COMPARISON OF ANTI-MICROBIAL NON-SUSCEPTIBILITY OF ANTIBIOTICS ON ESBL AND NON-ESBL PRODUCING

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1 Microbiology COMPARISON OF ANTI-MICROBIAL NON-SUSCEPTIBILITY OF ANTIBIOTICS ON ESBL AND NON-ESBL PRODUCING Escherichia coli ISOLATED FROM URINE SAMPLES Soni Shrestha 1*, Nabin Kishor Bimali 1, Reena Baidya 2, Binod Lekhak 1 1 Department of Microbiology, GoldenGate International College, Battisputali, Kathmandu, Nepal 2 B & B Hospital Pvt. Ltd., Gwarko, Lalitpur, Nepal *Correspondence: soni.shresthabimali@gmail.com Abstract Escherichia coli is the commonly isolated organisms in urinary tract infections, however the treatment is being troublesome due to rapid development of antimicrobial resistant organisms. This study was carried out from Nov 5, 2012 to May 2, 2013 in B & B Hospital Pvt. Ltd., Gwarko, Nepal with aim to isolate E. coli from urine collected from UTI suspected patients and observe their antimicrobial status. During the study period, a total of 1787 urine samples, regardless of age, sex and sex, from the patients were collected and processed using standard microbiological techniques. E. coli was isolated from 283(15.84%) samples; 156(55.12%) were ESBL and 138(48.76%) were multidrug resistant organisms (MDRO). ESBL organisms were significantly resistant (p-value <0.05) over non-esbl to cephalosporins (cefotaxime, ceftriaxone and ceftazidime) and fluoroquinolones (norfloxacin, ofloxacin, ciprofloxacin) whereas to other antibiotics namely amoxicillin, trimethoprim/ sulphamethoxazole, nitrofurantoin, amikacin, gentamicin, chloramphenicol, pipercillin/ tazobactam, imipenem, meropenem, cefoperazone/ sulbactam the resistance was not significantly different between ESBLs and non-esbls. Among ESBL, imipenem was obtained the most effective drug (R=3.8%) in vitro followed by amikacin (R=5.1%) and cefoperazone/ sulbactam (R=5.8%) whereas among non-esbl, imipenem and amikacin were the most effective drug in vitro followed by cefoperazone/ sulbactam and piperacillin/ tazobactam. The distribution of ESBL and MDRO was statistically significant (p-value <0.05) sex-wise whereas statistically insignificant (p-value>0.05) in between inpatients vs outpatients and different age-interval. And the distribution of MDR among ESBL and non-esbl was statistically significant. A high proportion of ESBL and MDR E. coli regardless of sex, age groups, inpatient vs outpatient reflects urgency to wisely picking up of proper antibiotics for the treatment of infections. Strategy to control the rapid proliferation of such resistant organisms is recommended. Keywords: E. coli, ESBL, MDR Introduction Urinary tract infection (UTI), infections that can occur at any site in the urinary tract, but usually involves bladder, kidneys, or prostate, are the most common bacterial infections in humans (Engleberg et al. 2007). Millions of people are affected by office, 1.5 million emergency room visits, and 300,000 hospital admissions annually only in the United States (Mittal et al. 2009). A wide array of microorganisms may be involved in UTI; though Escherichia coli is the most predominant pathogen, other organisms commonly involved are Proteus mirabilis, Klebsiella spp., other members of Enterobacteriaceae, Staphylococcus saprophyticus and enterococci (Tille 2014). These infections are classified based on the condition of the host; uncomplicated infections affect otherwise healthy individuals and are most commonly caused by uropathogenic Escherichia coli (UPEC), whereas complicated infection affect patients with underlying difficulties, such as urinary tract abnormality or catheterization, and are commonly caused by species such as Proteus mirabilis (Nielubowicz and Mobley 2010).

2 Although UTI can be easily treated with antibiotics, the development of several resistance mechanisms has worsened the scenario, extendedspectrum beta-lactamase being one of them (ESBL) (Kang et al. 2012). ESBLs are group of enzymes that have the ability to hydrolyse and cause resistance to the oxyiminocephalosporins (cefotaxime, ceftazidime, ceftriaxone, cefuroxime, and cefepime) and monobactams (aztreonam), but not the cephamycins (cefoxitin, cefotetan) or carbapenems (imipenem, meropenem, doripenem, ertapenem) (Rupp and Fey 2003). These ESBL-producing genes are present in the plasmid and (Pitout 2010) which also contains genes responsible for resistance of aminoglycosides, fluoroquinolones, or cotrimoxazole (Isenberg 2004). ESBLs causes multidimensional impact, i.e. they increase in the hospital stay, or the condition may even be worse in cases when ESBL detection method is not included, also the cost of treatment will be more (Cosgrove and Carmeli 2003). This study aims to find out the non-susceptibility of different antibiotics on ESBL -producing and - non-producing E. coli isolates from urine samples. Materials and Methods This descriptive study was carried out at B & B Hospital Pvt. Ltd., Gwarko, Lalitpur, Nepal, from Nov 5, 2012 to May 2, 2013, in collaboration with GoldenGate International College (GGIC). Outpatients and inpatients, regardless of sex and age, visiting the pathology department of hospital with request form for urine culture were included in this study. Patients recruited for this research work had no any underlying conditions or systemic disease that could be predisposing the host to bacterial infection. Sample processing Clean-catch mid-stream urine (CC-MSU), ml, collected adequately in clean, dry, sterile and leak-proof container was accepted and further processed. Semi-quantitative technique (with a loop of 4mm, holds 0.01 ml) of culture was performed on MacConkey agar (MA) and Blood agar (BA), the plates were incubated for 35±2 C for 24 to 48 hours. Once the colony obtained, observation was done for significant growth (>10 5 cfu/ml) (PHE 2016). Further investigation for the identification of E. coli was based on Gram staining and conventional biochemical tests (catalase and oxidase test, Indole test, methyl red and Voges-proskauer test, citrate test, TSI test, urease test, and oxidativefermentative test) (Tille 2014). Antimicrobial susceptibility testing (AST) Antimicrobial susceptibility testing of isolates was performed by modified Kirby Bauer disc diffusion method using the standard guidelines and interpretive criteria as per CLSI (2013). Antibiotics (HiMedia, Mumbai) used in this study are amoxycillin (10 µg), ceftriaxone (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), norfloxacin (10 µg), ofloxacin (5 µg), ciprofloxacin (5 µg), nitrofurantioin (300 µg), cotrimoxazole (25 µg), amikacin (30 µg), gentamicin (10 µg), cholramphenicol (30 µg), piperacillin/ tazobactam (100/10 µg), imipenem (10 µg), meropenem (10 µg) and cefoperazone/sulbactam (75/30 µg). ESBL testing ESBL testing was performed by combined disc methods, as per CLSI guidelines (CLSI 2013). Screening for ESBL production was performed using three cephalosporins, namely, cefotaxime, ceftazidime, and ceftriaxone. The screen positive isolates were subjected to confirmation assay where cefotaxime and ceftazidime were used in alone and with clavulanic acid. The zone of inhibition for the ceftazidime and cefotaxime discs were compared to those of the ceftazidime plus clavunic acid and cefotaxime plus cefotaxime clavunic acid combination discs. An increase presence of clavunic acid was concluded as confirmed ESBL producers. Quality control Quality control was performed by using Escherichia coli ATCC and Klebsiella pneumoniae ATCC for AST and ESBL testing. Analysis of data Antimicrobial susceptibility results by disc diffusion were analyzed by using WHONET 5.6 software. Other Statistical analysis was done by using SPSS 19.0 for windows.

3 Results A total of 1,787 urine samples were processed, with standard microbiological technique, during the study period. Among them 844 were from male patients and 943 were from female patients (Ratio 1:1.12). Of the total urine sample 462 samples were from inpatients and remaining 1325 samples were from outpatients. E. coli were isolated from 283 urine samples; 168 (59.36%) isolates were from female patients whereas 115 (40.64%) were from male patients. The distribution of E. coli according to sex was obtained statistically significant (p-value < 0.05). Of total, 225 (78.7%) of those samples were obtained from outpatients whereas the remaining, i.e. 58 (21.31%) were from inpatients (p-value <0.05). Table 1. Demographic distribution of E. coli isolates. Total Samples Processed = 1787 Total growth of E. coli = 283 Distribution of E. coli according to: Sex Male 115(40.64%) p-value Female 168(59.36%) Ward Outpatients 225(79.51%) p-value Inpatients 58(20.49%) Age-interval (yrs) Male Female < > No great difference was obtained in incidence of E. coli isolation between male and female patients in age-interval below 15 and over 45. However, in age group between 15 through 45, there was comparably high incidence of E. coli obtained in female than male. ESBL testing Among the three different cephalosporins employed, different results were obtained from all three; cefotaxime screened 169 isolates as possible ESBL producer, whereas ceftazidime and ceftriaxone screened 175 and 159 isolates as possible ESBL producers respectively. During confirmatory test, cefotaxime + cefotaxime/ clavulanic acid combination confirmed 147 screening positive isolates as ESBL producers whereas ceftazidime + ceftazidime + ceftazidime/ clavulanic acid combination confirmed 151 screening positive isolates as ESBL producers (table 2). Antimicrobial susceptibility pattern of isolates The frequency of antimicrobial non-susceptibility for 16 selected antimicrobial agents against E. coli has been illustrated in table 3. Higher rate of resistance was observed to amoxycillin (98.59%) followed by ceftazidime, ofloxacin, ciprofloxacin, cotrimoxazole, cefotaxime, norfloxacin and ceftriaxone whereas lower resistance was observed to imipenem followed by amikacin. ESBLproducing E. coli were significantly more resistant to norfloxacin, ofloxacin, and ciprofloxacin, but susceptible to chloramphenicol over ESBL nonproducing strains (p < 0.05). Non-susceptibility percentage of different antibiotics by hospital ward and sex is illustrated in table 4. Isolates from inpatients showed statistically significant higher resistance than those form outpatients to cephalosporins, fluoroquinolone, aminoglycosides, nitrofurantoin, piperacillin/ tazobactam, and cefoperazone/ sulbactam (p-value <0.05). Similarly, isolates from male showed statistically significant higher resistance than those from female to ceftriaxone, cefotaxime, ceftazidime, norfloxacin, ofloxacin, ciprofloxacin and nitrofurantoin. To none of the antibiotics, outpatients were significantly resistant over inpatients, nor female over male.

4 Table 2. Efficiency of different cephalosporin/ cephalosporin-clavulanic acid in ESBL confirmation. Combination used for ESBL confirmatory test Total screening positive isolates Confirmed ESBL isolates No. of missed ESBL isolates Total ESBL isolates Cefotaxime (30 µg)+ Cefotaxime/Clavulanic acid (30/10 µg) Ceftazidime (30 µg) Ceftazidime/ Clavulanic acid (30/10 µg) Distribution of MDR E. coli among ESBL producers and non-producers Isolates resistance to three or more antibiotics of different class were regarded as multidrug resistance (Magiorakos et al. 2012). Among total 283 isolates, 113 were such isolates which were both ESBL-producing as well as MDR, 43 were ESBL-producing but not MDR, 25 were MDR, but non-esbl, whereas 102 isolates were neither ESBL-producing nor MDR. A significant association was found among ESBL production and multidrug resistance (p-value= 0.000) (table 5). Table 3. Antimicrobial non-susceptibility of ESBL and non-esbl producing E. coli (n= 283). Antibiotics Non-susceptible (%) ESBL Non-ESBL p-value* (n=156) (n=127) Amxycillin 279 (98.59) Ceftriaxone 159 (56.18) Cefotaxime 169 (59.72) Ceftaxidime 175 (61. 84) Norfloxacin 165 (58.30) Ofloxacin 175 (61.84) Ciprofloxacin 172 (60.78) Nitrofurantoin 36 (12.72) Cotrimoxazole 171 (60.42) Amikacin 14(4.95) Gentamicin 60 (21.20) Chloramphenicol 40 (14.13) Piperacillin/Tazobactam 33 (11.66) Imipenem 12 (4.24) Meropenem 33 (11.66) Cefoperazone/Sulbactam 19 (6.71)

5 Table 4. Antimicrobial non-susceptible (%) of E. coli isolates in relation to ward and sex. Antibiotics Ward p-value Sex p-value Inpatient Outpatient Female Male (n=58) (n=225) (n=168) (n=115) Amxycillin Ceftriaxone * * Cefotaxime * * Ceftaxidime * * Norfloxacin * * Ofloxacin * * Ciprofloxacin * * Nitrofurantoin * * Cotrimoxazole Amikacin * Gentamicin * Chloramphenicol Piperacillin/Tazobactam * Imipenem Meropenem Cefoperazone/Sulbactam * * represents significant p-value. Table 5. Distribution of MDR E. coli among ESBL producers and non-producers. ESBL Non-ESBL Total p-value MDR Non-MDR Total Distribution of ESBL and MDR E. coli among ward, sex and age-interval The distribution of ESBL E. coli among ward, sex and age-interval has been illustrated in Table 6. There were 36 and 120 ESBL E. coli among inpatient and outpatient respectively (p >0.05); 74 and 82 ESBL E. coli among male and female respectively (p < 0.05) and 8, 68 and 80 ESBL E. coli among age-interval of <15, and >45 respectively (p >0.05). The distribution of MDR E. coli among ward, sex and age-interval is illustrated in Table 6. There were 34 and 104 MDR E. coli among inpatient and outpatient respectively (p >0.05); 68 and 70 MDR E. coli among male and female respectively (p <0.05) and 7, 62 and 69 MDR E. coli among age-interval of <15, and >45 respectively (p >0.05). Discussion In this present study, the distribution of E. coli isolates from urine sample and their nonsusceptibility to sixteen different antibiotics was observed. Also, the non-susceptibility of ESBL producing and non-producing isolates to those antibiotics was analyzed statistically. Further, the association between multidrug resistance and ESBL production was observed and finally, the distribution of ESBL and MDR E. coli among wards (inpatients/ outpatients), sex (male/female) and in different age interval was observed. E. coli were isolated more from female patients in comparison to male patients, which was statistically significant.

6 Table 6. Distribution of ESBL and MDR E. coli among ward, sex and age-interval. Isolates number ESBL (%) Non- ESBL (%) p-value MDR (%) Non-MDR (%) p- value Ward Inpatient 58 36(62.1) 22(37.9) (58.6) 24(41.4) Outpatient (53.3) 105(46.7) 104(46.2) 121(53.8) Sex Male (64.4) 41(35.6) (59.1) 47(40.9) Female (48.8) 86(51.2) 70(41.7) 38(58.3) Age-interval yr < (53.3) 7(46.7) 7(46.7) 8(53.3) (48.5) 72(51.5) (44.3) 78(55.7) > (62.5) 48(37.5) 69(53.9) 59(46.1) Similar results were observed previously by other studies also from Nepal (Jha and Yadav 1992; Dhakal et al. 1999; Chhetri et al. 2001; Chander and Shrestha 2013), India (Ahmed et al. 2012) and Nigeria (Oladeinde et al. 2011). Women are more prone to UTI due to several reasons; urethra is shorter and closer to anus, also they lack the bacteriostatic properties of prostatic secretions (Leigh et al. 1990). Isolation of E. coli in male and female was observed incomparable in age group <15 (8 in females and 7 in males) and age group >45 (63 in females and 65 in males), whereas in age-interval of there was a great difference in isolation of E. coli in female and male (97 in female and 43 in male). This may be due to the sexually active period of female (Rowe and Juthani-Mehta 2013). Also the isolation of E. coli in between inpatients and outpatients was obtained statistically significant (p-value 0.025); from outpatients 225 (16.98%) and from inpatients 58 (14.35%) E. coli were isolated. This observation shows that the prevalence of ESBL producing E. coli is increasing in community. A high percentage (55.12%) of ESBL-producing E. coli was observed in this study. Similar result was observed by Wani et al. (2009), who observed % of the E. coli isolates as ESBL producers. In contrast, study conducted at similar time in another tertiary hospital of Kathmandu, Nepal by Chander and Shrestha (2013) observed only % (60/444) E. coli as ESBL producers, similar low prevalence of ESBL E. coli (11.60%) was also reported earlier by Pokharel et al. (2006). ESBL distribution among ward, sex and age-interval was also observed in this study; the distribution was obtained statistically significant sex-wise, however statistically insignificant ward-wise and among different age-intervals. The incidence of ESBLproducers among inpatients and outpatients was statistically insignificant (p-value = 0.092). However, higher percentages of isolates among inpatients were ESBL-producers in comparison to outpatients (32.07% vs 53.33%), similar results were previously observed by Umadevi et al. (2011). Similar to Rajan and Prabavathy (2012) who observed higher percentage of ESBL E. coli over age of 50 (67.5%), this study also detect higher percentage of ESBL in age-interval of more than 45 (62.5%). However, in age interval of <15 and 15-45, there were 48.57% and 53.33% ESBL E. coli among the E. coli isolates in those respective age-intervals. Among 16 antibiotics used for susceptibility testing, high level of non-susceptibility was observed against amoxycillin (98.59 %), followed by cephalosporins and fluoroquinolones. Similarly, cotrimoxazole also exhibited nonsusceptibility of %. In contrast, antibiotics namely, imipenem, amikacin, cefoperazone/

7 sulbactam, meropenem, piperacillin/ tazobactam, nitrofurantioin, chloramphenicol, gentamicin demonstrated low level of non-susceptibility (4.24%, 4.95%, 6.71%, 11.66%, 11.66%, 12.72%, 14.13%, and 21.20% respectively). High level of non-susceptibility among cephalosporins and fluoroquiole is due to the high percentage of ESBL observed in this study. The plasmid harboring the genes for ESBL also harbors the genes for the resistance of fluoroquinolones (Isenberg 2004). In this study, significant difference was observed in non-susceptibility to ESBL compared to non-esbl among antibiotics of cephalosporins, fluoroquinolones and choloramphenicol. For other antibiotics used (above mentioned), the non-susceptibility among ESBL and non-esbl was not significant. Nitrofurantoin was observed more effective in vitro to E. coli in comparison to cephalosporin and fluoroquinolones, which is due to its limited impact on the normal gut flora and correspondingly limited selection of resistant organisms (Mavromanolakis et al. 1997). Treating of UTIs or other infections with broad spectrum antibiotics (cephalosporins, fluoroquinolones) may result into selection of drug-resistant organisms and infections with them on future (Paterson 2004; Ramphal and Ambrose 2006). Proper treatment should be understood in both the relation, i.e. proper management of the patients and containing the increasing resistance trend of the etiologic agents. Treatment of UTI is dependent infections; cystitis or pyelonephritis, complicated or non-complicated, and in vitro antimicrobial susceptibility testing of antibiotics to the involved organisms. Fluoroquinolones and cepahlosporins which cause selective pressure, possibly resulting into future or recurrent infections with multidrug resisting microbes should not be used in uncomplicated cases like cystitis (Gupta et al. 2011). Fluoroquinolones should be saved for the infections with high morbidity and mortality, i.e. pyelonephritis rather than cystitis (Foxman 2013). Fluoroquinolones or other beta-lactam drugs can also be recommended whenever the patients are allergic to first line drugs or during unavailability of other drugs. Nitrofurantoin, cotrimoxazole, fosfomycin, or pivmecillinam are recommended for the treatment of uncomplicated UTIs by joint guidelines from Infectious diseases Society of America (IDSA) and European Society for Microbiology and Infectious Diseases (Gupta et al. 2011). A total of 138 (48.76%) E. coli isolates in this study were multidrug resistance (MDR), which is lower in comparison to 55.8% as reported by Shakya et al. (2012). Furthermore, Shakya et al. (2012) also observed the increasing occurrence of MDR isolates every year in period of , 41 in 2006 to 88 in 2010; however no significant difference between year and occurrence of MDR. The occurrence of MDR isolates was found significantly greater in male in comparison to female; the result is in accordance to Baral et al. (2012). However, this study has limitations and do not put light on the risk factors behind it. The probable reason might be due to the higher incidence of UTIs in male in old age, as seen in this study (22.97% ); reaching to the old age any person may be in the circle of various risk factors like using mechanical devices, other underlying conditions like diabetes, use of antibiotics for other health problems etc. (Wright et al. 2000). In this study, high incidence of MDR was observed among inpatients over outpatients, however statistically insignificant. Previously Baral et al. (2012) had reported 85 % MDR among hospitalized patients and 36.7 % MDR among outpatients; the result was statistically significant. These results reflect widespread dissemination of MDR isolates in both hospital as well as community settings. With the aid of WHONET 5.6 antibiotic resistant profile of both ESBL and non- ESBL organisms were obtained. Altogether, 138 isolates were obtained to be MDR; 113 were ESBL and remaining 25 were non-esbl. The most encountered resistance profiles in this study were AMX CRO CTX CAZ NOR CIP OFX SXT, and AMX CRO CTX CAZ NOR CIP OFX SXT GEN, exhibited by 30 and 10 isolates respectively. The former are MDR with resistance to three different classes of antibiotics and later are MDR with resistance to four different classes of antibiotics. A variety of pattern was observed among E. coli isolates; some of the MDR were even resistant to all the 16 antibiotics exploited in this study. Both these profile were observed in ESBL producing organisms. Among non-esbl no any resistance profile was observed to contain more than two MDR isolates.

8 This study has some limitations too. The study do not mention about the status of other uropathogens and the type of ESBLs present in UPEC. However this study could not measure the actual prevalence of the urinary tract infections because of short duration of the study, this study supports the finding of Doi et al. (2003) that nitrofurantoin can be used as the drug of choice in UTI. In this study, 88.46% of ESBL E. coli and 85.83% of non-esbl E. coli were observed to be susceptible in vitro to nitrofurantoin, similar result with high susceptibility of E. coli isolates (98.1%) was observed by Doi et al. (2013). Conclusion A high percentage of ESBL-producing E. coli isolates shows an alarming condition since ESBL production is related with higher morbidity and mortality. Also, the observation that ESBL production is significantly higher in isolates from outpatients in comparison to inpatients necessitates the development of strategies to prevent the spread of such isolates in the environment. Although the drugs like cephalosporins and fluoroquinolones are not more effective in settings where there is high prevalence if ESBL producers, however nitrofurantoin is found to be promising for treatment, which is also regarded among the first ling drug for the treatment of cystitis. Also, high occurrence of UTI and MDR in age interval of >45 reflects that it may be due to several reasons like immunocompromised status, use of mechanical devices etc., so care should be given to prevent from UTI. Acknowledgements This study was reviewed and approved by Institutional Review Board, B & B Hospital, Lalitpur, Nepal. Laboratory facilities were provided by Pathology Laboratory, B & B Hospital and GoldenGate International College, Kathmandu, Nepal. References Ahmed S.M., Jakribettu R.P., Koyakutty S., Arya B., Shakir V.P.A Urinary tract infections an overview on the prevalence and the anti-biogram of gram negative uropathogens in a tertiary care center in North Kerala, India. J. Clin. Diagn. Res. 6: Baral P., Neupane S., Marasini B.P., Ghimire K.R., Lekhak B. and Shrestha B High prevalence of multidrug resistance in bacterial uropathogens from Kathmandu, Nepal. BMC Res. Notes. 5: Chander A. and Shrestha C.D Prevalence of extended spectrum beta lactamase producing Escherichia coli and Klebsiella pneumoniae urinary isolates in a tertiary care hospital in Kathmandu, Nepal. BMC Res. Notes. 6: Chhetri P.K., Rai S.K., Pathak U.N., Thapa J.B., Devkota K.C., Shrestha B.O. and Shrestha R.R Retrospective study on urinary tract infection at Nepal Medical College Teaching Hospital, Kathmandu. Nepal Med. Coll. J. 3: CLSI Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. CLSI document M100-S23. Wayne, PA: Clinical and Laboratory Standards Institute. Cosgrove S.E. and Carmeli Y The impact of antimicrobial resistance on health and economic outcomes. Clin. Infect. Dis. 36: Dhakal B.K., Pokharel B.M. and Basnyat S.R A Prospective Study of Urinary Tract Infection Based on Culture and Direct Microscopy of Urine Along with the Antibiotic Sensitivity Test of Urinary Pathogen. M.Sc. dissertation submitted to the Central Department of Microbiology, T.U, Kathmandu. Doi Y., Park Y.S., Rivera J.I., Adams-Haduch J.M., Hingwe A., Sordilo E.M., Lewis J.S., Howard W.J., Johnson L.E., Polsky B., Jorgensen J.H., Richer S.S., Shutt K.A. and Paterson D.L Communityassociated extended- lactamaseproducing Escherichia coli infection in the United States. Clin. Infect. Dis. 56: Engleberg N.C., DiRita V. and Dermondy T.S th ed. (eds. Lippincott Williams & Wikins). pp Foxman, B Extended- -lactamaseproducing Escherichia coli in the United States: Time to rethink empirical treatment for suspected E. coli infections? Clin. Infect. Dis. 56: Gupta K., Hooton T.M., Naber K.G., Wullt B., Colgan R., Miller L.G., Moran G.J., Nicolle L.E., Raz R., Schaeffer A.J. and Soper D.E International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases. Clin. Infect. Dis. 52: e103- e120. Isenberg, H.D Clinical Microbiology Procedures Handbook. 2 nd ed. ASM Press. Washington D.C. Jha V.C. and Yadav J.N Bacterial species isolated from urine of UTI suspected patients and their sensitivity to commonly available antibiotics. J Nepal Med. Assoc. 30:

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