Carbapenem-resistant Escherichia Coli Among the Hospitalized Patients with Urinary Tract Infections in a Tertiary Care Center of Nepal

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1 Original Article Carbapenem-resistant Escherichia Coli Among the Hospitalized Patients with Urinary Tract Infections in a Tertiary Care Center of Nepal Shrestha B*, Tada T**, Shrestha S*,Kattel HP***,Ohara H****, Kirikae T*****, Rijal BP******, Sherchand JB******, Pokhrel BM****** *PhD scholar, ***Microbiologist, ******Professor, Department of Microbiology, Institute of Medicine, Maharajgunj Medical Campus,Tribhuvan University, Kathmandu, Nepal,** Senior Scientist, *****Director, Department of Infectious Diseases, Research Institute, ****Director,,Department of International Medical Cooperation, National Center for Global Health and Medicine, Tokyo, Japan Abstract Introduction: Carbapenemases producing Enterobacteriaceae (CRE)has recently drawn widespread attention.the increasing number of carbapenemase producing organisms in urinary tract infection (UTI) leaves very few treatment options for clinicians.this study was designed to evaluate carbapenemases producing Escherichia coli(e. coli)amongst hospitalized subjects suffering from urinary tract infection in a tertiary care center of Kathmandu, Nepal, between December 2013 and July Methods: Standard microbiological techniques were used for isolation and identification of the isolates.the antimicrobial susceptibility of bacterial isolates was determined following Clinical and Laboratory Standard Institute (CLSI) recommended Kirby-Bauer Disc Diffusion method. The defining criterion in this study for an isolate to be multi-drug resistant, resistance to at least one agent in three or more than three different structural classes was taken. Results: Among 1624 hospitalized patients enrolled, a total of 454 (25%) isolates were recovered. Of these combined disc method performed to detect Metallo-β-Lactamase (MBL) mediated isolates; the most common isolate was E.coli (n=276, 61%). Out of 276 E. coli isolates, 156 (57%) were multi-drug resistant isolates and 21 (7.6 %) of the isolates were resistant to carbapenems. All of carbapenem-resistant E. coli (CRE) were MDR. Significant drug resistances were observed among CRE compared to other MDR E. coli. Conclusion: Our results demonstrate a high prevalence of carpabenemase producing E. coliin hospital settings.carbapenemases in E. coli is a majorthreat.early detection and surveillance of carbapenemase-producers routinely in clinical laboratoriesis necessary to facilitate effective infection control and to reduce the escalation of resistance. Key words: Carbapenem-resistant Escherichia coli, Metallo-β-lactamase, Multi-drug resistant Escherichia coli INTRODUCTION Urinary tract infections (UTIs) are one of the most common infectious diseases ranking next to upper respiratory tract infection. Urinary tract infections are often associated with significant morbidity and Correspondence : Basudha Shrestha ( basudha111@hotmail.com) Ph.D. Scholar, Department of Microbiology Institute of Medicine, Maharajgunj Medical Campus Tribhuvan University, Kathmandu, Nepal. mortality. Worldwide, about 150 million people are diagnosed with UTI each year, costing the global economy in excess of 6 billion dollars 1. In developing countries, including Nepal, the facilities for urine culture and antimicrobial susceptibility testing are still not sufficiently available, leading to improper diagnosis and irrational antibiotic treatment of UTI, which accelerates the emergence of multidrug resistant (MDR) strains.treatment of urinary tract infections is compromised worldwide by the emergence of bacteria that are resistant to multiple antibiotics 6 Gram negative Volume 14 Number 1 Jan-Jun

2 bacteria, especially the family Enterobacteriaceae are the common cause of both community and hospital acquired UTIs. Escherichia coli and Klebsiella pneumoniae are most commonly implicated among patients with UTI 2, 3. Previously, the emergences of MDR among enterobacteriaceae were mainly due to the production of enzymes, such as pencillinases, cephalosorinases, and extended spectrum β-lactamase (ESBL). However, recently carbapenemase production is one of the main mechanisms in the occurrence of drug resistance in the family of enterobacteriaceae. Carbapenem-resistant enterobacteriaceae (CRE) are difficult to treat because they have high levels of resistance to antibiotics, which capable of break down all β-lactam agents including carbapenems and make it ineffective. Carbapenem such as imipenem, meropenem, ertapenem, & doripenem are considered as the last resort antibiotics to treat ESBL producing enterobacteriaceae.unfortunately several studies have shown that the emergence of carbapenem resistant bacteria is increased throughout the world 4, 5.So this important drug may not be useful in future. Currently, increased burden of MDR organisms causing UTI compounded by harboring carbapenem resistance genes mainly among E. coli and K. pneumonia. These strains become a serious threat to public health, associated with high mortality rates and have the potential to spread widely. Infections are difficult, and in some cases impossible to treat and have been associated with mortality rates up to 50% 6, 7. The aetiology of UTI and the antibiotic resistance of uropathogens have been changing over the past years, both in community and health care associated infections. Current knowledge on the burden and antimicrobial susceptibility pattern of E. coli isolates is essential for appropriate therapy; since E. coli is the main cause of UTIs and possesses several mechanisms to destroy currently available antibiotics including carbapenems and the condition in Nepal is a little assessed. Therefore, the objective of this study was aimed to determine the prevalence of MDR and carbapenem-resistant E. coliisolates among hospitalized patients with UTI at a tertiary care center of Kathmandu, Nepal. METHODS Participants and clinical isolates: A cross-sectional study was conducted prospectively during the period from December 2013 to July Total of 1624 urine samples from hospitalized patients of Tribhuvan University Teaching Hospitalwas included in the study after obtaining approval from institution review board. Details of antibiotics used and clinical outcome of patients were collected. Samples were processed immediately using standard microbiological procedures as described by American Society for Microbiology (ASM) 8. Isolates were identified based on colony morphology on Blood agar, MacConkey agar, Gram staining and by standard biochemical tests 9. Antimicrobial susceptibility testing Antibiotic susceptibility testing was done by the modified Kirby-Bauer disk diffusion method in accordance with CLSI guidelines 10. The antibiotic discs used were amikacin (30 μg), amoxycillin (10 μg), amoxycillin/ clavulanic acid (20/10 μg), cefepime (30µg), cefoperazone /sulbactam (75/30 μg) ceftazidime (30 μg), cefoxitin (30 μg), ceftriaxone (30 μg), chloramphenicol (30µg), ciprofloxacin (5 μg), colistin sulphate (10 μg), doxycycline (30µg), levofloxacin (5 μg), gentamicin (10 μg), imipenem(10 μg), meropenem (10 μg), nitrofurantoin (300 µg), ofloxacin (5µg), piperacillin-tazobactam (100/10 μg), polymyxin- B (300 units) and tigecycline(5 μg). Screening for carbapenemase production E. coli isolates that showed the zone of inhibition 19 mm against meropenem and imipenem were suspected as carbapenemase producers 10. Phenotypic confirmation for metallo-β-lactamase production All carbapenemase producers E. coli were phenotypically confirmed for Metallo-β-lactamase (MBL) production. MBLin carbapenemase producing strains were detected as described by Yong et al. 12. A 0.5 M EDTA solution was prepared by dissolving g of disodium EDTA 2H2O (Sigmachemicals, Germany) in 1,000 ml of distilled water and adjusting it to ph 8.0 by using NaOH. The mixture was sterilized by autoclaving. One disc of imipenem (10 μg) alone 30 Volume 14 Number 1 Jan-Jun 2014

3 and one with imipenem (10 μg) in combination with EDTA were placed at a distance of 20 mm, from center to center, on a Muller Hinton agar plate inoculated with a bacterial suspension of 0.5 McFarland turbidity standards and incubated overnight at 35 C. The MBL producing strains showed a variation greater than 7 mm between the inhibition zone around imipenem discs alone and the inhibition zone around imipenem+ EDTA discs, and they showed a variation greater than 5mm between the inhibition zone around imipenem+edta discs and EDTA discs alone. P. aeruginosa ATCC was used as a negative control strain. RESULTS Out of 1624 urine samples,454 (25.0%) showed significant growth. The most common isolates were E. coli 276 (61.0%) followed by Enterococcus faecalis (14.0%) and Klebsiella pneumoniae 56 (12.0%) (Table1). All E. coli isolates were tested for antimicrobial susceptibility, 131 (47.0%) of them showed resistance to three or more classes of antibiotics(table 2) and considered as MDR isolates. Table 1: Microbiological profile of Urine S. No. Organisms No. of % 1 E. coli Enterococcus faecalis Klebsiella pneumoniae Klebsiella oxytoca Staphyloccusaureua Acinetobacter spp Burkholderia spp Proteus mirabilis Enterobacterclocae Pseudomonas aeruginosa Proteus vulgaris Table 2: Antibiotic susceptibility pattern of E. coli isolates (n=276) Antibiotics Sensitive Resistant No % No % Amoxycillin Cefixime Cefotaxime Cotrimoxazole Ciprofloxacin Nitrofurantoin Norfloxacin Ofloxacin Levofloxacin A total of 131 MDR E. coli isolates were analyzed for resistance ability against second line of antibiotics were found to resist most of the antimicrobial agents. Among first line antibiotics, MDR isolates were found to be 100% resistant toward amoxycillin, ceftriaxone, cefotaxime, cefixime.ciprofloxacin and cotrimoxazole. 80.0% of isolates were resistant to levofloxacin. Likewise, among second line of antibiotics, 100.0% of isolates were resistant to ceftazidime and ceftriaxone followed by amoxycillin/clavulanic acid (89.0%), cefoperazone/sulbactam (69.0%), piperacillin/ tazobactam (66.0%)and doxycycline (57.0%). Colistin sulphate, polymyxin-b and tigecycline were 100% effective followed by nitrofurantoin (95.0%), imipenem (84.0%), meropenem (82.0%), amikacin (76.0%) and gentamycin (73.0%) indicating these as the most potent antimicrobials (Table 3). Table 3: Antibiotic susceptibility pattern of MDR E. coli isolates with first and second line antibiotics Antibiotics Sensitive Resistant No % No % First line antibiotics Amoxycillin Cefixime Cefotaxime Ciprofloxacin Cotrimoxazole Levofloxacin Nitrofurantoin Norfloxacin Ofloxacin Second line antibiotics Amikacin Ampicillin/clavulanic acid Ceftazidime Ceftriaxone Cefoperazone/ sulbactam Colistin sulphate Doxycycline Volume 14 Number 1 Jan-Jun

4 Gentamycin Imipenem Meropenem Piperacillin/tazobactam Polymyxin-B Tigecycline MDR strains were tested for carbapenemase production by using phenotypic methods.a total of 23 E. coli isolateswere found to be resistance to carbapenems.after phenotypic confirmation of metallo-β-lactamase (MBL), the overall prevalence of MBL producing E. coli isolates was 8.0% (n=276) among all E. coli isolates and 16.0% (n=131). DISCUSSION The overall prevalence of MDR among E. coli isolates identified from patients with symptomatic UTI was 47.0%, which is similar with the results from previous study in Nepal (40.0%) 13 while it was higher than reports from other study in USA(19.1%) 14. However, it was lower than reports from Africa 15 [20-22].With regard to urinary tract infection among hospitalized patients, many researchers indicated its incidence as: 31%-47% The variation in prevalence of MDRE. coli isolates could be due to increase trend of MDR strains with time, difference in study period and study population. Besides, these bacteria are frequently difficult to treat because of both their intrinsic and acquired resistance to multiple groups of antimicrobial agents 2, 3. Among 276 E. coli isolates, 21(8.0%) were found to be carbapenemase producers. Comparable result were reported in studies from Pakistan (8.6%) 19, Turkey 20 (10.9%) and India 21 (12.9%). Indwelling medical devices are commonly used in hospitalized patients, which play a key role in the spread of infective agents. In any hospital setting, carbapenems are used as the last resort for treatment of MDRgramnegative bacterial infection. Antibiotic overuse is an important contributor for the emergence and spread of resistance; association between carbapenem consumption and resistance has been previously documented. 22. We observed all MBL producer E.coli were resistant to imipenem and meropenem.these isolates also demonstrated a high level of resistance to amoxycillin, the third and fourth generation cephalosporins, amikacin and gentamycin as well as to the beta-lactam/ beta-lactamase inhibitor combination testedin the study. These findings are similar with other reports The isolates were resistant to β- lactam antibiotics due to beta-lactamases, alterations in penicillin binding proteins, over-expression ofmultidrug efflux systems and decreased outer membrane permeability causedby the loss or reduced expression of porins 26 ; ENREF_26 resistant to fluoroquinolones due to efflux pumps and mutations affecting the DNA gyrase and topoisomeraseiv enzymes; similarly resistant to aminoglycosides is due to presence of aminoglycosidemodifying enzymes. 27 Nitrofurantoin seems to be the most effective antibiotic for first line treatment. The best choice for the second line treatment of carbapenemase producing E. coli wereto polymyxin-b, colistin, and tigecycline,as these isolates were 100.0% susceptible to these drugs but all these are parenteral antibiotics. CONCLUSION Our study has demonstrated a high level of multidrug resistance and increased trend in resistance to carbapenems in E.coli. The outcome of this study may be associated with excessive use of broad-spectrum antibacterial agents. Thus, early detection of the occurrence of carbapenemase enzyme will help in avoiding the development and the dissemination of these MDR strains. The awareness of the existence of carbapenemases initializes indication for the need for proper use of antibiotics to stem selective pressure and spread of MDR bacterial strains within these hospital and communities. Preventive measures like a continuous surveillance of various wards within the hospital and a strict implementation of infection control practices can go a long way in containing the menace of drug resistance in the health care settings. ACKNOWLEDGEMENT We extend our sincere appreciation to National Academy of Science and Technology (NAST) for providing PhD fellowshipto conduct thisresearch. We are thankful to entire patients who participated and helped us to complete this study.we would like to thank all the staff of department of microbiology at Tribhuvan University Teaching Hospital for their kind co-operation during this study. REFERENCES 1. Lee JB, Neild GH. Urinary tract infection. J Med. 2007; 35(8): Volume 14 Number 1 Jan-Jun 2014

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