Research Article EXTENDED SPECTRUM β-LACTAMASE PRODUCING MULTIDRUG-RESISTANT KLEBSIELLA SPECIES ISOLATED AT NATIONAL MEDICAL COLLEGE AND TEACHING HOSPITAL, NEPAL AAHUTI KUMARI UPADHYAY 1* AND PAWAN PARAJULI 2 1 Dept of microbiology, National Medical College and Teaching Hospital, Birgunj, Nepal, 2 Mycobacterial research laboratory, Leprosy Mission Nepal, Anandaban Hospital, Kathmandu, Nepal. Email: aahutiupadhyay4@yahoo.com Received: 24 June 2013, Revised and Accepted: 17 July 2013 ABSTRACT Introduction: Extended spectrum β- lactamases (ESBL) producing multidrug resistant (MDR) Klebsiella species resulting in limitation of therapeutic option. The present study has been undertaken to detect the presence of extended spectrum β- lactamases producing multidrug resistant Klebsiella species in various clinical specimens. Methodology: A total of 300 specimens including Urine, Pus and Blood were processed according to the standard methodology. Antibiotic susceptibility testing was done by Kirby Bauer disc diffusion method following Clinical and Laboratory standard institute (CLSI) guidelines. Combination disk method was done for the detection of extended spectrum β- lactamases producing isolates. Result: A total of 300 samples (200 urine, 45 bloods and 55 pus) were included in the study. Out of total samples proceed 110(36.7%) samples showed significant growth of Gram-negative bacteria. Amongst these significant growth of 65(59.1%) Klebsiella species, 45(69.2%) Klebsiella pneumonia and 20(30.8%) Klebsiella oxytoca were isolated. Amongst these total isolated Klebsiella species 30(66.7%) multidrug resistant Klebsiella pneumonia and 9(45.0%) multidrug resistant Klebsiella oxytoca were isolated. A total of 21(70.0%) Klebsiella pneumonia isolates and 4(44.4%) Klebsiella oxytoca isolates were found to be extended spectrum β- lactamases producers. Conclusion: This study shows that Klebsiella pneumonia and Klebsiella oxytoca recovered from clinical specimens in this region produces extended spectrum β- lactamases in much higher number. Such isolates are also resistant to Floroquinolones, Aminoglycosides, Tetracycline and Cotrimoxazole. Further studies to investigate the factors which determine the emergence and persistence of multidrug resistant extended spectrum β- lactamases producing Klebsiella species in this region and their impact on clinical and economic outcomes at such institutions would be useful. Keywords: Antibiotic susceptibility testing, MDR, ESBL, Combined disk assay, INTRODUCTION Among the human pathogenic bacteria Klebsiella species are very notable. They are gram negative, non motile, encapsulated lactose fermenting, facultative anaerobic and rod shaped bacterium found in the normal flora of the mouth, skin and intestine1. Klebsiella are ubiquitously present and reported worldwide. In recent years Klebsiella have become important pathogens in noscomial infections. Epidemic and endemic noscomial infections caused by Klebsiella species are leading causes of morbidity and mortality2. In addition to being the primary cause of respiratory tract infections like pneumonia, rhinoscleroma, ozaena, sinusitis and otitis it also causes infections of the elimentary tract like enteritis, appendicitis and cholycystitis. It is also commonly involved in acute pyelonephritis in pregnant women with urinary tract abnormalities such as urolithiases, hydronephrosis or congenital deformities. They are opportunistic pathogen and under certain conditions may cause serious infections3. Antibiotics are often used against diseases caused by Klebsiella species. But these pathogens are becoming increasingly antibiotic-resistant so that many are now labeled as Multi-drug resistant (MDR) Klebsiella species4-5. An increasing prevalence of multidrug resistant strains of K. pneumonia which possess extended spectrum beta-lactamases (ESBL) enzymes, encoded by plasmid-borne genes which confer resistance to broad spectrum Cephalosporins and other antibiotics used to treat serious infection has been widely reported6. Epidemic strains of Cephalosporin resistant K. pneumonia have been associated with increased morbidity and mortality in hospitalized patients7. Since 1983 noscomial outbreaks of ESBLs producing K. pneumonia infections in Europe, 8 the United States and South America have been described9-10. Between 1990 and 1992, 5% of K. pneumoniae clinical isolates produced ESBLs11. Multidrug-resistance contributes to unfavorable clinical-outcomes, impacts the utilization of hospital resources, and increases the burden of effective infection control practice and the overall health economic cost12, 6. Thus it is an important task for the researcher to find out alternative medicine. We have started our studies to identify ESBLs producing multidrug resistance Klebsiella species isolated from patients of National Medical College & Teaching Hospital, Nepal MATERIALS & METHODS Isolation and identification of Klebsiella species from different clinical laboratory samples. Collection of clinical samples: Clinical samples which include mid stream urine, blood, and pus were collected from prescribed patients. The samples were collected and labeled in medical laboratory unit of the hospital. These samples were analyzed within 30 minutes to 1 hour of collection. Isolation of Klebsiella species Samples were processed according to the sample nature type. All the samples were cultured in MacConkey Agar, Blood agar plates and incubated at 37°C for 24 hours. The sample where significant pure growth of pathogen obtained was included in this study. Isolates were subcultured and colonies were screened for gram negative isolates. MacConkey Agar media: This media is selective media for the growth of the lactose fermenting organisms in some times contaminating organisms also grows so, further identification is essential. Identification of Klebsiella species: A series of morphological and biochemical tests were performed to identify the suspected Klebsiella species isolates. The test included Gram staining, motility, oxidase activity, catalase production, oxidation-fermentation test different biochemical studies like sugar fermentation, IMViC test and urease production test. All tests were conducted according to the Bergey’s Manual of Determinative Bacteriology .13 Vol 6, Issue 4, 2013 ISSN - 0974-2441