Indian Journal of Biotechnology Vol 14, April 2015, pp 167-171 High quinolone resistance pattern among enteric pathogens isolated from patients with urinary tract infection Lesley R Varughese and Vikas Beniwal* Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala 133 207, India Urinary tract infection (UTI) is one of the most common infections that exists in all age groups. It is predominantly caused by members of the family Enterobacteriaceae, such as, Escherichia coli, Klebsiella, Enterobacter and Proteus. Quinolones have been routinely prescribed for the treatment of UTIs and this has led to a dramatic increase in antibiotic resistance by these uropathogens. The aim of the present study was to determine the quinolone resistance pattern in enteric pathogens isolated from UTI patients in Maharishi Markandeshwar University (MMU), Mullana, Haryana. Clinical samples were obtained from MMU Medical College. A total of 70 samples were screened, 60 isolates (41 and 19 from female and male, respectively) were confirmed as those belonging to the family Enterobacteriaceae. About 63.41% of the female patients belonged to the age group 21-40 yr, while men in the age group of 41-80 yr, were found to be more prone to UTI infections (68.4%). Minimum inhibitory concentration (MIC) of the isolates against ciprofloxacin, norfloxacin, ofloxacin and levofloxacin was determined. About 93% of the isolates were resistant to all the antibiotics. Further, 16% of the isolates exhibited MICs higher than 1000 mg/l against ciprofloxacin and 59% against norfloxacin. Keywords: Enteric, quinolones, resistance, urinary tract infection Introduction Urinary tract infection (UTI) is the colonization of microorganisms in urinary tract, predominantly caused by bacteria. UTI is existent in all age groups and has become as common as cold. About 60-80% of complicated and hospital acquired UTIs are caused by Gram-negative species 1,2. The occurrence of UTI varies with age and gender. Women are more inclined to develop UTI due to their anatomical features like short urethra, and other factors like pregnancy, use of diaphragms and sexual activity 3-5. Majority of the UTIs in elderly men are complicated owing to irregularities in the functioning of urinary tract like prostatic hypertrophy. Treatment of complicated UTIs becomes difficult when accompanied by risk factors like renal failure, calculi, catheters, renal transplantation, pregnancy and, most importantly, antimicrobial resistance 2,6. It is important to know the clinical history of patients as well as the microbial resistance pattern of a community before prescribing the antibiotics. The EAU (European Section for Infection in Urology) has described a new system for diagnosis whereby risk factors are detected to accomplish effective treatment. *Author for correspondence: Mobile: +91-9416768062; Fax: +91-1731274375 beniwalvikash@gmail.com Pregnancy, sexual behaviour, hormonal imbalance, diabetes, polycystic nephropathy, nephritis, catheters etc. are some of the risk factors that are to be considered in an UTI patient before prescribing antibiotics 7. The use of quinolone antibiotics for the treatment of complicated and uncomplicated urinary tract infections globally has led to the emergence and spread of resistance among enteric pathogens, especially Escherichia coli 8. Quinolones are a family of broad-spectrum antibiotics that kill bacteria by inhibiting DNA replication. Mutations in chromosomal genes that lead to alterations in the drug targets are found to be the major cause of quinolone resistance encountered in clinical isolates. Other mechanisms include overexpression of efflux pumps and plasmids that carry resistant genes 9. Ciprofloxacin and levofloxacin are the most commonly prescribed quinolones for UTI treatment 10, while norfloxacin is suggested for long term prophylaxis 11. Quinolones are recommended for cystitis and hospital acquired UTIs and also for patients with pyelonephritis if E. coli resistance is below 10% 7. Goettsch et al 12 correlated increased norfloxacin resistance by E. coli with increased medicament of quinolones for UTI patients in the Netherlands. It was also reported that resistance was high in elderly patients possibly due to cumulative exposure during their lifetime. Ciprofloxacin
168 INDIAN J BIOTECHNOL, APRIL 2015 resistance was observed to be high among women who had an earlier episode of UTI 13. In France, E. coli resistance was noticed against ofloxacin and ciprofloxacin in patients with acute pyelonephritis 14. High norfloxacin resistance among Gram-negative bacteria has also been reported in India, as it is routinely prescribed for UTIs 15. A detailed study in South India has revealed high resistance pattern to ciprofloxacin among the different members of Enterobacteriaceae other than E. coli, such as Klebsiella and Citrobacter species. A recent study in Jaipur showed E. coli as the most frequently isolated pathogen among UTI patients. It was also reported that females in the reproductive age group and elderly males were most susceptible to UTI 16. Quinolone resistance was reported to be higher for elderly patients, especially those with complications 17. However, this was not the condition a few years back. E. coli was sensitive to ofloxacin and norfloxacin and quinolones were preferred for empirical treatment when resistance to other antibiotics made treatment challenging 18,19. Presently, the steep rise in resistance pattern due to inappropriate usage has made quinolones ineffective. Antimicrobial susceptibility patterns may differ between geographical areas and it is imperative to be educated about the occurrence of uropathogens, their distribution in males and females, and their levels of resistance to quinolones 20. Keeping this in view, the aim of the present study was to determine the quinolone resistance pattern among enteric pathogens isolated from patients with UTI from a local hospital in Ambala, Haryana (India). Four quinolones, viz., norfloxacin (second generation-class I), ciprofloxacin, ofloxacin (second generation-class II) and levofloxacin (third generation), were selected to analyze their optimal activity towards uropathogens Materials and Methods Population under Study The present study was conducted on 70 patients who attended MMU Medical College, Mullana, Ambala from October 2012 to September 2013. Patient-specific data collected was age and gender. UTI patients were classified 3 age groups, 20, 21-40 and 41-80 yr. Collection and Processing of Samples Collection of samples was done in the Microbiology Laboratory of MMU Medical College. All the media used were obtained from HiMedia Laboratories Pvt. Ltd. The samples were inoculated onto cysteine lactose electrolyte deficient (CLED) media and MacConkey plates, followed by incubation for 24 h at 37 C. Identification of Enteric Pathogens The isolates were selectively plated onto Hi Touch Hexa Crome Flexi Plates (used for differentiation of Enterobacteriaceae), UTI plates and eosin blue methylene (EMB) plates for confirmation between lactose fermenting and non-lactose fermenting strains. Other biochemical tests done included triple sugar iron (TSI agar slants), mannitol motility nitrate (MMN medium) and amino acid decarboxylation (arginine, lysine and ornithine discs). Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing was performed by agar dilution method on Muller Hinton agar to determine the minimum inhibitory concentration (MIC) at varying concentrations of quinolone antibiotics as per guidelines 21. The MICs for each isolate were determined as per the interpretive standards defined by Clinical and Laboratory Standards Institute M100-S23 22. The antibiotics were obtained from Cipla Ltd, Mumbai. Statistical Analysis Statistical analysis was done by using SPSS statistical software, Release version 20. The results were considered significant if the probability was less than 5% (P < 0.05). Results Of 70 urine samples tested, 60 isolates (41 females/ 19 males) were identified as Gram-negative enteric pathogens and were used for further study. Females in the reproductive age group of 21-40 yr constituted 63.41% (26) of the total female population, while the larger fraction in males (68.4%) was made up of elderly patients in the age group of 41-80 yr. Table 1 outlines the age and gender wise distribution of uropathogens isolated from UTI patients in MMU Medical College, Mullana. It was observed that females were more susceptible to E. coli infection, while males were liable to Klebsiella and Enterobacter species infections. The percentage of pathogen occurrence is depicted in Fig. 1. E. coli was found to be the major uropathogen (56%). The distribution of pathogens in 41 females/19 males was: E. coli (68.3/31.6%)>Klebsiella spp. (7.3/31.6%)>Enterobacter spp. (7.3/21%)>non-lactose fermenters (NLFs) (9.8/10.5%)>Citrobacter spp. (7.3/5.3%).
VARUGHESE & BENIWAL: HIGH QUINOLONE RESISTANCE PATTERN IN ENTERIC PATHOGENS 169 Enteric pathogen Table 1 Age and gender wise distribution of uropathogens isolated from UTI patients Female Age group Male Age group 20 yr 21-40 yr 41-80 yr 20 yr 21-40 yr 41-80 yr Escherichia coli (34) 2.9 53 26.47-2.94 14.7 Klebsiella spp. (9) - 22.2 11.1 11.1 33.3 22.2 Enterobacter spp.(7) - 28.5 14.3 14.3 14.3 28.5 Citrobacter spp. (4) - 50 25 - - 25 NLFs (6) - 50 16.6 - - 33.3 p-values 0.374 0.003 0.003 0.182 0.188 0.001 Enteric pathogens No. of isolates (n=60) Table 2 Frequency of enteric pathogens resistant to quinolones Resistance to quinolones Ciprofloxacin Levofloxacin Ofloxacin Norfloxacin Escherichia coli (34) 91 88 91 88 Klebsiella spp.( 9) 100 100 100 100 Enterobacter spp.(7) 86 86 86 86 Citrobacter spp.(4) 100 100 100 100 NLFs (6) 83 83 83 83 p-values 0.00 0.00 0.00 0.00 n=total sample size; Susceptibility criteria of CLSI M100-S23 Fig. 1 Frequency distribution of enteric pathogens from UTI patients. The antimicrobial resistance pattern of all the isolates against the selected quinolones is shown in Table 2. Only 7% of the isolates were sensitive, while the majority of isolates (93%) were resistant to all the quinolones. Klebsiella and Citrobacter isolates were found to be 100% resistant against all the 4 quinolones studied. E. coli showed 90% resistance against ciprofloxacin and ofloxacin. Among the resistant uropathogens, 16 and 59% of the isolates exhibited MICs higher than 1000 mg/l against ciprofloxacin and norfloxacin, respectively. Further, Fig. 2 Resistance patterns of uropathogens. [Pattern: P1, N>C>O>L; P2, N>C>O=L; P3, N>C=O>L; P4, N=C>O>L; P5, N>C>L>O; P6, O>C; P7, N>O>C=L; P8, N>O>L>C; P9, N>O>C>L; P10- N>C=O] 100% of Klebsiella and 52% of E. coli strains exhibited MICs above 1000 mg/l for norfloxacin. These high rates of resistance were noted primarily among women (48%) in the age group of 21-40 yr, followed by elderly patients (39%) in the age group of 41-80 yr. About 56 UTI pathogens expressed 10 different resistance patterns (Fig. 2). Resistance towards ciprofloxacin was found to be lower than that towards norfloxacin in 86% of the strains. Similarly, resistance towards ofloxacin was higher than
170 INDIAN J BIOTECHNOL, APRIL 2015 levofloxacin resistance in 89% of strains. The patterns were arranged in decreasing order of resistance for each strain. About 5 resistance patterns were observed in E. coli. A single pattern N>C>O>L (norfloxacin>ciproflocain>ofloxacin>levofloxacin) was followed in all the five strains (54%). It was not unusual to see 58% E. coli isolates comprised with this pattern. Discussion The present study investigated the distribution and quinolone resistance pattern of pathogens isolated from UTI patients in MMU Medical College, Mullana, Haryana. Gram-negative bacilli, especially members of Enterobacteriaceae have been reported to be responsible for UTI infections in many parts of the world, like Latin America, Europe and India. E. coli has been stated to be the number one causative agent of UTIs all over the world 23. Of the 70 samples that we collected from UTI patients, 60 Gram-negative enteric pathogens were identified. E. coli was found to be the most prevalent in distribution pattern (57%). Other significant isolates included Klebsiella (15%), Enterobacter (12%), non-lactose fermenters (11.7%) and Citrobacter (6.7%). This trend in distribution was also observed from other parts of India 4,16. Distribution patterns on the basis of age and gender have significantly (p 0.005) proved that women in the reproductive age group (21-40 yr) as well both males and females in age group of 41-80 yr are susceptible to UTI infections. Anatomical factors and pregnancy are common causes for incidence of UTI in young women 3, while diabetes, kidney failure and use of catheters increase the chances of infection in older women. Prostrate infections, urinary stone and use of catheters are to blame for UTIs in males who are in the age group of 41-80 yr (p 0.005) 2. Several reports have expressed concern over the increase in resistance rates to quinolones 8,16. In our study, resistance was reported in 93% of the isolates and 92% of the isolates were resistant to all the four quinolones. Isolates with high resistance levels (>1000 mg/l) against ciprofloxacin (16%) and norfloxacin (59%) were isolated from women (21-40 yr) and elderly patients (41-80 yr). Our findings are consistent with other studies that associate increased resistance to increased prescription of quinolones 13,15. Of the 10 resistance patterns that were seen in 60 isolates (Fig. 2), N>C>O>L was followed in isolates from all the 5 species (54%). The second common pattern was N>C>O=L, although not observed in Klebsiella spp. These patterns support the high resistance trend that was visible throughout our study. Another interesting finding was that each species had its own individual resistance pattern. Our study generated data that could be used for local intervention and surveillance, as well for diagnosis of UTIs. Conclusion Bacterial resistance profile to quinolones was found to be quite high in the present study emphasizing the need to educate the public about ideal use of antimicrobials. Alternate therapy will also help to prevent high and multi-drug resistance. 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