole blood) collected
from 205 humans formed material for present study (Table 1). Human isolates of Brucella
spp. recovered from human (volunteers) blood samples were used for characterization in the
present work.
Table 1: Showing different human specimens used and investigation carried.
Sr. No. Specimen Total no. of
specimens collected Investigation
Number of
samples processed
1 Whole blood in
EDTA 100
Whole blood
PCR 100
Isolation/ Culture 60
2 Serum 205 RBPT 205
STAT 205
Total 305
Primers
PCR assays were carried out using six different primers (Table 2) for identifing Brucella
isolates at genus and species level and also for detecting Brucella in human blood samples.
BCSP31,[4]
IS711/AB and IS711/BM were obtained from M/s Bangalore Genei, Bangalore
(India). Whereas, BCSP31 (31F/31R), DNAJ/BM, DNAJ/AB were designed primers.[20]
Serological tests
Serum samples were processed for detection of Brucella abortus antibodies by Rose Bengal
Plate Test (RBPT) and Standard Tube Agglutination Test (STAT) using Brucella abortus
agglutinating antigen obtained from Division of Biological Products, Indian Veterinary
Research Institute (I.V.R.I.); Izatnagar, Uttar Pradesh.
Table 2: Oligonucleotide primers for Molecular Characterization of Brucella.
Sr.
No
Name of the
PCR Primer sequence Reference
1. BCSP31 PCR
B4 -5’ : TGG-CTC-GGT-TGC-CAA-
TAT-CAA-'3 Baily et al. (1992)
B5- 5’: CGC-GCT-TGC-CTT-TCA-GGT-
CTG-‘3
2. BCSP31 PCR
31F-5’:TGC-GCG-TAT-CGT-TCT-TGA-
AGC-CTA-3’ Mahajan (2010)
31R-5’:TAT-CGA-GCT-TGA-TGA-GCT-
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TGC-CCT-3’
3. DNAJ/AB PCR
F- 5’: GGA-TAT-TTT-CGG-CGA-GAT-
GA-3’ Mahajan (2010)
R- 5’: GTC-CTC-GAT-ACC-TGT-GGG-
AA-3’
4. DNAJ/BM PCR
F-5’:TAC-GAA-ACC-CTG-AAA-GAC-
CCG-CAA-3’ Mahajan (2010)
R-5’:ATA-TTG-ACC-GAA-AGC-GAA-
CGC-TCC-3’
5. IS711/AB PCR
F-5’:TGC-CGA-TCA-CTT-AAG-GGC-
CTT-CAT- 3’ Bricker and
Halling (1994) R-5’:GAC-GAA-CGG-AAT-TTT-TCC-
AAT-CCC- 3’
6. IS711/BM PCR
F-5’:TGC-CGA-TCA-CTT-AAG-GGC-
CTT-CAT- 3’ Bricker and
Halling (1994) R-5’: AAA-TCG-CGT-CCT-TGC-TGG-
TCT-GA- 3’
Isolation, identification and antibiotic susceptibility test of Brucella spp. isolates
Isolation of Brucella spp. was carried out from blood sample using biphasic media[30]
and
further identification was done using different tests.[28]
Antibiotic susceptibility test of
Brucella isolates was carried out by disc diffusion method on Brucella agar medium.[17]
Antibiotics discs of Tetracycline (30 mcg), Erythromycin (15 mcg), Doxycycline (30 mcg),
Ciprofloxacin (5 mcg), Streptomycin (10 mcg), Co-trimoxazole (23.75 mcg) and Cephotaxim
(30 mcg) from Hi media were used.
Molecular Detection of Brucella spp. Isolates
Six PCR assays were carried out using genus specific primers viz, BCSP31 (B4/B5) and
(31F/31R) and species specific primers viz, IS711/AB and IS711/BM, DNAJ/AB and
DNAJ/BM for identification of Brucella isolates. Direct detection of Brucella spp. in blood
samples carried out by PCR amplification of DNAJ/BM gene. The amplification products
generating from all the PCR assays were evaluated by agarose gel electrophoresis in 1.5 %
agarose gel stained with ethidium bromide.[7]
Molecular Detection of Brucellosis in human blood samples
A total of 100 whole blood samples (34 seropositive + 66 seronegative) were investigated for
molecular detection of Brucella spp. directly in the extracted DNA from blood samples using
DNAJ/BM PCR assay.
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RESULTS AND DISCUSSION
Seroprevalence of Brucellosis in Humans
A total of 205 human sera were examined for presence of Brucella antibodies in which,
overall 34 cases were revealed seropositive together by RBPT (Plate 1) and STAT showing
16.58% seroprevalence of Brucellosis in Western Maharashtra and Konkan region.
Seroprevalence of brucellosis was observed higher in Western Maharashtra region (23.52%)
as compared to Konkan region (9.09%) as shown in table no. 3. RBPT (12.68%) and STAT
(14.14%) showed almost equal efficacy in detecting Brucella antibodies. However, three sera
samples which were positive in STAT were found negative by RBPT.
Table 3: Results of detection of Brucella antibodies by RBPT and STAT.
Region No. of samples
tested
Number of positive
samples (%) by RBPT
Number of positive
samples (%) by STAT
Konkan 154 14 (9.09) 19 (12.33)
Western
Maharashtra
51 12 (23.52) 10 (19.60)
Total 205 26 (12.68) 29 (14.14)
The seroprevalence of a present investigation (16.58%) correlated with the 15.72%; 15.69%;
19.83% seroprevalence reported.[1,2,31]
Similar efficacy of RBPT and STAT in detecting
Brucella antibodies was reported by some authours;[1,14,33]
however being IgM isotype of
antibody predominantly involved in agglutination reactions at neutral pH[25]
the false positive
reactions in STAT may occur due to such cross-reacting antibodies.[26]
Conventional characterization of Brucella isolates
Cultural isolation of Brucella spp. employing biphasic media (Plate 2) was attempted from
total 60 (22 seropositive + 38 seronegative) whole blood samples of human. Out of 60
samples processed for isolation, only 4 Brucella isolates (6.6%) could be recovered, of which
3 were from serologically positive and one was from serologically negative brucella sample.
Out of 4 human Brucella isolates recovered during present investigation, 3 were identified as
Brucella abortus whereas one isolate could not be identified upto its species level (Table 4).
Biotyping of 4 Brucella abortus isolates was attempted. The combined results of
conventional method and IS711/ AB PCR assay were used for detecting biotypes; however
from the results (Table 5) the biotype of any of the isolate could not be traced.
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Table 4: Characterization of Brucella isolates.
Sr. No. Name of tests
Isolates Reference strains
B.
abort
us
HM
N-
001
B.
abort
us
HM
N-
002
B.
abort
us
HM
N-0
03
B.
spp.
HM
N-
004
B.
abort
us
stra
in 5
44
B.m
elit
ensi
s
Rev
-1
1. Indole test - - - - - -
2. Urease test + + + + - +
3. Nitrate reduction + + + + + +
4. Catalase + + + + + +
5. Oxidase + + + + + +
6. Acriflavine test All isolates formed
homogeneous suspension
Homogeneous suspension.
Homo generous.
Susp. suspension
7. Reaction with anti-
Brucella std. serum + + + + + -
+ : Positive, - : Negative.
Table 5: Biotyping of Brucella isolates.
Sr.
No.
Isolates
CO2
requirement
H2S
production
Growth on media
containing
IS711
(AB)
PCR
Biotype Thionin* Fuchsin*
1. B. abortus
HMN - 001 + + + + +
B. abortus
biotype:
unidentified
2. B. abortus
HMN - 002 + + + + +
B. abortus
biotype:
unidentified
3. B. abortus
HMN - 003 + + + + +
B. abortus
biotype:
unidentified
4. B. spp.
HMN - 004 + + + + -
Brucella
spp.
Isolation of Brucella spp. from clinical specimens is considered to be the gold standard since
it is a direct evidence of infection; further, several groups working in India have attempted
isolation of Brucella spp. from different clinical specimens with varying degrees of success.
The blood samples have proved to be the useful specimen for isolation of Brucella spp. in
human beings and several researchers have successfully attempted isolation of Brucella spp.
from human blood samples for detection of Brucellosis.[34,27]
The probable reason for low % of isolation could be due to presence of few Brucella
organisms per unit of blood reducing chances of recovery of bacteria from blood, since the
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blood samples were not specifically collected critically at height of temperature. Moreover, it
has been suggested that in chronically infected cases when antibody levels are vanishing or in
case of recent exposure to low dose of brucella where there is no sufficient period for
development of immune response.[10]
Therefore reports of lower percentages viz. 0.77%,
8.33% & 0% of isolation of Brucella spp. from blood samples have been published[16,24,31]
which correlated with present study.
Amongst the above Brucella species known to infect humans, Brucella melitensis is the most
commonly reported,[21]
however Brucella abortus is also known to infect humans.[35]
The
disease in human is characterized by an acute bacteraemic phase and irregular fever, followed
by chronic stage that may extend over many years and may involve many tissues.[21]
In the present investigation, Brucella abortus species was found to be predominant in human
brucellosis. From the history of persons, it is revealed that brucellosis in occupationally
exposed animals seems to be existed in chronic or subclinical form in population, wherein no
severe acute symptoms of brucellosis observed in positive persons. Although, recently Br.
abortus spp. is not reported commonly in humans, there are past reports available, indicating
zoonotic importance of Br. Abortus.[32,35]
In antibiotic susceptibility test, all 4 isolates found sensitive to tetracycline, doxycycline,
ciprofloxacin, streptomycin and cephotaxim, whereas 2 isolates showed intermediate
sensitivity to erythromycin and one to co-trimoxazole. (Plate 3).
Table 6: Antibiotic susceptibility test of Human isolates.
Sr.
No Brucella Isolates
Antibiotics used
Tet
racy
clin
e
(10 m
cg)
Doxycy
clin
e
(30 m
cg)
Ery
thro
myci
n
(15 m
cg)
Cip
rofl
ox
aci
n
(30 m
cg)
Str
epto
my
cin
(10 m
cg)
Co-t
rim
oxa
zole
(10 m
cg)
Cep
hota
xim
(30 m
cg)
1. B. abortus HMN- 001 S S S S S S S
2. B. abortus HMN- 002 S S S S S I S
3. B. abortus HMN- 003 S S I S S I S
4. B. spp. HMN- 004 S S I S S I S
S = Sensitive, I = Intermediate
There is not yet resistance problem for classically recommended antibiotics targeted
to Brucella species reported, however antibiotic susceptibility patterns of Brucella spp.
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appear to vary geographically.[5]
Similar findings as in present study of not showing drug
resistance and effectiveness of above antibiotics in brucellosis was reported.[22,8]
The
emergence of drug resistance did not observe; may be due to lack of awareness for diagnosis
and treatment of brucellosis.
Molecular Detection of Brucella spp. isolates
All 4 human isolates and the reference strains B. abortus 544 and B. melitensis Rev 1 as
positive controls were included in PCR assay (Plate 4-8). Out of 4 isolates tested by genus
specific PCR assay BCSP31 published and newly designed primers, all isolates showed
specific amplification product of 223 bp and 428 bp respectively. Moreover, DNAJ/AB and
DNAJ/BM PCR also yielded 388 bp and 503 bp amplification products respectively in all
isolates including reference strains B.abortus 544 and B. meletensis Rev 1 confirming identity
of isolates as members of genus Brucella. In B. abortus specific IS711/AB PCR, out of 4
Brucella isolates, 3 showed an amplification product of 498 bp confirming their identity as B.
abortus. However, one isolate of Brucella did not yield abortus specific amplification
product. All isolates and reference strains were also simultaneously subjected to B. melitensis
specific IS711/BM PCR where in none of the isolate produced amplicon of 731 bp as
generated except reference strain B. melitensis Rev 1 suggesting that none of the isolates
belonged to Br. meletensis species.
A number of PCR assays targeting different regions of Brucella DNA viz. IS711[6]
heat shock
proteins like DnaJ[11]
etc. have been developed for identification of Brucella at genus and
species level. Sensitivity of PCR assay in detection of brucellosis has been reported by many
workers. The results of our study are in agreement with authors who employed IS711/AB and
IS711/BM PCR assays for identification of Brucella abortus and meletensis and found it to
be efficacious.[12,19,2]
The results of present investigations are concordant with the findings of
workers who used DNAJ PCR assays for detecting Brucella genus.[11,20]
Molecular Detection of Brucellosis from blood samples
A total of 100 whole blood samples (34 seropositive + 66 seronegative) were investigated for
molecular detection of Brucella spp. by DNAJ/BM PCR. An amplification product of 503 bp
was observed in 48 (48%) samples confirming the infection of human due to Brucella
species. Of 34 serologically positive samples, 32 (94.11%) yielded Brucella spp. specific
amplification product in PCR while two seropositive humans did not exhibit any
amplification. Out of 39 PCR positive blood samples, only 4 Brucella spp. (10.25%) isolates
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Rajashree et al. World Journal of Pharmacy and Pharmaceutical Sciences
were recovered. PCR assay was found to be most sensitive followed by serological and
cultural methods in detection of human brucellosis during present investigation. Our results
correlated with the findings of investigators who suggested PCR to be of value in detection of
Brucella spp. from blood.[18]
As for other fastidious pathogens, amplification of DNA by PCR offers an alternative way of
diagnosis of brucellosis. Now a days genetic characterization using DNA based molecular
technique has been pursued for the rapid identification of Brucella. PCR has been applied to
detect Brucella DNA in varieties of clinical samples including tissues (mainly aborted
foetuses and associated maternal tissues), blood, milk and semen.[18,15,13]
The PCR based
methods are not only advantageous in being more sensitive than conventional methods in
diagnostic identification of the pathogen but can also facilitate characterization and typing of
the organism for studying epidemiology of infection.[3]
Besides being rapid and specific the
added advantage of these techniques is non-requirement of handling of virulent cultures
thereby reducing the safety concerns.
1 2 3 4 5 6 7
1000 bp
200 bp
100 bp
223 bp
1 2 3 4 5 6 7
Lane 1 : 100 bp ladder, 2: B. abortus 544, 3: HMN 001, 4: HMN 002
5: HMN 003 , 6:HMN 004 and 7: Negative control
300 bp
Plate 4: BCSP 31 (B4/B5) PCR. Plate 3: Antibiotic Sensitivity
Test of Brucella isolates
Plate 1: Detection of brucella
antibodies by Rose Bengal Plate Test.
Plate 2: Colonies of Brucella
spp. on biphasic media.
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Rajashree et al. World Journal of Pharmacy and Pharmaceutical Sciences
1 2 3 4 5 6 7
Lane 1 : 100 bp ladder, 2: B. abortus 544, 3: HMN 001, 4:
HMN 002 5: HMN 003 , 6:HMN 004 and 7: Negative control
1000 bp
400 bp
100 bp
428 bp500 bp
1 2 3 4 5 6 7
1000 bp
300 bp
100 bp
400 bp 388 bp
Lane 1 : 100 bp ladder, 2: B. abortus 544, 3: HMN 001, 4:
HMN 002 5: HMN 003 , 6:HMN 004 and 7: Negative control
4321 5 6 7
1000 bp
100 bp
400 bp500 bp 503 bp
Lane 1 : 100 bp ladder, 2: B. abortus 544, 3: HMN 001, 4: HMN 002
5: HMN 003 , 6:HMN 004 and 7: Negative control
1000 bp
500 bp
100 bp
498 bp400 bp
1 2 3 4 5 6 7
Lane1:100 bp ladder, 2: B. abortus 544, lane 3 , 5 & 6: Br.
abortus positive samples.. Lane 7: negative control
1 2 3 4 5 6 7
CONCLUSIONS
Overall seroprevalence of Brucellosis was found to be 16.58%. Serological prevalence of
human brucellosis was found relatively higher in Western Maharashtra region (23.52%) as
compared to Konkan region (9.09%). Brucella abortus spp. was found to be predominant in
human brucellosis. Biotype 2 of Brucella abortus spp. was found prevalent in animal
brucellosis. In Antibiotic susceptibility test no marked difference in antibiogram pattern
amongst the isolates was found and none of the isolate showed resistance to any of antibiotics
tested in in vitro antibiotic susceptibility testing. PCR assays were found to be most sensitive
followed by serological and cultural methods in detection of human brucellosis.
Plate 8. IS711/AB PCR. Plate 7: DNAJ/BM PCR assay of
Brucella spp.
Plate 6: DNAJ/AB PCR
assay of Brucella spp.
Plate 5: BCSP 31 (31F/31R) PCR.
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