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- 1. BACTERIAL PYODERMABACTERIAL PYODERMA DURING SUMMER MONTHS ATDURING 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 BY Mr.VISHNUPRASADMr.VISHNUPRASAD CHIEF MICROBIOLOGISTCHIEF MICROBIOLOGIST MIMS , CALICUTMIMS , CALICUT
- 2. INTRODUCTIONINTRODUCTION Pyoderma is a generic term used to describe aPyoderma 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 TerritoryIn indigenous communities in the Northern Territory the prevalence has been reported at between 10 andthe prevalence has been reported at between 10 and 70%.70%. In Australian Aboriginal communities and those livingIn Australian Aboriginal communities and those living in the Pacific region have generally had the highestin 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%.
- 3. Pyodermas,which can be defined as anyPyodermas,which can be defined as any purulent skin disease are mainly classifiedpurulent 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
- 5. Most commonly these lesions are produced byMost commonly these lesions are produced by staphylococcal and streptococcal species .staphylococcal and streptococcal species . Pyoderma is important not only because of itsPyoderma is important not only because of its local effects as skin infection,but morelocal effects as skin infection,but more importantly because the primary pathogenimportantly because the primary pathogen underlying skin infection in Aboriginal children isunderlying 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 anGAS infections of the skin are believed to be an important factor in acute post-streptococcalimportant factor in acute post-streptococcal glomerulonephritis(APSGN) and acuteglomerulonephritis(APSGN) and acute rheumatic fever (ARF).rheumatic fever (ARF).
- 6. Determinants of high rates ofDeterminants 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 factorssocio-economic factors
- 7. Institution of appropriate treatment including timelyInstitution of appropriate treatment including timely recognition and a prompt bacterial diagnosis of suchrecognition 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 regionAntibiotic 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.
- 8. 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.
- 9. MATERIALS AND METHODSMATERIALS AND METHODS Materials RequiredMaterials Required For StainingFor Staining Clean glass slide, standard wire loop (0.01 ml capacity), CrystalClean 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 plateBlood 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.
- 10. 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 sampleLesion was swabbed with alcohol and sample collected using a sterile swab.collected using a sterile swab. Material from the intact pustualr lesion was collectedMaterial 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 tubeThe swabs were carefully logged into a tube containing transport media.containing transport media. The specimens were transported to the laboratoryThe specimens were transported to the laboratory and processed within 2 hours .and processed within 2 hours .
- 11. 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 wasOne of the swabs collected during the study was rubbed over a clean grease free glass slide andrubbed over a clean grease free glass slide and conducted Grams staining.conducted Grams staining.
- 12. ∞ Grams stainingGrams staining Prepared the smear and heat fixed.Prepared the smear and heat fixed. Flooded the smear with (crystal violet) primary stainFlooded 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 byAdded the decolourising solution (ethanol) drop by drop until the violet colour just disappears.drop until the violet colour just disappears. Washed with water.Washed with water. Flooded the smear with counter stain (safranin) andFlooded 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.
- 13. Hanging Drop Method (Motility Test)Hanging Drop Method (Motility Test) ♦ A clean coverslip was taken and Vaseline was appliedA 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 atOne 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 theA 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 bacterialThe edge of the drop was examined for bacterial movement.movement.
- 14. 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 with1mm), 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, oropaque, 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 andE.Coli : Colonies are large, thick, moist, smooth and are hemolytic on blood agar.are hemolytic on blood agar.
- 15. On Mac Conckey AgarOn Mac Conckey Agar S. aureus : Colonies are smaller that are pink due toS. aureus : Colonies are smaller that are pink due to lactose fermentation.lactose fermentation. P. aeruginosa : Grows on media forming, non-lactoseP. aeruginosa : Grows on media forming, non-lactose fermenting colonies.fermenting colonies. E.Coli : Colonies are bright pink due to lactoseE.Coli : Colonies are bright pink due to lactose fermentation.fermentation. Klebsiella : It forms lactose fermenting pink colonies.Klebsiella : It forms lactose fermenting pink colonies.
- 16. 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 fermentationSugar fermentation Coagulase testCoagulase test 1.1. Tube coagulase testTube coagulase test 2.2. Slide coagulase testSlide coagulase test
- 17. Antibiotic Sensitivity TestingAntibiotic Sensitivity Testing Preparation of incoculumPreparation of incoculum Media :MHAMedia :MHA Inoculating agar plates.Inoculating agar plates. Antibiotic discsAntibiotic discs Penicillin GPenicillin G AmpicillinAmpicillin GentamycinGentamycin VancomycinVancomycin AzlocillinAzlocillin LinezolidLinezolid RifampicinRifampicin
- 19. Reading of Antibiotic SensitivityReading of Antibiotic Sensitivity Sensitivity was read after overnight incubation ofSensitivity was read after overnight incubation of plate at 37°C. The zone of inhibition around the discplate at 37°C. The zone of inhibition around the disc was measured by using scale.was measured by using scale.
- 20. RESULTRESULT No. of Patien ts Male Female Nature of infection Sterile Single infecn. Mixed infecn. 100 56 44 20 72 08 Table I
- 21. Table – II Age & 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
- 22. Processing and Characterisation of Isolates S.aureus E.coli Pseudomonas Klebsiella Strep.Pyo gene s Proteus Enterococcus 1° Analysis Gram 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- motil e Motile Non-Motile 2° Analysis On Mac Conkey Agar Smaller & Lactose fermenti ng pink colonie s Bright pink lactos-e fermenti ng colonie s Non-lactose fermentin g colonies Lactose fermenting colonies Smooth,colo urless colonie s Tiny,deep Pink colonies On Blood Agar Large & hemolyt ic Hemolytic Hemolytic Non-Hemolytic
- 23. 3° Analysis Biochemical Tests +ve +ve +ve +ve -ve Oxidase -ve +ve -ve -ve Indole -ve +ve -ve -ve +ve Methylred +ve +ve -ve -ve +ve Voges- Proska eur +ve -ve -ve +ve -ve Citrate -ve -ve +ve +ve Urease +ve -ve -ve +ve -ve +ve Nitrate +ve +ve +ve
- 24. Coagulase +ve -ve -ve -ve Phosphatas e +ve +ve Sugar fermentation Ferment sugars ,produce acid, no gas Ferme nt sugar with acid, no gas Ferment sugar with acid and gas production Ferment sugars forming acid, no gas Ferment sugars (mannitol, sucrose, sorbitol, aesculin)
- 28. Staphylococci in mac conkey & blood agarStaphylococci in mac conkey & blood agar
- 29. METHYL RED TESTMETHYL RED TEST INDOLE TESTINDOLE TEST
- 30. CITRATE TESTCITRATE TEST VP TESTVP TEST
- 31. OXIDASE TESTOXIDASE TEST CATALASE TESTCATALASE TEST
- 32. UREASE TESTUREASE TEST COAGULASE TESTCOAGULASE TEST
- 34. Table – III Organisms 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
- 35. Staphylococcus aureus E. coli Pseudomonas Klebsiella Streptococcus pyogenes Proteus Enterococcus Mixed infection
- 36. ANTIBIOTIC SENSITIVITY TESTANTIBIOTIC SENSITIVITY TEST
- 37. Table - IV Antibiotic 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
- 39. Table V Antibiotic sensitivity of E.Coli Drug used Sensitive (No.of org.) Intermediate (no.) Resistant (No). 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
- 41. Table VI Antibiotic sensitivity of Pseudomonas Drug used Sensitive (No.of org.) Intermediate (no.) Resistant (No). 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
- 43. Table VII Antibiotic sensitivity of Klebsiella Drug used Sensitive (No.of org.) Intermediate (no.) Resistant (No). 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
- 45. Table VIII Antibiotic sensitivity of Strep. pyogenes Drug used Sensitive (No.of org.) Intermediate (no.) Resistant (No). Oxacillin (Ox) 3 -- -- Gentamicin (G) 3 -- -- Vancomycin (Va0 3 -- -- Rifampicin ( R ) 3 -- -- Pencillin ( P ) 3 -- -- Azlocillin (Az) 3 -- -- Linezolid (Lz) 3 -- --
- 47. Table IX Antibiotic sensitivity of Proteus Drug used Sensitive (No.of org.) Intermediate (no.) Resistant (No). Meropenem 3 -- -- Peiperacillin + Tazobactum 3 -- -- Netillin 2 -- 1 Ampicillin -- -- 3 Cefazidine 3 -- -- Cefepime 2 1 -- Norflaxacin 1 2 --
- 49. Table X Antibiotic sensitivity of Enterococcus Drug used Sensitive (No.of org.) Intermediate (no.) Resistant (No). Rifampicin ( R ) 2 -- -- Vancomycin (Va) 2 -- -- Oxacillin (Ox) -- -- 2 Pencillin (P) -- -- 2 Linezolid (Lz) 2 -- -- Azlocillin (Az) -- -- 2 Cefeperazone + Sulphactum (Cfs) 1 1 --
- 51. Summary and ConclusionSummary and Conclusion The main aim of the study was to isolate predominant organisms causingThe 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 fromA total of 100 pus swabs collected from patients with pyoderma from MIMS hospital, Calicut during April to May 2010 were included in the studyMIMS 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 andThe organisms were isolated by cultivation on Mac Conkey Agar and Blood Agar .Gram staining, Motility test and further biochemical tests wereBlood 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, IMViCThe 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%).
- 52. • 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.