Conjugal transfer of PMQR from uropathogenic E.coli under high ciprofloxacin selection pressure generates gyrA mutation.

Incidence of high fluoroquinolone resistance has been rising rapidly worldwide. Resistance against fluoroquinolones can be either chromosomal or plasmid mediated. Plasmid mediated quinolone resistant(PMQR) genes impart low level of resistance against fluoroquinolones but provides favorable background for selection of additional chromosomally encoded resistance mechanisms. In the natural habitat, conjugal transfer of PMQR genes provides a vehicle for dissemination of fluoroquinolone resistance. Our study indicated successful transmission of PMQR determinants(aac(6')-Ib-cr, qnrA, qnrB, qnrS, oqxB) from ciprofloxacin resistant clinical uropathogenic E.coli(UPEC) isolates to susceptible E.coliJ53Azide-resistant strain in vitro in presence of high ciprofloxacin (5 µg/ml) selection pressure generating transconjugants that exhibited varied MIC(25-800µg/ml) towards the drug with acquired mutations Ser83Leu and Asp87Asn in the quinolone resistant determining regions(QRDR) in gyrA. Therefore this is a first study of its kind that identified high rate of gyrA mutations among transconjugants selected under high ciprofloxacin pressure resulting from bacterial conjugation a common phenomenon in natural habitat along with PMQR gene transmission which imposes a major public health concern.

Incidence and risk of co-transmission of plasmid-mediated quinolone resistance and extended-spectrum ß-lactamase genes in fluoroquinolone-resistant uropathogenic Escherichia coli: a first study from Kolkata, India.

OBJECTIVES: Co-resistance to fluoroquinolones and ß-lactams results in treatment complications for uropathogenic Escherichia coli (UPEC) infections. This study aimed to detect the coexistence and co-transmission of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum ß-lactamase (ESBL) genes in UPEC from Kolkata, India. METHODS: Escherichia coli was detected biochemically from culture-positive urine samples. Antimicrobial resistance and ESBL production were confirmed by disk diffusion assay. Transfer of PMQR and ESBL genes was performed using azide-resistant E. coli J53 as recipient. PCR was conducted to identify PMQR and ESBL genes, plasmid incompatibility types, insertion sequences, integrons and ERIC-PCR patterns. RESULTS: PMQR determinants were detected in 50.0% (35/70) of ciprofloxacin-resistant isolates, with ESBL production in 42.9% (15/35) and a ß-lactamase inhibitor-resistant phenotype in 51.4% (18/35). The highest co-occurrence (37.1%; 13/35) and co-transmission of aac(6')-Ib-cr with blaTEM, blaCTX-M and blaOXA was observed. Among the conjugal plasmids, replicon types FrepB/FrepB+F1B were predominant, with rare incidences of A/C, N, X, I1, FIIS, L/M and H1. Distribution of integrons and ISEcp1 and IS26, either alone or in combination, irrespective of PMQR and ESBL gene types was observed. Discrete ERIC-PCR profiles indicated that acquisition of PMQR and ESBLs and their dissemination may be attributed to horizontal gene transfer. CONCLUSION: This study demonstrates for the first time the risk of co-transmission of fluoroquinolone and ß-lactam resistance amongst UPEC from Kolkata, posing a major public-health threat and limiting treatment options. Monitoring at the molecular level is necessary to design appropriate prescription policies to combat the alarming rise in drug resistance amongst these uropathogens.

Phylogenetic background of E. coli isolated from asymptomatic pregnant women from Kolkata, India.

INTRODUCTION: Asymptomatic bacteriuria (ABU) in pregnancy generates medical complications. E. coli is the common etiologic agent responsible for ABU-associated infections. This study aimed to identify the phylogenetic background and drug resistance in asymptomatic E. coli from a pregnant population. METHODOLOGY: E. coli was confirmed biochemically from culture-positive urine samples collected from asymptomatic pregnant women. Phylogenetic typing was done by polymerase chain reaction (PCR). The isolates were subjected to antibiotic susceptibility testing and extended-spectrum beta-lactamase (ESBL) production. Statistical significance was determined using SPSS 17.0 software. RESULTS: Bacteriuria was observed in 113 (22.6%) of 500 asymptomatic pregnant females. E. coli was reported in 44 (38.9%) of 113 isolates. The mean age-wise distribution was 25.14 ± 4.63. Although pathogenic phylogroup B2 was predominant (54.5%), incidence of non-pathogenic phylogroup B1 (27.3%) was found to be statistically significant (p ≤ 0.001), and B1 and B2 were correlated with respect to total ABU population. Antibiotic sensitivity against ampicillin (34.1%), ceftazidime (50%), cefotaxime (47.7%), ciprofloxacin (47.7%), amikacin (86.4%), nitrofurantion (79.5%), and co-trimoxazole (36.4%) was observed. Multidrug resistance (MDR) and ESBL production was reported in 26 (59.1%) of 44 and 18 (69.2%) of the 26 MDR isolates, respectively. A significant distribution of phylogroup B1 (p = 0.03) with drug resistance was also observed. CONCLUSIONS: This is the first study that reported significant incidence of non-pathogenic phylogroup B1 in asymptomatic E. coli with high incidence of MDR isolated from pregnant women in Kolkata, India.  These varied resistance patterns present major therapeutic and infection control challenges during pregnancy.

Multidrug-Resistance and Extended Spectrum Beta-Lactamase Production in Uropathogenic E. Coli which were Isolated from Hospitalized Patients in Kolkata, India.

BACKGROUND AND OBJECTIVE: Urinary Tract Infections (UTIs) are mostly caused by Escherichia coli. The appropriate therapy demands a current knowledge on the antimicrobial susceptibility pattern amongst these pathogens, as an inappropriate use of antibiotics may lead to complications and treatment failure. The UTIs which are caused by multidrug resistant Extended-Spectrum Beta-Lactamase (ESBL) producing bacteria further pose a severe problem, as the treatment options are limited. The aim of this study was to identify the pattern of multi drug resistance amongst the uropathogenic E. coli (UPEC) isolates which were obtained from hospitalized patients. MATERIALS AND METHODS: Forty UPEC were isolated from 200 urine samples of hospitalized patients who were clinically suspected for UTIs. Antimicrobial susceptibility screening was performed by using 16 antibiotics, by the Kirby Bauer disk diffusion technique. The isolates which were resistant to the third generation cephalosporins were subjected to the ESBL confirmatory test by using drug and drug-inhibitor combination disks by following the CLSI guidelines. RESULTS: All the 40 isolates except three were multidrug resistant. They showed the highest sensitivities for nitrofurantoin (72.5%) and amikacin (70%). A high level of resistance was observed against ampicillin (97.5%), nalidixic acid and cefelexin (95%), amoxicillin (92.5%), cotrimoxazole (82.5%) and ciprofloxacin (80%) respectively. Thirty different antibiotic resistance patterns were observed against the different antibiotics. Twenty-eight out of the 40 isolates were resistant to the third generation cephalosporins. However, the phenotypic test for the ESBL confirmation indicated that eighteen out of the twenty-eight isolates were ESBL producers and that eleven different drug resistance patterns were observed amongst them. CONCLUSIONS: Therefore, this study accounts for the varied multidrug resistance pattern amongst the uropathogenic E. coli which were isolated from hospitalized patients in Kolkata, an eastern region of India. Nitrofurantoin and amikacin should be assigned as potent drugs to treat this infection in this region of the country. These varied resistance patterns present major therapeutic and infection control challenges and they suggest a heterogeneous population of the uropathogenic E. coli isolates which circulate in this sector of India.

Molecular Characterization of Uropathogenic Escherichia coli: Nalidixic Acid and Ciprofloxacin Resistance, Virulent Factors and Phylogenetic Background.

BACKGROUND AND OBJECTIVE: A proficient pathogen should be virulent, resistant to antibiotics, and epidemic. However, the interplay between resistance and virulence is poorly understood. Perhaps, the most commonly accepted view is that resistance to quinolones is linked to a loss of virulence factors. However, the low virulent phylogenetic groups may be more prone to acquire resistance to quinolones. The aim of this study was to identify and characterise the Nalidixic Acid (NA) and ciprofloxacin (CIP) resistant uropathogenic Escherichia coli (UPEC) isolates with respect to virulence and phylogenetic background, from hospital settings in Kolkata, an eastern region in India. Research based on these bacterial populations will help in understanding the molecular mechanisms underlying the association between resistance and virulence, that in turn, may help in managing the future disseminations of UTIs in their entirety. MATERIAL AND METHODS: One hundred and ten E. coli isolates were screened against NA and CIP using Kirby-Bauer disk diffusion technique, following CLSI guidelines. Prevalence of virulent factor genes and distribution of phylogenetic groups amongst the isolates was determined by PCR, using gene specific primers against the different virulent factors and DNA markers (chuA, yjaA and DNA fragment, TSPE4.C2) respectively. Statistical analysis of the data was performed using SPSS software. RESULTS: Resistance to both NA and CIP was reported in 75.5 % of the isolates which were analysed. The virulent determinants, papC, pap GII, papEF, afa, cnf1, hlyA and iroN were significantly predominant in the drug susceptible than the resistant isolates. A significant reduction of phylogroup B2 in NA (85.7% versus 64.6%, χ(2)P<0.001) and CIP (85.2 % versus 61.4%, χ(2)P<0.001) resistant UPEC isolates, followed by increase in predominance of non-B2 phylotypes (group D and group B1), were observed. CONCLUSION: This is the first report from India that has indicated possible evidence on horizontal gene transfer from pathogenic to commensal strains and selection of the latter, on extensive usage of this group of antimicrobials in hospital settings, where these drugs were routinely prescribed for treating urinary tract infection. Therefore, this information necessitates surveillance programs and administration of effective strategies, to put an end to random prescription policies involving this group of antimicrobials.

Effect of subinhibitory concentrations of ciprofloxacin on Mycobacterium fortuitum mutation rates.

OBJECTIVES: Fluoroquinolones have found a place in the management of mycobacterial diseases including tuberculosis. It has been previously shown that subinhibitory concentrations of quinolones increase the mutation rate in Escherichia coli and staphylococci. The purpose of this study is to extend this observation to mycobacteria and to quantify mutation rates. METHODS: The mutation rate in Mycobacterium fortuitum to ciprofloxacin, levofloxacin, moxifloxacin, rifampicin, erythromycin and gentamicin resistance was determined when grown with and without various sub-MIC concentrations of ciprofloxacin. RESULTS: M. fortuitum exposed to 1/2 MIC ciprofloxacin had an increase in the mutation rate of between 72- and 120-fold when selected on quinolones or other antimycobacterial antibiotics. Smaller, but significant increases in mutation rate were seen when the organism was exposed to lower concentrations (1/4 MIC and 1/8 MIC). CONCLUSIONS: These data show that sub-MIC concentrations of fluoroquinolone significantly increase mutation rates and these data suggest that care must be taken to ensure that bacteria are not exposed to subinhibitory concentrations when adding quinolones to a regimen used to treat mycobacterial infection.