Research - MS (Thesis)

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Title: Prevalence of Extended Spectrum Β- Lactamase Producing Klebsiella Pneumoniae in Septicemic Children and Clonal Variation between Regional Isolates

Author: A.N.M. Mehedi Hasan

Supervisor: Md. Abdul Hakim and S.K. Saha

Co-Supervisor: Md. Nuruddin Mahmud and Sharmin Sultana

Research Field: Medical Microbiology

Batch: 11th

Session: 2010-2011


Abstract

Invasive bacterial diseases in children less than 5 years of age are priority public health problems with significant morbidity and mortality especially in the developing part of the world like Bangladesh. It has been shown in several studies that K. pneumoniae to be one of the most important etiological agents of septicemia in children as the premature skin is vulnerable to sepsis. This study was a consequence research of Invasive Bacterial Diseases Surveillance (IBD) funded by WHO (World Health Organization) and coordinated by the Department of Microbiology, Dhaka Shishu (Children) Hospital. The current research investigated the frequency of sepsis in children under 5 years of age in Chittagong Maa-Shishu-O-General Hospital (CMSOGH) and Dhaka Shishu Hospital (DSH) due to the pathogenic bacterium K. pneumoniae focusing specially on determination of the antimicrobial susceptibility pattern and evaluation of prevalence of the ESBL (Extended Spectrum β-Lactamase) producing K. pneumoniae isolates. This research work also tried to detect the presence of ESBL producing genes in those K. pneumoniae isolates and scan them at molecular level to observe the clonal similarities and dissimilarities between the isolates from two different regions of Bangladesh.
During the study period from January 2011 to May 2012, from children aged 0-59 months a total of 1739 and 1612 blood cultures were performed at CMSOGH and DSH, respectively. Only 4.14% of these cultures yielded true bacterial growth at CMSOGH while it was 8.2% at DSH. Among the total bacterial isolates of the respective places, 27 at CMSOGH and 9 at DSH were primarily confirmed as Klebsiella pneumoniae, taking their cultural, morphological and biochemical characteristics into consideration. The confirmed K. pneumoniae were preserved in -80ºC freezer. Later we conducted our current research work with available 20 isolates from CMSOGH and all the 9 isolates from DSH. These 29 isolates were further confirmed as K. pneumoniae by API (Analytical Profile Index) 20E scoring for more reliable identification of the isolates. The antimicrobial susceptibility patterns of the K. pneumoniae isolates were investigated and the results were interpreted using the current CLSI (Clinical Laboratory Standard Institute) guidelines. All the isolates were found to be resistant to ampicillin (AMP) and cotrimoxazole (SXT). Imipenem emerged as the most potent antibiotic against K. pneumoniae as 97% of the isolates were sensitive to it. One K. pneumoniae isolate (CM18) was found to be resistant against all the twelve antibiotics used in this study while the rest isolates of CMSOGH were resistant to at least 6 antibiotics. About 90% isolates were resistant to at least one of the third generation cephalosporins in both CMSOGH and DSH.
The current research work also explored the ESBL producing ability of the K. pneumoniae isolates. Based on their antimicrobial susceptibility data, the primarily suspected ESBL producers were subjected to confirmatory test, the double disc synergy (DDS) test. Alarmingly 70% and 45% of the total K. pneumoniae isolates of CMSOGH and DSH, respectively were finally appeared as true ESBL producers. These ESBL producing isolates were resistant to more antimicrobial agents than non-ESBL producers. All the ESBL producers of the two hospitals under investigation were resistant to ampicillin, ceftriaxone and ceftazidime. This prevalence of ESBL producer in K. pneumoniae isolates causing septicemia is a matter of great concern as antibiotic resistance among them is escalating day by day.
The study then went one step ahead and tried to detect the ESBL producing genes using multiplex PCR. The ESBL producing genes that had been detected were ‘blaTEM’, ‘blaSHV’, ‘blaOXA-9’ and ‘blaAmpC’. Around 85% of the previously confirmed ESBL producers of CMSOGH carried both blaTEM and blaSHV while only ‘blaSHV’ gene was detected in the ESBL producing K. pneumoniae isolates of DSH. Isolates having both the blaTEM and blaSHV genes were multidrug resistant.
The PFGE typing used in this study to characterize the ESBL producers, showed various DNA banding profiles. The banding profiles were in no way similar or related to each other indicating their independent origin. This clonal variation among the isolates from two different regions of Bangladesh is in concordance with some conventional thoughts about the community acquired infections, distinct nature of the etiological agents isolated from different regions and the epidemiology of antibiotic resistance.
The disease burden due to Klebsiella pneumoniae among the children under 5 years of age is high in this country and it is increasing very rapidly. So it is high time to think about the future drug of choice to save our children. This study detected the drug resistance pattern and ESBL producers by conventional and molecular methods which will play the role of usher in controlling septicemic diseases by providing a clear insight to antimicrobial therapy and inhibiting spread of resistance due to indiscriminate use of antibiotics in Bangladesh. Successful implementation of the results of this study may present a future Bangladesh free of child death.