Phenotypic Characterization of Multidrug-resistant Escherichia Coli with Special Reference to Extended-spectrum-beta-lactamases and Metallo-beta-lactamases in a Tertiary Care Center

Introduction: The increasing reports on extended-spectrum-beta-lactamase and metallo-betalactamase producing Escherichia coli have addressed a potential threat to global health since it is found to be highly resistance to most of the currently available antibiotics including carbapenems. The present study was aimed to determine the antibiogram of extended-spectrum-beta-lactamase and metallo-beta-lactamase producing MDR E. coli isolates from various clinical samples. Methods: This was a cross-sectional study conducted over a period of seven months (December 2013 to July 2014) at bacteriology laboratory of Tribhuvan University Teaching Hospital. A total of 250 clinical specimens (urine, pus, sputum, blood, body fluid, bile, tissue and central venous pressure line tip) were processed from inpatients, with multidrug-resistant Escherichia coli infections. Standard microbiological techniques were used for isolation and identification of the isolates. The presence of extended-spectrum-beta-lactamase was detected by phenotypic confirmatory test recommended by Clinical and Laboratory Standards Institute and imipenem (IMP) /EDTA combined disc method was performed to detect metallo-beta-lactamase mediated resistance mechanism. Results: We found high level of beta lactamase mediated resistance mechanism as part of multidrug resistance. Among 250 MDR isolates, 60% isolates were extended-spectrum-beta-lactamase producers and 17.2% isolates were metallo-beta-lactamase producers. Co-existence of extended-spectrum-betalactamase and metallo-beta-lactamase identified in 6.8% isolates. Conclusions: Beta-lactamase mediated resistance mechanisms are accounting very high in the multidrug resistant isolates of E. coli. Therefore, early detection of beta lactamase mediated resistant strains and their current antibiotic susceptibility pattern is necessary to avoid treatment failure and prevent the spread of MDR.  Keywords: e. coli; extended-spectrum-β-lactamase; metallo-β-lactamase; multidrug-resistance

High burden of antimicrobial resistance among gram negative bacteria causing healthcare associated infections in a critical care unit of Nepal

Abstract Background Healthcare associated infections (HCAI) and antimicrobial resistance are principal threats to the patients of intensive care units and are the major determining factors for patient outcome. They are associated with increased morbidity, mortality, excess hospitalization and financial costs. The present study is an attempt to investigate the spectrum and antimicrobial resistance of bacterial isolates involved in healthcare associated infections (HCAI) in the patients of a critical care unit at a tertiary care university hospital in Kathmandu, Nepal. Methods A laboratory based study was conducted over the period of 15 months (January 2014 to March 2015) among the patients of intensive care unit of Tribhuvan University Teaching Hospital, Kathmandu, Nepal. Clinical specimens from patients with suspected healthcare-associated infection were processed and bacterial isolates were identified with standard microbiological methods. Antimicrobial susceptibilities of the isolated strains were determined according to the CLSI guidelines and β-lactamases (ESBL, AmpC, MBL and KPC) were detected by various phenotypic tests. Results One hundred and forty nine clinical specimens received from 135 patients suspected of HCAI (out of 491 patients) were found with significant bacterial growth. Specimens were from patients suspected of hospital-acquired pneumonia (16%, 79/491), bloodstream infections (5.7%, 28/491), surgical site infections (4.7%, 23/491), and urinary tract infections (3.9%, 19/491). Acinetobacter spp., Klebsiella spp., Escherichia coli and Burkholderia cepacia were the leading bacterial pathogens. Extremely high level of drug resistance (95.8%) along with the production of β-lactamases (ESBL; 43.7%, AmpC; 27.5%), MBL; 50.2% and KPC; 4.2%) was observed among Gram negative bacterial isolates. Conclusion Healthcare associated infections are very common in our ICU. Gram negative bacterial pathogens are major culprits associated with these infections and there is alarming state of drug resistance among these isolates. Continuous surveillance and establishment of preventive and control measures of healthcare associated infections are urgently needed in our setting

Extended-Spectrum β-Lactamase (ESBL) Genotypes among Multidrug-Resistant Uropathogenic Escherichia coli Clinical Isolates from a Teaching Hospital of Nepal

Urinary tract infections (UTI) represent the most common bacterial infections among patients visiting outpatient clinics of healthcare centers in Nepal. However, treatment of such infections is compounded by emergence and spread of multidrug-resistant uropathogens associated with extended-spectrum β-lactamases (ESBLs). In this study, we aimed to investigate the burden of antimicrobial resistance and occurrence of ESBL genes among clinical isolates of uropathogenic Escherichia coli at a tertiary care teaching hospital of Nepal. During the study period, we processed a total of 1,626 urinary tract specimens, isolated significant bacterial pathogens, and investigated their antimicrobial susceptibilities. Escherichia coli (n = 154), the predominant pathogen associated with UTI, was further investigated for the existence of ESBL enzymes by using conventional phenotypic as well as molecular approaches. Among suspected cases of UTI, we found that 15.2% were having UTI and female patients of the reproductive age group were more affected (p<0.05). Escherichia coli (154, 62.1%) was the key uropathogen, and majority (∼64.9%) of them were multidrug resistant (MDR). Among MDR E. coli isolates, 40.3% were producing extended-spectrum β-lactamases (ESBLs). bla-TEM (83.8%), bla-CTX-M (66.1%), and bla-SHV (4.8%) were common ESBL genotypes. Nitrofurantoin, gentamycin, and imipenem were the most effective antibiotics for ESBL-producing Escherichia coli isolates. It indicates that the high rates of multidrug-resistant Escherichia coli are frequent causes of UTI in our hospital. Nitrofurantoin and aminoglycosides are the most useful first-line drugs to be used in the cases of UTI. We recommend the regular investigation of drug resistance among all isolates and develop a useful antibiotic prescription policy in our country

Inadequate Empirical Antibiotic Therapy in Hospital Acquired Pneumonia

Introduction: Inadequate empirical antibiotic therapy for HAP is a common phenomena and one of the indicators of the poor stewardship. This study intended to analyze the efficacy of empirical antibiotics in the light of microbiological data in HAP cases. Methods: Suspected cases of HAP were followed for clinico-bacterial evidence, antimicrobial resistance and pre and post culture antibiotic use. The study was taken from February,2014 to July, 2014 in department of Microbiology and department of Respiratory medicine prospectively. Data was analyzed by Microsoft Office Excel 2007. Results: Out of 758 cases investigated, 77(10 %) cases were HAP, 65(84%) of them were culture positive and 48(74 %) were late in onset. In early onset cases, isolates were Acinetobacter 10(42%), Escherichia coli 5(21%), S.aureus 4(17%), Klebsiella 1(4%) and Pseudomonas 1(4%). From the late onset cases Acinetobacter 15(28%), Klebsiella 17(32%) and Pseudomonas 13(24%) were isolated. All Acinetobacter, 78% Klebsiella and 36% Pseudomonas isolates were multi drug resistant. Empirical therapies were inadequate in 12(70%) of early onset cases and 44(92%) of late onset type. Cephalosporins were used in 7(41%) of early onset infections but found to be adequate only in 2(12%) cases. Polymyxins were avoided empirically but after cultures were used in 9(19%) cases. Conclusions: Empirical antibiotics were vastly inadequate, more frequently so in late onset infections. Use of cephalosporins empirically in early onset infections and avoiding empirical use of polymyxin antibiotics in late onset infections contributed largely to the findings. Inadequate empirical regimen is a real time feedback for a practitioner to update his knowledge on the local microbiological trends. Keywords: empirical therapy; hospital acquired pneumonia

Inadequate Empirical Antibiotic Therapy in Hospital Acquired Pneumonia

Introduction: Inadequate empirical antibiotic therapy for HAP is a common phenomena and one of the indicators of the poor stewardship. This study intended to analyze the ef cacy of empirical antibiotics in the light of microbiological data in HAP cases.  Methods: Suspected cases of HAP were followed for clinico-bacterial evidence, antimicrobial resistance and pre and post culture antibiotic use. The study was taken from February,2014 to July 2014 in department of Microbiology and department of Respiratory medicine prospectively. Data was analyzed by Microsoft Of ce Excel 2007.  Results: Out of 758 cases investigated, 77(10 %) cases were HAP, 65(84%) of them were culture positive and 48(74 %) were late in onset. In early onset cases, isolates were Acinetobacter 10(42%), Escherichia coli 5(21%), S.aureus 4(17%), Klebsiella 1(4%) and Pseudomonas 1(4%). From the late onset cases Acinetobacter 15(28%), Klebsiella 17(32%) and Pseudomonas 13(24%) were isolated. All Acinetobacter, 78% Klebsiella and 36% Pseudomonas isolates were multi drug resistant. Empirical therapies were inadequate in 12(70%) of early onset cases and 44(92%) of late onset type. Cephalosporins were used in 7(41%) of early onset infections but found to be adequate only in 2(12%) cases. Polymyxins were avoided empirically but after cultures were used in 9(19%) cases.  Conclusions: Empirical antibiotics were vastly inadequate, more frequently so in late onset infections. Use of cephalosporins empirically in early onset infections and avoiding empirical use of polymyxin antibiotics in late onset infections contributed largely to the  ndings. Inadequate empirical regimen is a real time feedback for a practitioner to update his knowledge on the local microbiological trends. Keywords: empirical therapy; hospital acquired pneumonia

Antimicrobial susceptibility pattern of Shigella spp. isolated from children under 5 years of age attending tertiary care hospitals, Nepal along with first finding of ESBL-production

Abstract Background Shigella is an important cause of bacterial gastroenteritis in resource-poor countries. The treatment of shigellosis mostly requires antibiotics. However, the increase of multidrug resistance along with emergence of extended-spectrum β-lactamase and ciprofloxacin resistance among Shigella spp. has challenged the situation. This study was conducted to determine the distribution of species and antibiotic susceptibility pattern of Shigella species isolated from stool specimen among children less than 5 years of age in Nepal. Results Out of total 717 stool samples collected, 15 cases of Shigella spp. was isolated which includes 12 S. flexneri and 3 S. sonnei. Multidrug resistance was found among 13(86%) of the isolates. One of the isolates of S. flexneri was found to be ESBL-producer with MIC >256 mg/L for cefixime. Conclusion The high occurrence of multidrug resistance among Shigella spp. along with a case of ESBL-production for the first time in Nepal alarms the concerns about dissemination of the resistant isolates. So, systemic monitoring of the antimicrobial susceptibility pattern of Shigella spp. is becoming crucial to guide therapy

Isolation, speciation and antifungal susceptibility testing of Candida isolates from various clinical specimens at a tertiary care hospital, Nepal

Abstract Background Candida species are responsible for various clinical infections ranging from mucocutaneous infection to life threatening invasive diseases along with increased resistance to antifungal drugs has made a serious concern. Resistance to antifungal agents has increased during the last decade. Thus, identification of Candida up to species level and its antifungal susceptibility testing has a paramount significance in the management of Candidal infections. The aim of the study was to speciate Candida species and to determine antifungal susceptibility pattern of Candida species to antifungal agents. Methods A total of 100 consecutive Candida species were isolated from 1248 clinical specimens over 7 months period. Growths on Sabouraud dextrose agar were evaluated for colony appearance, macroscopic examination, Gram staining, germ tube test and urea hydrolysis test. Further, they were processed for Candida speciation on CHROMagar. Antifungal susceptibility testing was performed as recommended by Clinical and Laboratory Standards Institute (CLSI) M44-A document. Results Out of 100 Candida isolates, Candida albicans (56%) was the most common species. Among the non-albicans Candida species, Candida tropicalis (20%) was the predominant isolate followed by Candida glabrata (14%). Regarding antifungal susceptibility pattern, Candida species were more susceptible to clotrimazole (82%) followed by fluconazole (64%) and miconazole (44%). Conclusions Candida albicans was the predominant species responsible for various Candidal infections. Among commonly used antifungal drugs clotrimazole, miconazole and fluconazole were most effective

Carriage of Extended-Spectrum-β-Lactamase- and AmpC-β-Lactamase-Producing Enterobacteriaceae (ESBL-PE) in Healthy Community and Outpatient Department (OPD) Patients in Nepal

Background. Extended-spectrum β-lactamase (ESBL)- and AmpC-β-lactamase-producing Enterobacteriaceae have recently emerged as a public threat in the treatment of nosocomial as well as community-acquired infections. Very little information is currently available about its existence in Nepal. We, therefore, aim to determine the prevalence of ESBL and AmpC-β-lactamase-producing Enterobacteriaceae and also to determine their drug resistance pattern. Methods. During a 6-month period (November 2014–April 2015), a total of 190 stool specimens from 190 participants were obtained from different population. Of the total 260 fecal isolates, 152 from outpatient department (OPD) and 108 from healthy volunteer were collected. Stool specimens were cultured and enterobacterial isolates were subjected to antimicrobial susceptibility tests according to the standard microbiologic guidelines. ESBL was screened using ceftazidime (CAZ, 30 μg) and cefotaxime (CTX, 30 μg) disks and confirmed by double-disk synergy test. AmpC-β-lactamase enzyme production was detected by the aminophenylboronic acid inhibitor-based detection method. Antibiotic susceptibility test was performed for ESBL-positive isolates as per the Kirby-Bauer disk diffusion method, and interpretation was done according to CLSI (Clinical and Laboratory Standard Institute). Results. The prevalence of ESBL, AmpC-β-lactamases, and coproducer (ESBL + AmpC-β-lactamase) producing Enterobacteriaceae in OPD participants were 30.92%, 18.4%, and 13.81%, respectively, while 25%, 6.4%, and 1.8% in healthy population. ESBL-producing E. coli was 70.2% followed by K. pneumoniae (12.7%), and among AmpC-β-lactamase producer, E. coli were detected in half of the isolates (14/28, 50.0%) among OPD patients. Similarly, E. coli remained the most frequent ESBL producers 21/27 (77.8%) followed by K. pneumoniae 4/27 (14.21%) in healthy participants, and K. pneumoniae 5/7 (71.42%) and C. freundii 2/7 (28.57%) were detected highest among AmpC-β-lactamase-producing isolates. All isolates were highly sensitive (100%) to imipenem in both OPD and healthy participants. Conclusion. Our study revealed a high prevalence of ESBL- and AmpC-β-lactamase-producing enteric pathogen in Nepalese OPD and healthy population. The significant increase of these isolates and increased rate of drug resistance indicates a serious threat that stress the need to implement the surveillance system and a proper control measure so as to limit the spread of ESBL-producing Enterobacteriaceae (ESBL-PE) in both OPD as well as in community. Therefore, healthcare providers need to be aware that ESBL- and AmpC-β-lactamase-producing strains are not only circulating in hospital environments but also in the community and should be dealt with accordingly