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BACTERIAL PYODERMA BACTERIAL PYODERMA DURING SUMMER MONTHS AT DURING SUMMER MONTHS AT MALABAR REGION OF KERALAMALABAR REGION OF KERALA

BYBY VIJI ANAND NARAYANVIJI ANAND NARAYAN

M.Sc MICROBIOLOGYM.Sc MICROBIOLOGY E.M.E.A KONDOTTYE.M.E.A KONDOTTY

GUIDED BYGUIDED BYMr.VISHNUPRASADMr.VISHNUPRASAD

CHIEF MICROBIOLOGISTCHIEF MICROBIOLOGISTMIMS , CALICUTMIMS , CALICUT

INTRODUCTIONINTRODUCTION

Pyoderma is a generic term used to describe a Pyoderma is a generic term used to describe a clinical diagnosis of superficial bacterial skin infections. clinical diagnosis of superficial bacterial skin infections.

In indigenous communities in the Northern Territory In indigenous communities in the Northern Territory the prevalence has been reported at between 10 and the prevalence has been reported at between 10 and 70%.70%. In Australian Aboriginal communities and those living In Australian Aboriginal communities and those living in the Pacific region have generally had the highest in the Pacific region have generally had the highest burden ,often in the range of 40 to 90%. burden ,often in the range of 40 to 90%.

Pyodermas,which can be defined as any Pyodermas,which can be defined as any purulent skin disease are mainly classified purulent skin disease are mainly classified as:as:

Primary infectionsPrimary infections

Secondary infectionsSecondary infections

Cutaneus involvement in systemic bacterial infectionsCutaneus involvement in systemic bacterial infections

Infections due to unusual organismsInfections due to unusual organisms

IMPETIGOIMPETIGO PUSTULE PUSTULE

Most commonly these lesions are produced by Most commonly these lesions are produced by staphylococcal and streptococcal species .staphylococcal and streptococcal species .

Pyoderma is important not only because of its Pyoderma is important not only because of its local effects as skin infection,but more local effects as skin infection,but more importantly because the primary pathogen importantly because the primary pathogen underlying skin infection in Aboriginal children is underlying skin infection in Aboriginal children is a Group A Streptococcus or GAS.a Group A Streptococcus or GAS.

GAS infections of the skin are believed to be an GAS infections of the skin are believed to be an important factor in acute post-streptococcal important factor in acute post-streptococcal glomerulonephritis(APSGN) and acute glomerulonephritis(APSGN) and acute rheumatic fever (ARF). rheumatic fever (ARF).

Determinants of high rates of Determinants of high rates of pyoderma are : pyoderma are :

CrowdingCrowding

inadequate water supplyinadequate water supply

humidity,poor education and poor hygienehumidity,poor education and poor hygiene

socio-economic factors socio-economic factors

Institution of appropriate treatment including timely Institution of appropriate treatment including timely recognition and a prompt bacterial diagnosis of such recognition and a prompt bacterial diagnosis of such lesions in these common dermatoses is a must .lesions in these common dermatoses is a must .

Antibiotic resistant strain produces risks with treatment. Antibiotic resistant strain produces risks with treatment.

Antibiotic sensitivity pattern differs from region to region Antibiotic sensitivity pattern differs from region to region and even within the same region,with progress of time. and even within the same region,with progress of time.

AIM AND OBJECTIVEAIM AND OBJECTIVE

To isolate the aerobic organisms causing pyoderma. To isolate the aerobic organisms causing pyoderma.

To identify the isolated organisms. To identify the isolated organisms.

To study theantibiotic susceptibility of the isolates. To study theantibiotic susceptibility of the isolates.

MATERIALS AND METHODSMATERIALS AND METHODSMaterials RequiredMaterials Required

For StainingFor Staining Clean glass slide, standard wire loop (0.01 ml capacity), Crystal Clean glass slide, standard wire loop (0.01 ml capacity), Crystal

violet, ethanol solution, counter stain.violet, ethanol solution, counter stain.

For MotilityFor Motility Standard wire loop, petroleum jel, cavity slide, coverslipStandard wire loop, petroleum jel, cavity slide, coverslip

For CultureFor Culture Blood Agar, Mac Conkey Agar, Peptone water, MHA plate Blood Agar, Mac Conkey Agar, Peptone water, MHA plate

standard wire loop of 0.01 ml capacity. standard wire loop of 0.01 ml capacity.

For Further identificationFor Further identification Biochemical Media.Biochemical Media.

For Further identificationFor Further identification Biochemical Media.Biochemical Media.

Collection of specimenCollection of specimen specimen collected, mainly was pus using the swab.specimen collected, mainly was pus using the swab.

Lesion was swabbed with alcohol and sample Lesion was swabbed with alcohol and sample collected using a sterile swab. collected using a sterile swab.

Material from the intact pustualr lesion was collected Material from the intact pustualr lesion was collected after rupturing it .after rupturing it .

The swab was swirled on to the infected area.The swab was swirled on to the infected area.

The swabs were carefully logged into a tube The swabs were carefully logged into a tube containing transport media. containing transport media.

The specimens were transported to the laboratory and The specimens were transported to the laboratory and processed within 2 hours .processed within 2 hours .

Processing of SampleProcessing of Sample∞ Macroscopic examinationMacroscopic examination The appearance of the specimen was noted.The appearance of the specimen was noted.

∞ Microscopic examinationMicroscopic examination One of the swabs collected during the study was One of the swabs collected during the study was

rubbed over a clean grease free glass slide and rubbed over a clean grease free glass slide and conducted Grams staining. conducted Grams staining.

∞ Grams stainingGrams staining Prepared the smear and heat fixed.Prepared the smear and heat fixed.

Flooded the smear with (crystal violet) primary stain Flooded the smear with (crystal violet) primary stain and allowed to react for 1 minute.and allowed to react for 1 minute.

Washed with water.Washed with water.

Added the decolourising solution (ethanol) drop by drop Added the decolourising solution (ethanol) drop by drop until the violet colour just disappears.until the violet colour just disappears.

Washed with water.Washed with water.

Flooded the smear with counter stain (safranin) and Flooded the smear with counter stain (safranin) and allowed it to react for 30 seconds.allowed it to react for 30 seconds.

Gram positive organisms appear in violet colour .Gram positive organisms appear in violet colour . Gram negative organisms appear in pink colour. Gram negative organisms appear in pink colour.

Hanging Drop Method (Motility Test)Hanging Drop Method (Motility Test)♦ A clean coverslip was taken and Vaseline was applied A clean coverslip was taken and Vaseline was applied

on the four corners of the coverslip. on the four corners of the coverslip.

♦ One drop of 4-6 hours old broth culture was placed at One drop of 4-6 hours old broth culture was placed at the centre of coverslip by using a sterile loop. the centre of coverslip by using a sterile loop.

♦ A clean and dried cavity slide inverted gently over the A clean and dried cavity slide inverted gently over the coverslip so that concavity faces the drop. coverslip so that concavity faces the drop.

♦ The slide was turned over carefully .The slide was turned over carefully .

♦ The edge of the drop was examined for bacterial The edge of the drop was examined for bacterial movement.movement.

Culture MethodsCulture Methods On blood agar.On blood agar. Staphylococcus aureus : Colonies are large (2-4mm), Staphylococcus aureus : Colonies are large (2-4mm),

circular, convex, smooth, shiny and are hemolytic.circular, convex, smooth, shiny and are hemolytic.

Streptococcus pyogenes : Colonies are small (0.5 – Streptococcus pyogenes : Colonies are small (0.5 – 1mm), circular, semitransparent, low convex discs with 1mm), circular, semitransparent, low convex discs with an area of clear hemolysis around them.an area of clear hemolysis around them.

Pseudomonas aeruginosa : Colonies are large, Pseudomonas aeruginosa : Colonies are large, opaque, irregular colonies with a distinctive musty, or opaque, irregular colonies with a distinctive musty, or earthy smell and hemolytic on blood agar.earthy smell and hemolytic on blood agar.

E.Coli : Colonies are large, thick, moist, smooth and E.Coli : Colonies are large, thick, moist, smooth and are hemolytic on blood agar.are hemolytic on blood agar.

On Mac Conckey AgarOn Mac Conckey AgarS. aureus : Colonies are smaller that are pink due to S. aureus : Colonies are smaller that are pink due to lactose fermentation.lactose fermentation.

P. aeruginosa : Grows on media forming, non-lactose P. aeruginosa : Grows on media forming, non-lactose fermenting colonies.fermenting colonies.

E.Coli : Colonies are bright pink due to lactose E.Coli : Colonies are bright pink due to lactose fermentation.fermentation.

Klebsiella : It forms lactose fermenting pink colonies.Klebsiella : It forms lactose fermenting pink colonies.

Biochemical testsBiochemical tests Catalase productionCatalase production Oxidase reactionOxidase reaction Indole ProductionIndole Production Methyl red test (MR)Methyl red test (MR) Voges – Proskauer Test (VP)Voges – Proskauer Test (VP) Citrate utilization testCitrate utilization test Nitrate reduction testNitrate reduction test Urease testUrease test Sugar fermentation Sugar fermentation Coagulase testCoagulase test

1.1. Tube coagulase testTube coagulase test2.2. Slide coagulase testSlide coagulase test

Antibiotic Sensitivity TestingAntibiotic Sensitivity Testing Preparation of incoculumPreparation of incoculumMedia :MHAMedia :MHA Inoculating agar plates.Inoculating agar plates.Antibiotic discsAntibiotic discs

Penicillin G Penicillin G Ampicillin Ampicillin Gentamycin Gentamycin Vancomycin Vancomycin Azlocillin Azlocillin LinezolidLinezolid RifampicinRifampicin

OxacillinOxacillin Netillin Netillin Norfloxacin Norfloxacin Meropenem Meropenem Piperacillin+Tazobactum Piperacillin+Tazobactum Amikacin Amikacin Aztreonem Aztreonem Cefepime Cefepime Cefazidine Cefazidine Cefoperazone+Sulbactum Cefoperazone+Sulbactum CiprofloxacinCiprofloxacin

Reading of Antibiotic SensitivityReading of Antibiotic Sensitivity

Sensitivity was read after overnight incubation of Sensitivity was read after overnight incubation of plate at 37°C. The zone of inhibition around the disc plate at 37°C. The zone of inhibition around the disc was measured by using scale.was measured by using scale.

RESULTRESULT

No. of Patients

Male Female

Nature of infection

Sterile Single infecn. Mixed infecn.

100 56 44 20 72 08

Table I

Table – IIAge & Sex – wise distribution of patients

Age group (yr)Age group (yr) Number of casesNumber of cases

MaleMale FemaleFemale TotalTotal

Below 1 yr.Below 1 yr. -- 22 22

1-101-10 22 44 66

11-2011-20 22 33 55

21-3021-30 55 77 1212

31-4031-40 1010 99 1919

41-5041-50 66 44 1010

51-6051-60 1212 55 1717

61-7061-70 1010 44 1414

71-8071-80 66 66 1212

81-9081-90 33 00 33

100100

Processing and Characterisation of Isolates

S.aureusE.coli Pseudomonas Klebsiella Strep.Pyo

genes

Proteus Enterococcus

1° AnalysisGram Stain

Gram +ve Cocci

Gram –ve bacilli

Grame-ve bacilli

Gram +ve cocci

Gram +ve cocci

Grame –ve bacilli

Gram+ve cocci

Motility Non-motile Motile Motile Non-motile Non-motile

Motile Non-Motile

2° AnalysisOn Mac

Conkey Agar

Smaller & Lactose fermenting pink colonies

Bright pink lactos-e fermenting colonies

Non-lactose fermenting colonies

Lactose fermenting colonies

Smooth,colourless colonies

Tiny,deep Pink colonies

On Blood Agar

Large & hemolytic

Hemolytic Hemolytic Non-Hemolytic

3° AnalysisBiochemical

Tests

+ve +ve +ve +ve -ve

Oxidase -ve +ve -ve -ve

Indole -ve +ve -ve -ve +ve

Methylred +ve +ve -ve -ve +ve

Voges-Proskaeur

+ve -ve -ve +ve -ve

Citrate -ve -ve +ve +ve

Urease +ve -ve -ve +ve -ve +ve

Nitrate +ve +ve +ve

Coagulase +ve -ve -ve -ve

Phosphatase

+ve +ve

Sugar fermentation

Ferment sugars ,produce acid, no gas

Ferment sugar with acid, no gas

Ferment sugar with acid and gas production

Ferment sugars forming acid, no gas

Ferment sugars (mannitol, sucrose, sorbitol, aesculin)

GRAM STAININGGRAM STAININGStaphylococciStaphylococci

GRAM STAININGGRAM STAININGStreptococciStreptococci

GRAM STAININGGRAM STAININGE.coliE.coli

Staphylococci in mac conkey & blood agarStaphylococci in mac conkey & blood agar

METHYL RED TESTMETHYL RED TEST INDOLE TESTINDOLE TEST

CITRATE TESTCITRATE TEST VP TESTVP TEST

OXIDASE TESTOXIDASE TEST CATALASE TESTCATALASE TEST

UREASE TESTUREASE TEST COAGULASE TESTCOAGULASE TEST

SUGAR FERMENTATIONSUGAR FERMENTATION

Table – IIIOrganisms isolated and distribution

Sl.No. Organism isolated Total No. %

1. Staphylococcus aureus 48 60

2. E.coli 13 16.25

3. Pseudomonas 10 12.50

4. Klebsiella 8 10

5. Streptococcus pyogenes 3 3.75

6. Proteus 3 3.75

7. Enterococcus 2 2.5

Mixed infection rates

8. Staphylococcus & Pseudomonas 2 2.5

9. Staphylococcus & klebsiella 1 1.25

10. Proteus & Klebsiella 1 1.25

11. E.coli & Enterococcus 1 1.25

12. E.coli & S.Pyogrenes 1 1.25

13. Enterococcus & Klebsiella 1 1.25

14. Klebsiella & Pseudomonas 1 1.25

95

Staphylococcus aureus

E. coli

Pseudomonas

Klebsiella

Streptococcus pyogenes

Proteus

Enterococcus

Mixed infection

ANTIBIOTIC SENSITIVITY TESTANTIBIOTIC SENSITIVITY TEST

Table - IVAntibiotic Sensitivity of S.aureus

Drug used Sensitive (No.of org.) Intermediate (no.) Resistant (No).

Pencillin (P) 37 -- 11

Vancomycin (Va) 48 -- --

Azlocillin (Az) 40 3 5

Linezolid (Lz) 48 -- --

Rifampicin ( R ) 47 -- 1

Oxacillin (Ox) 35 1 12

Gentamycin (G) 36 -- 13

0

5

10

15

20

25

30

35

40

45

50

Peni

cilli

n (P

)

Vanc

omyc

in(V

a)

Azl

ocill

in (A

z)

Line

zolid

(Lz)

Rifa

mpi

cin

®

Oxa

cilli

n(O

x)

Gen

tam

ycin

(G)

SensitiveResistant

Table VAntibiotic sensitivity of E.Coli


Netillin (Nt) 10 2 1

Norfloxacin (Nx) 5 1 7

Gentamicin (G) 10 -- 3

Ampicillin (A) -- -- 13

Meropenem (Mem) 9 4 --

Piperacillin + Tazobactum (PT) 13 -- --

Amikacin (AK) 9 2 2

02468

101214

Net

illin

Nor

floxa

cin

Gen

tam

ycin

Am

pici

llin

Mer

open

em

Pipe

raci

llin+

Tazo

bact

um

Am

ikac

in

SensitiveResistant

Table VI Antibiotic sensitivity of Pseudomonas


Netillin (Nt) 7 -- 3

Aztreonem (Atm) 4 1 5

Norfloxacin (Nx) 7 2 1

Meropenem (Mem) 7 -- 3

Gentamicin (G) 9 1 1

Cefeoune (Cpm) 6 1 3

Amikacin (Ak) 6 -- 1

0123456789

Net

illin

Azt

reon

am

Nor

floxa

cin

Mer

open

em

Gen

tam

ycin

Cef

epim

e

Am

ikac

in

SensitiveResistant

Table VIIAntibiotic sensitivity of Klebsiella


Meropenem (Mem) 7 1 --

Cefepime (cpm) 3 -- 5

Gentamicin (G) 6 -- 1

Piperacillin + Tazobactum (PT) 7 1 --

Cefazidine (Ca) 3 -- 5

Ciprofloxacin (Ci) 3 3 2

Amikacin (AK) 6 1 1

01234567

Mer

open

em

Cef

epim

e

Gen

tam

ycin

Pipe

raci

llin+

Tazo

bact

um

Cef

acid

ine

Cip

roflo

xaci

n

Am

ikac

in

SensitiveResistant

Table VIII Antibiotic sensitivity of Strep. pyogenes


Oxacillin (Ox) 3 -- --

Gentamicin (G) 3 -- --

Vancomycin (Va0 3 -- --

Rifampicin ( R ) 3 -- --

Pencillin ( P ) 3 -- --

Azlocillin (Az) 3 -- --


0

0.5

1

1.5

2

2.5

3

Oxa

cilli

n

Gen

tam

ycin

Vanc

omyc

in

Rifa

mpi

cin

Peni

cilli

n

Azl

ocill

in

Line

zolid

SensitiveResistant

Table IXAntibiotic sensitivity of Proteus


Meropenem 3 -- --

Peiperacillin + Tazobactum 3 -- --

Netillin 2 -- 1

Ampicillin -- -- 3

Cefazidine 3 -- --

Cefepime 2 1 --

Norflaxacin 1 2 --

00.5

11.5

22.5

3

Mer

open

emPi

pera

cilli

n+Ta

zoba

ctu

Net

illin

Am

pici

llin

Cef

izid

ine

Cef

epim

e

Nor

floxa

cin

SensitiveResistant

Table XAntibiotic sensitivity of Enterococcus


Rifampicin ( R ) 2 -- --

Vancomycin (Va) 2 -- --

Oxacillin (Ox) -- -- 2

Pencillin (P) -- -- 2


Azlocillin (Az) -- -- 2

Cefeperazone + Sulphactum (Cfs) 1 1 --

00.20.40.60.8

11.21.41.61.8

2

Rifa

mpi

cin

Vanc

omyc

in

Oxa

cilli

n

Peni

cilli

n

Line

zolid

Azl

ocill

in

Cef

aper

azon

e+Su

lbac

tu

SensitiveResistant

Summary and ConclusionSummary and Conclusion

The main aim of the study was to isolate predominant organisms causing The main aim of the study was to isolate predominant organisms causing bacterial skin pyoderma.bacterial skin pyoderma.

A total of 100 pus swabs collected from patients with pyoderma from A total of 100 pus swabs collected from patients with pyoderma from MIMS hospital, Calicut during April to May 2010 were included in the study MIMS hospital, Calicut during April to May 2010 were included in the study in which 80 samples showed bacterial growth.in which 80 samples showed bacterial growth.

The organisms were isolated by cultivation on Mac Conkey Agar and The organisms were isolated by cultivation on Mac Conkey Agar and Blood Agar .Gram staining, Motility test and further biochemical tests were Blood Agar .Gram staining, Motility test and further biochemical tests were performed to characterize the isolates.performed to characterize the isolates.

The biochemical tests conducted are catalase test, Oxidase test, IMViC The biochemical tests conducted are catalase test, Oxidase test, IMViC test, Urease, test, Nitrate test, Sugar fermentation and coagulase test. test, Urease, test, Nitrate test, Sugar fermentation and coagulase test.

Then the isolates are studied for their antibiotic sensitivity.Then the isolates are studied for their antibiotic sensitivity.

The predominant organism isolated was Staphylococcus aureus (60%) The predominant organism isolated was Staphylococcus aureus (60%) followed by E.coli (16.25%). followed by E.coli (16.25%).

• The antibiotic sensitivity studies showed that Vancomycin and Linezolid are the most effective antibiotic drug against Staphylococcus aureus and Netillin and Gentamicin against E. coli.

• Other organisms such as Klebsiella, Pseudomonas, Strep.pyogenes, Proteus, Enterococcus and mixed cultures of these organisms were also isolated.

Thank youThank you

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