Mutations In Quinolone Resistance-determining Regions Of gyrA Research Articles

Changing trends of antimicrobial susceptibility and resistance mechanisms to quinolones in typhoidal salmonellae isolated from India in last 5 years

Background: Antimicrobial resistance in enteric fever is a major therapeutic challenge and there is a need to monitor the pattern of resistance to the antityphoidal agents and detection of molecular mechanisms of resistance.The study was designed to monitor the antimicrobial susceptibility of Salmonella enterica serovars Typhi and Paratyphi A isolated from patients of enteric fever during 2014 to 2019 and detection of antimicrobial resistance mechanisms to quinolones. Methods and materials: Total 281 strains isolated from January 2014 to September 2019 were selected for the study. Antimicrobial susceptibility was done as per CLSI guidelines (2019) for amoxicillin, chloramphenicol, co-trimoxazole, ciprofloxacin, ceftriaxone and azithromycin. Minimum inhibitory concentration (MIC) determination for ciprofloxacin, ceftriaxone and azithromycin were done by E-Test method according to manufacturer's (ABs Biodisk, Sweden) instructions. DNA sequencing was done for gyrA, gyrB, parC and parE genes to find mutations in quinolone resistance determining region (QRDR). Results: Out of total 281 strains included in the study 214 (76.2%) were S. Typhi and 67 (23.8%) were S. Paratyphi A. As per CLSI 2019, antimicrobial susceptibility for chloramphenicol, ampicillin and cotrimoxazole was 0.7% (2/281), 1.42% (4/281) and 1.06% (3/281) respectively. Only 6/214 (2.8%) were susceptible for S. Typhi and no strain was found to be susceptible in S. Paratyphi A. Azithromycin susceptibility was 100% in S. Typhi, for S. Paratyphi there is no breakpoint available. Ciprofloxacin MIC50 and MIC90 values found to be 0.25 μg/mL and 1 μg/mL in S. Typhi and 0.5 μg/mL and 6 μg/mL in S. Paratyphi A. Ceftriaxone MIC50 and MIC90 values found to be 0.023 μg/mL and 0.5 μg/mL in both S. Typhi and S. Paratyphi A. For azithromycin MIC50 and MIC90 were 2 μg/mL and 6 μg/mL for S. Typhi and 4 μg/mL and 12 μg/mL for S. Paratyphi A respectively. All quinolone resistance strains had mutations in QRDR of gyrA and parC genes. Conclusion: Ciprofloxacin is no longer a drug of choice for treatment of enteric fever in India. Although susceptibility to ceftriaxone is 100% but MIC is creeping towards resistance. Azithromycin can be a promising treatment option for uncomplicated enteric fever.

Co-Incidence of Type II Topoisomerase Mutations and Efflux Expression in High Fluoroquinolone Resistant Enterococcus faecalis Isolated from Urinary Tract Infections.

IntroductionEnterococcus faecalis is one of the most common pathogens in urinary tract infections (UTIs). Fluoroquinolones have been frequently used to treat E. faecalis UTIs, and the emergence of fluoroquinolone-resistant E. faecalis strains has recently been reported in several countries. This study aimed to elucidate the mechanisms involved in fluoroquinolone resistance in clinical E. faecalis isolates by analyzing mutations in quinolone- resistance-determining regions (QRDRs) of gyrA and parC and investigating the role of some efflux pumps.MethodsIn total, 70 clinical E. faecalis isolates collected from UTIs were identified by phenotypic and genotypic methods. Antimicrobial susceptibility testing was performed and multidrug-resistant (including ciprofloxacin resistant) isolates were studied for minimum inhibitory concentrations to ciprofloxacin, levofloxacin, and ofloxacin. In the following, mutations in QRDRs of gyrA and parC and expression of EfrA, EfrB, and EmeA efflux pumps were investigated in 20 high-level ciprofloxacin resistant and two ciprofloxacin susceptible isolates.ResultsHigh-level resistance to ciprofloxacin was detected in 97.5% of isolates. Sequencing of QRDRs revealed that 65% and 75% of isolates carried mutations in gyrA and parC, respectively. The presence of efflux genes was detected in all studied isolates, but expression of efrA, emeA, and efrB was demonstrated in 50%, 40%, and 30% of resistant isolates, respectively. Neither QRDR mutation nor the expression of efflux genes showed any significant association with MIC.ConclusionCo-incidence of mutation and efflux gene expression in more than half of isolates (13/20) suggests that both mechanisms may play a role in fluoroquinolone resistance. The other unknown mechanisms including different efflux pumps and probably other QRDRs mutations may contribute to fluoroquinolone resistance in E. faecalis.

High prevalence of fluoroquinolone-resistant Escherichia coli strains isolated from urine clinical samples.

SummaryBackgroundFluoroquinolone resistant Escherichia coli isolates have become an important challenge in healthcare settings in Iran. In this study, we have determined Fluoroquinolone resistant E. coli isolates (from both outpatients and inpatients) and evaluated mutations of gyrA and parC within the quinolone resistance-determining regions (QRDR) of these clinical isolates.Materials and methodsClinical isolates were recovered from the urine sample of patients with urinary tract infections admitted at Alzahra hospital, Iran, between September and February 2013. We assessed antimicrobial susceptibility of all isolates and determined mutations in QRDR of gyrA and parC genes from 13 fluoroquinolone-resistant isolates by DNA sequencing.ResultsA total of 135 E. coli strains were obtained from 135 patients (91 outpatients and 44 inpatients). The resistance rate of fluoroquinolones (Ciprofloxacin, Norfloxacin and Ofloxacin) among our strains was 45.2%. Two E. coli isolates were shown just a single mutation, but other isolates possessed 2-5 mutations in gyrA and parC genes. Mutations in the QRDR regions of gyrA were at positions Ser83 and Asp87 and parC at positions Ser80, Glu84, Gly78.ConclusionsCiprofloxacin is the most common antimicrobial agent used for treating urinary tract infections (UTIs) in healthcare settings in Iran. Accumulation of different substitutions in the QRDR regions of gyrA and parC confers high-level resistance of fluoroquinolones in clinical isolates.

Detection of mutations in gyrB using denaturing high performance liquid chromatography (DHPLC) among Salmonella enterica serovar Typhi and Paratyphi A.

Fluoroquinolone resistance is mediated by mutations in the quinolone-resistance determining region (QRDR) of the topoisomerase genes. Denaturing high performance liquid chromatography (DHPLC) was evaluated for detection of clinically important mutations in gyrB among Salmonella. Salmonella Typhi and S. Paratyphi A characterised for mutation in QRDR of gyrA, parC and parE were studied for mutation in gyrB by DHPLC and validated by sequencing. The DHPLC analysis was able to resolve the test mutant from isolates with wild type gyrB and distinguished mutants from other mutant by peak profile and shift in retention time. Three sequence variants were detected at codon 464, and a novel mutation Ser→Thr was also detected. gyrB mutation was associated with non classical quinolone resistance (NALS-CIPDS) in 34 isolates of S. Typhi only and was distinct from classical quinolone resistance associated with gyrA mutations (NALR-CIPDS). DHPLC is effective for the detection of mutation and can reduce the need for sequencing to detect clinically significant gyrB mutations. KF993966, KF993965 and KF993964.

Molecular characterization and antimicrobial resistance properties of uropathogenic Escherichia coli isolated from elderly patients in a district tertiary hospital in shanghai

Objective To investigate the molecular epidemiology and antimicrobial resistance status of uropathogenic Escherichia coli (UPEC) in senior population in Putuo District, Shanghai. Methods A total of 72 UPEC strains were isolated from elderly inpatients with urinary tract infections in Putuo Hospital, Shanghai University of Traditional Chinese Medicine from January 2013 to March 2015. The strains were characterized by multi-locus sequence typing (MLST). The β-lactamase gene and the plasmid mediated quinolone resistance (PMQR) gene were detected, and the mutations of quinolone resistance-determining regions (QRDR) in gyrA and parC genes were demonstrated. In vitro drug susceptibility test was performed. Continuous variables were compared using t test and categorical variables were compared using chi-squared test or Fisher exact test. Results The UPEC strains showed different resistance rates to ciprofloxacin, cefotaxime and trimethoprim-sulfamethoxazole, which were 76.4%, 73.6% and 65.3%, respectively. UPEC still remained highly sensitive to imipenem, meropenem, amikacin and piperacillin-tazobactam. Among 72 isolates, 55(76.4%) of 49(68.1%) extended-spectrum β-lactamase (ESBL)-positive strains harbored blaCTX-M genes. Among the 55 ciprofloxacin resistant strains, 51 (92.7%) had three or four mutations in QRDR of gyrA and parC genes. The hot-spot mutations of QRDR were located at amino acid position 83 and 87 in gyrA gene and at positions 80 and 84 in parC gene. Forward analysis by MLST showed that the most frequent sequence types (ST) were ST131 (18/72, 25.0%), ST1193(7/72, 9.7%), ST405 (7/72, 9.7%), ST38 (5/72, 6.9%) and ST648 (3/72, 4.2%). ST131 isolates were predominant in ST which caused community-onset urinary tract infections. Multiple drug-resistance were detected in ST131, ST405, ST38 and ST648 which were mainly producing blaCTX-M ESBL. Conclusions Community-acquired multiple drug-resistant UPEC strains such as ST131 clone are prevalent in elderly patients. Thus, monitoring of molecular epidemiology would be beneficial to prevent the prevalence of multiple drug-resistant UPEC. Key words: Uropathogenic Escherichia coli; Elderly patients; MLST; Antimicrobial resistance; gyrA; parC; ESBL; ST131

In vitro selection of resistance to pradofloxacin and ciprofloxacin in canine uropathogenic Escherichia coli isolates

This study explored and compared the mechanisms and selective concentration of resistance between a 3rd (pradofloxacin) and 2nd (ciprofloxacin) generation fluoroquinolone. Pradofloxacin- and ciprofloxacin-resistant mutants were selected by stepwise exposure of Escherichia coli (E. coli) to escalating concentrations of pradofloxacin and ciprofloxacin. The sequence of the quinolone resistance determining region (QRDR) and the transcriptional regulator soxS were analyzed, and efflux pump AcrAB-TolC activity was measured by quantitative real-time reverse transcription-PCR (qRT-PCR). First-step mutants reduced the fluoroquinolone sensitivity and one mutant bore a single substitution in gyrA. Four of six second-step mutants expressed ciprofloxacin resistance, and displayed additional mutations in gyrA and/or parC, while these mutants retained susceptibility to pradofloxacin. All the third-step mutants were fluoroquinolone resistant, and each expressed multidrug resistance (MDR) phenotypes. Further, they displayed resistance to all antibacterials tested except cefotaxime, ceftazidime and meropenem. The number of mutations in QRDR of gyrA and parC correlated with fluoroquinolone MICs. Mutations in parC were not common in pradofloxacin-associated mutants. Moreover, one second- and one third-step ciprofloxacin-associated mutants bore both mutations at position 12 (Ala12Ser) and 78 (Met78Leu) in the soxS gene, yet no mutations in the soxS gene were detected in the pradofloxacin-selected mutants. Altogether, these results demonstrated that resistance emerged relatively more rapidly in 2nd compared to 3rd generation fluoroquinolones. Point mutations in gyrA were a key mechanism of resistance to pradofloxacin, and overexpression of efflux pump gene acrB played a potential role in the emergence of MDR phenotypes identified in this study.

Assessing the molecular basis of transferable quinolone resistance in Escherichia coli and Salmonella spp. from food-producing animals and food products

Enterobacteriaceae resistant to quinolones frequently arise in animals, being easily disseminated through the food-chain. The aim of this study was to investigate the presence of plasmid-mediated quinolone resistance (PMQR) determinants in Salmonella spp. (n=183) and Escherichia coli (n=180) isolates, collected from food-producing animals and food products among swine, poultry, rabbits and cattle. All isolates were subjected to antimicrobial susceptibility testing and molecular screening of PMQR determinants. β-Lactamase-encoding genes, and the quinolone resistance determining region (QRDR) of gyrA, gyrB, parC and parE genes were also investigated in PMQR-positive isolates. Plasmid characterization was performed by conjugation, followed by replicon-typing. Genetic relatedness of PMQR-positive E. coli was examined by Multilocus Sequence Typing, while Salmonella was previously serotyped. The association of mobile genetic elements and PMQR was investigated through PCR mapping assays. Overall, 4.1% (15/363) isolates harbored qnrB2 (n=3), qnrB19 (n=3), and qnrS1 (n=9) genes. All but one isolate presented one to four mutations in QRDR of gyrA or parC genes, which is consistent with the range of MIC values detected (0.19–64mg/L) for ciprofloxacin; 60% (9/15) of qnr-harboring isolates were non-susceptible to β-lactam antibiotics which was justified by the presence of β-lactamases from TEM (TEM-1, n=8; TEM-135, n=1) and SHV (SHV-108, n=1) families. Analysis of mobile genetic elements revealed that qnr genes were detected nearby relevant genetic elements like intI1, ISEcl2, IS26 and ISCR1 and enclosed in diverse Inc. type plasmids. This study illustrated the existence of Qnr-producing E. coli and Salmonella from food-producing animals, associated to specific mobile elements that might mediate their transference between species and among distinct settings.

Detection of mutations in the gyrA and parC genes in Escherichia coli isolates carrying plasmid-mediated quinolone resistance genes from diseased food-producing animals

In this study, the prevalence of plasmid-mediated quinolone resistance (PMQR) was investigated in 495 Escherichia coli isolates from diseased food-producing animals in Guangdong province, China. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analysed for mutations in 55 isolates harbouring only oqxAB and all isolates harbouring other PMQR genes. Overall, 282 (57.0 %) E. coli isolates had at least one PMQR gene. oqxAB was detected in 215 isolates and predominated the PMQR genes, followed by qnrS (63 isolates), aac(6')-Ib-cr (56 isolates), qnrB (39 isolates) and qepA (18 isolates). qnrA, qnrC and qnrD were not found in any of the isolates. The rates of resistance to ciprofloxacin, enrofloxacin, levofloxacin and nalidixic acid were 75.2, 81.0, 70.5 and 97.4 %, respectively, among the 495 isolates. Eight types of mutation in gyrA were detected in 154 PMQR-positive isolates, and 147 isolates were found to have mutations in parC. PFGE analysis indicated that the PMQR-positive E. coli isolates were genetically diverse. This study demonstrated that the number of mutations in QRDRs of gyrA and/or parC was significantly associated with the MICs of quinolones (P<0.01). The rates of resistance to ciprofloxacin, enrofloxacin and nalidixic acid in PMQR-positive isolates were significantly higher than those in PMQR-negative isolates (P<0.05). In addition, the prevalence of oqxAB had significant Spearman correlation coefficients in relation to the MICs of all four tested quinolones (P<0.01).

Characterization ofEscherichia coliIsolates from Healthy Food Handlers in Hospital

Recent studies have reported that Escherichia coli in fecal samples of healthy humans could also serve as important reservoirs of drug-resistant bacteria. Limited data are available for E. coli-resistant profiles of healthy food handlers in hospitals who provide food service to inpatients and hospital staffs. E. coli isolates were recovered from hospital healthy food handlers, and one random selected isolate from each food handler was subjected to antimicrobial susceptibility testing, phylogenetic typing, and screening for antimicrobial-resistant mechanisms by polymerase chain reaction amplification. Ciprofloxacin-resistant isolates were further characterized by mutation analysis in the quinolone resistance determining regions (QRDRs) of GyrA and ParC. And extended-spectrum β-lactamase (ESBL) producing isolates were screened for bla(CTX-M) by polymerase chain reaction amplification and DNA sequence analysis. In total, more than 50% (47/92) of E. coli isolates from healthy food handlers showed multidrug-resistant profiles and 50% (46/92) isolates carried intI. Resistance prevalence of the B2 phylogenetic group was significantly lower than that of the non-B2 groups for all tested antimicrobials (p < 0.05) except chloramphenicol and tetracycline. Seven isolates of phylogenetic group A (n = 3) and D (n = 4) produced ESBL, and 12 isolates of phylogenetic group A (n = 5), B2 (n = 2), and D (n = 5) were resistant to ciprofloxacin. Transferable quinolone resistance determinants were identified in four isolates. Point mutations in QRDRs of GyrA or ParC were identified among 59 out of 62 E. coli isolates showing decreased susceptibility or resistance to ciprofloxacin. Genes encoding CTX-M enzyme were identified in seven ESBL-producing isolates. The preponderance in hospital food handlers of multidrug-resistant E. coli makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food service and limit inpatient therapeutic options.

Prevalence and Antimicrobial Susceptibility ofCampylobacter coliIsolates from Swine

Swine is a common source of Campylobacter coli human gastroenteritis, for the treatment of which erythromycin and fluoroquinolones are recommended. The prevalence of antimicrobial-resistant C. coli differs significantly depending on countries. We investigated the prevalence of C. coli in swine from a farm in Buan-gun, Korea in 2010, and determined antimicrobial susceptibility of the isolates. Rectal swab specimens were used to inoculate Campylobacter Preston media and incubated microaerophilically at 42℃ for 48 h. The species were identified by phenotypic tests and by detecting hipO and glyA genes. PCR was used to detect mutations of A2074C in 23S rRNA gene, and quinolone resistancedetermining region (QRDR) of gyrA, which are associated with high level resistance to erythromycin, and with ciprofloxacin, respectively. Antimicrobial susceptibility was determined by the disk diffusion and agar dilution tests. Of the 100 specimens, 55 (55%) yielded C. coli, and 23 of them (41.8%) had A2074G mutation. A2074G mutated isolates showed the lowest MIC90 of imipenem, while those of ampicillin and clindamycin were relatively low. The majority of both A2074G mutation-positive and -negative isolate were susceptible to ampicillin, cefotaxime, and chloramphenicol. All isolates were resistant to ciprofloxacin, and had mutation in QRDR of gyrA. In conclusion, C. coli was detected in 55% of swine, and A2074G mutation was detected in 41.8% of the isolates. All isolates had gyrA mutation-mediated ciprofloxacin resistance.

First Molecular Characterization of Fluoroquinolone Resistance in Aerococcus spp

Aerococcus urinae and Aerococcus sanguinicola are emerging Gram-positive pathogens responsible for urinary tract infections, especially in elderly patients (2). Although they seem to be intrinsically susceptible to fluoroquinolones (3, 8, 10), acquired fluoroquinolone resistance has not been yet reported. Resistance to fluoroquinolones in Gram-positive bacteria is mainly due to point mutations in the quinolone-resistance determining regions (QRDRs) of the GyrA and GyrB subunits of the DNA gyrase and QRDRs of ParC and ParE subunits of the topoisomerase IV (4). Decreased accumulation of fluoroquinolones is a second resistance mechanism that is mediated by the overexpression of efflux pump systems (4). Since QRDR sequences of A. urinae and A. sanguinicola are not available, the aim of this study was to elucidate the mechanisms associated with the fluoroquinolone resistance. Nineteen A. urinae and 8 A. sanguinicola urinary isolates, previously identified by 16S rRNA sequencing, were studied (2). The MICs of ofloxacin, ciprofloxacin, levofloxacin, and moxifloxacin were established using the Etest method (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar supplemented by 5% horse blood. The MICs of ciprofloxacin were also determined in the presence of reserpin, an efflux pump inhibitor, incorporated in the medium (10 μg/ml) (11). Against A. urinae isolates, moxifloxacin (MIC50, 0.12 μg/ml) was 4- and 16-fold more active than ciprofloxacin/levofloxacin (MIC50, 0.5 μg/ml) and ofloxacin (MIC50, 2 μg/ml), respectively (Table ​(Table1).1). Against A. sanguinicola isolates, moxifloxacin (MIC50, 0.25 μg/ml) was also 4- and 16-fold more active than ciprofloxacin/levofloxacin (MIC50, 1 μg/ml) and ofloxacin (MIC50, 4 μg/ml), respectively (Table ​(Table1).1). The potent activity of moxifloxacin against Aerococcus spp. is concordant with data previously reported (8). Finally, active efflux did not seem to play a major role in fluoroquinolone resistance in aerococci, since the MICs of ciprofloxacin were similar in the absence or presence of reserpin (Table ​(Table11). TABLE 1. Susceptibility to fluoroquinolones and mutations in QRDRs of gyrA, gyrB, parC, and parE genes of A. urinae and A. sanguinicola Following the use of degenerate primers, the DNA fragments corresponding to QRDRs of GyrA, GyrB, ParC, and ParE were amplified using standard PCR conditions with novel specific primers (Table ​(Table2).2). The sequences of GyrA, GyrB, ParC, and ParE of A. urinae were 100%, 98%, 88%, and 93% identical to those of A. sanguinicola, respectively. In A. urinae, a serine residue and a glutamate residue were found at positions 84 and 88 (corresponding to 83 and 87 in Escherichia coli numbering) in GyrA and also at positions 79 and 83 (corresponding to 80 and 84 in E. coli numbering) in ParC, as described in Enterococcus faecalis (9). The unique difference with A. sanguinicola was the presence of an aspartate residue at position 83 in ParC, as described in Streptococcus pneumoniae (9). Whereas all susceptible strains possessed no mutation, at least one mutation was found in gyrA and/or parC in all four resistant strains (Table ​(Table1).1). Except for S79T, similar amino acid changes in hot spot positions of ParC have been identified in other Gram-positive bacteria; these are S79R in E. faecalis and Enterococcus faecium, E83K in E. faecium and Staphylococcus aureus, and D78N and D83G in S. pneumoniae (1, 5, 7, 9). Concerning GyrA, an identical mutation (S84L) has also been identified in S. aureus, E. faecium, and Streptococcus agalactiae (6, 9). These findings suggest that topoisomerase IV seems to be the primary target of fluoroquinolones in Aerococcus spp., as previously described in other Gram-positive bacteria (4, 9). TABLE 2. Deoxynucleotide primers used in this study

Molecular analysis of fluoroquinolone-resistance in Escherichia coli on the aspect of gyrase and multiple antibiotic resistance (mar) genes.

We analyzed the fluoroquinolone resistance mechanism of 28 isolates of ciprofloxacin-resistant E. coli from patients who received ciprofloxacin as a regimen of a selective gut decontamination. Isolates distinctive by infrequent restriction site polymerase chain reaction (IRS-PCR) were subjected to Hinf I restriction fragment length polymorphism analysis, single-stranded conformation polymorphism (SSCP), and nucleotide sequencing of the quinolone resistance determining region (QRDR) in gyrA. Double mutations in QRDR of gyrA (Ser83 Leu and Asp87Asn) were found from most of the strains. Nucleotide sequencing of the marR locus showed that 18 out of 28 (64%) ciprofloxacin-resistant E. coli strains had three types of base change in marR loci: a double-base change at nucleotides 1628 and 1751, or 1629 and 1751: and a single-base change at 1751. However, all the mutated strains showed no tolerance to cyclohexane test, suggesting the mutation in the marR region had no influence on overexpression of the MarA protein. In conclusion, mutation in gyrA was the main mechanism of ciporfloxacin resistance in E. coli from patients with selective gut decontamination. Therefore, mutation in the mar region did not influence the levels of ciprofloxacin resistance in our isolates.