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Isolation And Antibiotic Susceptibility Pattern Of Extended Spectrum Β-Lactamases Producing Klebsiella Pneumoniae From Human Clinical Specimen

By: Muhammad Tahir Ishaq (2013-va-443) | Dr. Jawad Nazir.
Contributor(s): Dr. Muhammad Zubair Shabbir | Dr. Aqeel Javeed.
Material type: materialTypeLabelBookPublisher: 2015Description: 60p.Subject(s): Department of MicrobiologyDDC classification: 2366-T Dissertation note: Klebsiella species particularly K. pneumoniae are important opportunistic nosocomial pathogens causing a variety of infections including urinary tract infections (UTIs), pneumonia, septicemia and wound infections. Epidemic and endemic nosocomial infections caused by K. pneumonia species are leading causes of morbidity and mortality throughout the world. Irrational use of antibiotics is creating antibiotic resistance problems in Pakistan. Excessive use of β-lactam antibiotics is responsible for production of ESBL enzymes by Gram negative bacteria especially K. pneumoniae. A total of 150 samples including urine, pus and sputum were processed for the isolation and evaluation of the ESBL enzyme producing K. pneumoniae as well as their antimicrobial sensitivity pattern. Samples were cultured on cystine lactose electrolyte deficient (CLED), blood, chocolate and McConky agar. Isolates were identified by culture characters, staining reaction followed by biochemical testing through API-20E index system. ESBL production potential was assessed by double disc diffusion method. Out of total 50 urine samples, K. pneumoniae was isolated from 20 samples. Among these 20 isolates, 6 were confirmed as ESBL producers. However, within 50 sputum and pus samples, 13 and 15 isolations were possible out of which 5 and 6 were positive for ESBL production, respectively. Frequency distribution for isolation of K. pneumoniae from urine, pus and sputum samples did not significantly vary from each other. Similarly, ESBL producing potential of all K. pneumoniae isolates also did not significantly vary as p values are higher than 0.05. A total of 17 ESBL producing K.pneumoniae isolates were tested for their antibiotic susceptibility pattern against 12 commonly used antibiotics i.e. Amoxacillin, Ampicillin, Aztreonam, Ceftazidime, Cefatoxime, Ceftriaxone, Cefixime, Imipenem, Meropenem, Ciprofloxacin, Gentamycin and Amikacin. All of the tested K. pneumoniae isolates showed 100 % sensitivity against amikacin, imipenem and meropenem while found to be completely resistant against rest of the antibiotics. Molecular confirmation of ESBL production was done through PCR. DNA samples were extracted from ESBL producing isolates. Amplification of the plasmid DNA region (TEM gene) from the DNA samples was done through PCR. The resulting PCR product was run on 1% agarose gel through horizontal gel electrophoresis. Three samples from urine (S1, S4 and S5) and two samples from sputum (S7 and S8) produced required bands while none of the other samples produced any band. High proportion of ESBL producing K. pneumoniae isolates in present study is an alarming scenario. These organisms are resistant to conventional antibiotics and might spread in the environments thus pose serious threat to the health of human and animal population. Only clinical specimen submitted to the diagnostic lab were tested in present study. Further extensive studies are needed to bridge the gaps in knowledge of antibiotic resistance pattern of K. pneumoniae.
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Klebsiella species particularly K. pneumoniae are important opportunistic nosocomial pathogens causing a variety of infections including urinary tract infections (UTIs), pneumonia, septicemia and wound infections. Epidemic and endemic nosocomial infections caused by K. pneumonia species are leading causes of morbidity and mortality throughout the world. Irrational use of antibiotics is creating antibiotic resistance problems in Pakistan. Excessive use of β-lactam antibiotics is responsible for production of ESBL enzymes by Gram negative bacteria especially K. pneumoniae.
A total of 150 samples including urine, pus and sputum were processed for the isolation and evaluation of the ESBL enzyme producing K. pneumoniae as well as their antimicrobial sensitivity pattern. Samples were cultured on cystine lactose electrolyte deficient (CLED), blood, chocolate and McConky agar. Isolates were identified by culture characters, staining reaction followed by biochemical testing through API-20E index system. ESBL production potential was assessed by double disc diffusion method. Out of total 50 urine samples, K. pneumoniae was isolated from 20 samples. Among these 20 isolates, 6 were confirmed as ESBL producers. However, within 50 sputum and pus samples, 13 and 15 isolations were possible out of which 5 and 6 were positive for ESBL production, respectively. Frequency distribution for isolation of K. pneumoniae from urine, pus and sputum samples did not significantly vary from each other. Similarly, ESBL producing potential of all K. pneumoniae isolates also did not significantly vary as p values are higher than 0.05.
A total of 17 ESBL producing K.pneumoniae isolates were tested for their antibiotic susceptibility pattern against 12 commonly used antibiotics i.e. Amoxacillin, Ampicillin, Aztreonam, Ceftazidime, Cefatoxime, Ceftriaxone, Cefixime, Imipenem, Meropenem, Ciprofloxacin, Gentamycin and Amikacin. All of the tested K. pneumoniae isolates showed 100 % sensitivity against amikacin, imipenem and meropenem while found to be completely resistant against rest of the antibiotics.
Molecular confirmation of ESBL production was done through PCR. DNA samples were extracted from ESBL producing isolates. Amplification of the plasmid DNA region (TEM gene) from the DNA samples was done through PCR. The resulting PCR product was run on 1% agarose gel through horizontal gel electrophoresis. Three samples from urine (S1, S4 and S5) and two samples from sputum (S7 and S8) produced required bands while none of the other samples produced any band.
High proportion of ESBL producing K. pneumoniae isolates in present study is an alarming scenario. These organisms are resistant to conventional antibiotics and might spread in the environments thus pose serious threat to the health of human and animal population. Only clinical specimen submitted to the diagnostic lab were tested in present study. Further extensive studies are needed to bridge the gaps in knowledge of antibiotic resistance pattern of K. pneumoniae.

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