Thesis presentation shreejeet

Presentation and Viva-voce examination on

Bacterial Etiology of Wound Infection and Antibiotic Susceptibility Pattern of the Isolates

A

DissertationSubmitted to the Department of Microbiology

Kantipur College of Medical Sciences(Affiliated to Tribhuvan University)

In Partial Fulfillment of the Requirements for the Award of the

Degree of Master of Science in Microbiology (Medical)

ByShreejeet Shrestha

Department of MicrobiologyKantipur College of Medical Sciences

(Affiliated to Tribhuvan University) Sitapaila, Kathmandu, Nepal

2010

Introduction

• The presence of microorganisms in a wound is not unusual but not all wounds support the same range and number of species, the outcome of wound infection depends on interaction of complex host and microbial factors.

• Since wound colonization is most frequently polymicrobial involving numerous microorganisms that are potentially pathogenic, any wound is at some risk of becoming infected.

• Wound infections may occur as a result of penetrating trauma from plants, animals, guns, knives or other various objects and are mainly caused by overcrowding, lack of general cleanliness, poor socioeconomic condition and lack of education too.

• Wound infections are one of the most common hospital acquired infections and are an important cause of morbidity and account for 70-80% mortality.

10/12/14 2Kantipur College of Medical Sciences,

Sitapaila

Introduction

• Wound infections can be caused by different groups of microorganisms like bacteria, fungi and protozoa. However, different microorganisms exist in polymicrobial communities especially in the margins of wounds and in chronic wounds

• Most commonly isolated aerobic microorganisms include Staphylococcus aureus, Coagulase-negative staphylococci (CoNS), Enterococci, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Proteus mirabilis, Acinetobacter etc

• Infected wounds may not yield pathogens by culture owing to the fastidious nature of some pathogens, or if the patients has received an antimicrobial therapy

• The widespread uses of antibiotics, together with length of time over which they have been available have led to major problems of resistance pathogens contributing to morbidity and mortality.


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Objective

• General Objective

To study the bacterial etiology of wound infection and their antibiotic susceptibility pattern among the patients visiting B and B Hospital.

• Specific Objectives

To isolate and identify the bacterial pathogens from patients with wound infections.

To describe the distribution pattern of bacterial pathogens in relation to age and sex of the patients.

To describe the seasonal distribution of bacterial pathogens among the patients.

To study the antibiotic susceptibility pattern of the isolated bacterial pathogen.


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Method and Methodology

• Study site and period : The study was carried out in microbiology laboratory, B and B Hospital from September 2010 to August 2011.

• Study population: A total of 1164 wound samples (swab, pus and aspirates) were collected for culture and antibiotic sensitivity test was performed. The patients with age of 1 year to 94 years were enrolled in the study. The types of wound included were surgical wounds, trauma, burns, ulcers, and other pyogenic wounds.

• Specimen collection: Wound pus and wound swab were collected on a sterile swab or in a stoppered syringe without contamination. Two samples were taken from each patient, one for culture and another for direct Gram stain. The sample was taken to the laboratory for further processing following standard microbiological laboratory procedures.

• Sample processing and isolation of organism:

Macroscopic examination: Colour, odour and granules content were noted


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Microscopic examination: The smear was gram stained and was examined by using high dry (40X) and oil immersion (100X) microscopy.

Culture of specimen: The specimen was inoculated on Nutrient agar, MacConkey agar and Blood agar and incubated at 37 C for 24 to 48 hrs ̇aerobically.

Identification of bacteria: Identification of the significant bacterial isolates was done by using microbiological techniques as described in the Bergey’s manual that involves colony characteristics, staining reaction and various biochemical properties.

Antimicrobial susceptibility testing: Antimicrobial Susceptibility test was performed by Kirby-Bauer disk diffusion method following Clinical and Laboratory Standard Institute guidelines (CLSI, 2010).


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Isolates showing resistance to two or more different classes of antibiotic tested were considered as MDR.

Preservation of the MDR isolates: MDR isolates in pure culture were preserved in 20% glycerol containing Tryptic Soya broth and kept at -70°C

Quality control: Accuracy of the overall procedure was monitored by using S. aureus ATCC 25923 and E. coli ATCC 25922 as reference strain.

Data analysis: Data were analysed by using WHONET 5.6.


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Results


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Table 1: Age and Gender based distribution of patients with wound infection

Gender\

Age

Male Female

Total Positive Prevalence Total Positive Prevalence

<15 58 29 50.0% 25 13 52.0%

15-30 317 151 47.6% 141 75 53.1%

30-45 282 146 51.7% 67 25 37.3%

>45 206 105 50.9% 68 29 42.6%

Total 863 431 49.9% 301 142 47.1%

Results

Figure : Types and distribution of samples


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Results

Figure : Distribution of positive cases on season basis


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ResultsBacterial isolates No. of isolate (N=573) Inpatients

(N=608)Outpatients (N=556)

Gram positive Bacteria (N=146)

S. aureus 135 (92.46%) 87 48 CoNS 3 (2.05%) 2 1

β-haem. Streptococci 3 (2.05%) 2 1Non haem. Streptococci 5 (3.42%) 3 2Total 146 (25.48%)

Gram negative isolate (N=427)E. coli

120(28.10%) 54 66

Pseudomonas spp. 132(30.91) 91 41Klebsiella. Spp 41(9.60%) 28 13Enterobacter spp. 60(14.05%) 36 24P. mirabilis 3(0.70%) 3 0P. vulgaris 2(0.46%) 0 2Acinetobacter spp. 61(14.28%) 39 22Citrobacter spp. 8(1.87%) 4 4Total 427 (74.52%)Total 573 349 (57.40%) 224 (40.29%)

Table : Types of bacteria


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ResultsAntibiotics Gram

positive(N=146)

Gram negative (N=427)

Total (N=573)

S. aureus (N=135)

Gentamycin 25 (17.12%) 214 (50.11%) 239 (41.71%) 19 (14.07%)

Amikacin 4 (2.73%) 146 (34.19%) 150 (26.17%) 4 (2.96%)

Ofloxcin 47 (32.19%) 251 (58.78%) 298 (52.00%) 38 (28.14%)

Ciprofloxacin 52 (36.61%) 260 (60.88%) 312 (54.45%) 44 (32.59%)

Amoxycillin 14 (9.58%) 357 (83.60%) 371 (64.74%) 13 (9.62%)

Cotrimoxazol 11 (7.53%) 240 (56.20%) 251 (43.80%) 10 (7.40%)

Chloramphenicol NT 260 (60.88%) ND NT

Erythromycin 48 (32.87%) NT ND 43 (31.85%)

Oxacillin 48 (32.87%) NT ND 46 (34.07%)

Penicillin NT NT ND 111 (82.22%)

Vancomycin NT NT ND 10 (7.40%)

Table : Antibiotic sensitivity pattern of the bacterial isolates


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Conclusion

• Culture positivity in wound infection accounts about 50% of the cases irrespective of their demographic status.

• Once infected it’s a problem for management as the etiological agents isolated (S. aureus, E. coli, P. aurogenesa etc) are resistant to most of commonly used antibiotics, so antibiotics like Amikacin and Vancomycin (for S. aureus) can be used for treatment and control.


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Recommendation

• Anaerobic bacteria also accounts in wound infection so, anaerobic culture should be performed to establish exact prevalence of bacterial wound infection.

• Proper disinfectants should be used while collecting wound specimen to minimize commensals contamination.

• New antibiotics or high level antibiotics should be screened as common antibiotics are usually seen to be resistant.

• The emergence of antibiotic resistance strains possess problem in the management of wound infection. Thus, further molecular studies should be recommended to reveal the real scenario.


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Acknowledgement

• Supervisors

• Faculty member Kantipur College of Medical Sciences

• Laboratory staff Kantipur College of Medical Sciences

• Staffs of Microbiology department, B and B hospital

• Family and friends


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Health & Medicine

Thesis presentation shreejeet