Mechanisms of fluoroquinolone resistance among Escherichia coli isolates from urinary tract infections in Thailand.

Characterization of the quinolone resistance mechanism in foodborne Salmonella isolates with high nalidixic acid resistance

BackgroundFluoroquinolones are commonly recommended as treatment for urinary tract infections (UTIs). The development of resistance to these agents, particularly in gram-negative microorganisms complicates treatment of infections caused by these organisms. This study aimed to investigate antimicrobial resistance of different Enterobacteriaceae species isolated from hospital- acquired and community-acquired UTIs against fluoroquinolones and correlate its levels with the existing genetic mechanisms of resistance.MethodsA total of 440 Enterobacteriaceae isolates recovered from UTIs were tested for antimicrobial susceptibility. Plasmid-mediated quinolone resistance (PMQR) genes and mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were examined in quinolone-resistant strains.ResultsAbout (32.5%) of isolates were resistant to quinolones and (20.5%) were resistant to fluoroquinolones. All isolates with high and intermediate resistance phenotypes harbored one or more PMQR genes. QnrB was the most frequent gene (62.9%) of resistant isolates. Co-carriage of 2 PMQR genes was detected in isolates (46.9%) with high resistance to ciprofloxacin (CIP) (MICs > 128 μg/mL), while co-carriage of 3 PMQR genes was detected in (6.3%) of resistant isolates (MICs > 512 μg/mL). Carriage of one gene only was detected in intermediate resistance isolates (MICs of CIP = 1.5–2 μg/mL). Neither qnrA nor qnrC genes were detected. The mutation at code 83 of gyrA was the most frequent followed by Ser80-Ile in parC gene, while Asp-87 Asn mutation of gyrA gene was the least, where it was detected only in high resistant E. coli isolates (MIC ≥128 μg/mL). A double mutation in gyrA (Lys154Arg and Ser171Ala) was observed in high FQs resistant isolates (MIC of CIP < 128 μg/mL).ConclusionFQs resistance is caused by interact between PMQR genes and mutations in both gyrA and parC genes while a mutation in one gene only can explain quinolone resistance. Accumulation of PMQR genes and QRDR mutations confers high resistance to FQs.

Prevalence and Mechanism of Fluoroquinolone Resistance in Escherichia coli Isolated from Swine Feces in Korea

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6′)-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25–2 μg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 μg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25–2 μg/mL), thus providing insights into mechanisms underlying CIP resistance.

Urinary Tract Infections (UTIs) remain a significant global health concern, with the pathogen spectrum and antibiotic resistance patterns critically influencing clinical management. This study aimed to analyze the microbial profile of UTIs from 2022 to 2024, focusing on Escherichia coli (E. coli), the predominant pathogen, by evaluating its antibiotic resistance phenotypes, biofilm formation capability, and mutations in GyrA, ParC and Plasmid-Mediated Quinolone Resistance (PMQR) genes prevalence. Midstream urine samples from UTI patients were collected and analyzed for microbial identification. E. coli isolates were tested for antibiotic susceptibility, particularly to levofloxacin, the most frequently used antibiotic in our hospital. Biofilm formation was assessed, and mutations in GyrA, ParC and PMQR genes were sequenced to determine resistance mechanisms.The pathogen spectrum revealed that Enterobacteriaceae were the most prevalent (44.42%), with E. coli being the dominant species. Over 70% of E. coli isolates exhibited resistance to levofloxacin, and 58.97% (46/78) demonstrated biofilm-forming ability. Among levofloxacin-resistant strains, 75.64% (59/78) showed high-level resistance (MIC ≥ 8μg/mL). The most common mutations in GyrA were Ser83Leu (89.74%) and Asp87Asn (71.79%), while Ser80Ile (74.36%) was predominant in ParC. PMQR genes were detected in 17.95% (14/78) of isolates. The elevated prevalence of quinolone-resistant E. coli in urinary tract infections within this region, combined with intricate resistance gene mutations and generally strong biofilm-forming capabilities, underscores the critical necessity for rational antibiotic stewardship.

Emergence of a new mutation and its accumulation in the topoisomerase IV gene confers high levels of resistance to fluoroquinolones in Escherichia coli isolates

Escherichia coli is a common commensal bacterial species of humans and animals that may become a troublesome pathogen causing serious diseases. The aim of this study was to characterize the quinolone resistance phenotypes and genotypes in E. coli isolates of different origin from one area of the Czech Republic. E. coli isolates were obtained from hospitalized patients and outpatients, chicken farms, retailed turkeys, rooks wintering in the area, and wastewaters. Susceptibility of the isolates grown on the MacConkey agar with ciprofloxacin (0.05 mg/L) to 23 antimicrobial agents was determined. The presence of plasmid-mediated quinolone resistance (PMQR) and ESBL genes was tested by PCR and sequencing. Specific mutations in gyrA, gyrB, parC, and parE were also examined. Multilocus sequence typing and pulsed-field gel electrophoresis were performed to assess the clonal relationship. In total, 1050 E. coli isolates were obtained, including 303 isolates from humans, 156 from chickens, 105 from turkeys, 114 from the rooks, and 372 from wastewater samples. PMQR genes were detected in 262 (25%) isolates. The highest occurrence was observed in isolates from retailed turkey (49% of the isolates were positive) and inpatients (32%). The qnrS1 gene was the most common PMQR determinant identified in 146 (56%) followed by aac(6′)-Ib-cr in 77 (29%), qnrB19 in 41 (16%), and qnrB1 in 9 (3%) isolates. All isolates with high level of ciprofloxacin resistance (>32 mg/L) carried double or triple mutations in gyrA combined with single or double mutations in parC. The most frequently identified substitutions were Ser(83)Leu; Asp(87)Asn in GyrA, together with Ser(80)Ile, or Glu(84)Val in ParC. Majority of these isolates showed resistance to beta-lactams and multiresistance phenotype was found in 95% isolates. Forty-eight different sequence types among 144 isolates analyzed were found, including five major clones ST131 (26), ST355 (19), ST48 (13), ST95 (10), and ST10 (5). No isolates sharing 100% relatedness and originating from different areas were identified. In conclusion, our study identified PMQR genes in E. coli isolates in all areas studied, including highly virulent multiresistant clones such as ST131 producing CTX-M-15 beta-lactamases.

Impact on quinolone resistance of plasmid-mediated quinolone resistance gene and mutations in quinolone resistance-determining regions in extended spectrum beta lactamase-producing Klebsiella pneumoniae isolated from urinary tract infection patients.

Molecular characterization of fluoroquinolone-resistant Escherichia coli from broiler breeder farms

Sir, Serratia marcescens, once considered to be an innocuous and non-pathogenic organism, is now an important cause of hospital-acquired infections. This organism is associated with respiratory tract infections, urinary tract infections, septicemia, meningitis, and wound infections [1, 2]. S. marcescens infections are difficult to treat because of high resistance to a wide variety of antibiotics, including cephalosporins, fluoroquinolones, and aztreonam [2]. Fluoroquinolones are broad-spectrum bactericidal antimicrobial agents that are used to treat various bacterial infections. Although S. marcescens infections are frequently treated with fluoroquinolones, the incidence of fluoroquinolone resistance continues to increase in clinical settings in China [2]. Fluoroquinolone resistance is mainly caused by chromosomal mutations affecting the quinolone resistance-determining region (QRDR) of gyrA and gyrB, which encode DNA gyrase subunits, and parC and parE, which encode topoisomerase IV subunits [3]. Moreover, plasmid-mediated quinolone resistance (PMQR) genes have been reported in gram-negative bacteria, including S. marcescens, and include the qnr, qep, and oqx systems [4]. The major cause of fluoroquinolone resistance is chromosomal mutation. The acquisition of PMQR genes alone results in a low level of fluoroquinolone resistance and does not lead to minimum inhibitory concentrations (MICs) exceeding the threshold of these agents [3]. We isolated a S. marcescens strain, designated as GN0780, from the wound drainage fluid of a 66-yr-old male patient who had femoral fractures and was admitted to the Department of Orthopedics at the People's Hospital of Huangshan (Huangshan, China) in 2011. The MICs of ciprofloxacin (CIP), levofloxacin (LVX), gatifloxacin (GAT), and nalidixic acid (NAL) exceeded the resistance thresholds proposed by the Clinical and Laboratory Standards Institute (2012) (Table 1) [5]. Surprisingly, direct DNA sequencing of the QRDRs did not reveal any mutations in gyrA, gyrB, parC, or parE when compared with the wild-type strain (Table 1). We then screened for the PMQR genes qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, qepA, and oqxAB by PCR. None of the PMQR genes were identified in S. marcescens GN0780 (Table 1). Table 1 Fluoroquinolone susceptibility, QRDR mutations, and PMQRs in Serratia marcescens GN0780 Quinolones were introduced into clinical practice in the late 1960s. Although quinolone resistance was described soon after their introduction, the transmission mechanism of quinolone resistance was confirmed only in 1998. To date, five different PMQR mechanisms have been described in the literature, including target protection (Qnr), quinolone modification (AAC(6')-Ib-cr), plasmid-encoded efflux systems (e.g., QepA or OqxAB), effect on bacterial growth rates, and natural transformation. Although PMQRs usually result in only a slight increase in the MICs of quinolones, they show an additive effect and may thus facilitate the acquisition of full quinolone resistance [6]. Several clinical bacterial isolates that have been reported to express phenotypic resistance do not exhibit corresponding genotypic mutations. This phenomenon has recently been studied in a clinical isolate of Escherichia coli HUE1 from Japan [7]. The authors suggested that the fluoroquinolone resistance in this HUE1 isolate, which does not have mutations in the QRDR, is caused by the coexistence of oqxAB and qnrS. OqxAB and QnrS increase the MIC of CIP by approximately 32-fold and 64-fold, respectively. However, other mechanisms may also be associated with fluoroquinolone resistance in HUE1. Chopra and Galande [8] isolated an Acinetobacter baumannii mutant, designated as strain AB-7, which exhibited a CIP MIC of 16 mg/L. However, no mutation was detected in QRDRs, and no PMQR genes were present in AB-7. Our findings are consistent with the case of AB-7; S. marcescens GN0780 showed no mutation in QRDRs, and no PMQR gene was detected. We speculate that other mechanisms may be associated with fluoroquinolone resistance in GN0780; therefore, further investigations are needed. Our findings suggest an exception to the well-accepted mechanism of resistance to fluoroquinolones. These results also underscore the need of achieving deeper understanding of the mechanisms of action and evolution of resistance to conventional antibiotics. In conclusion, we present the first report of fluoroquinolone resistance in S. marcescens lacking PMQR genes and mutations in QRDRs.

Sir, Target changes via alterations of DNA gyrase and/or topoisomerase IV, and reduced intracellular drug accumulation, either by reduced drug permeability and/or increased efflux activity, are the two major mechanisms that mediate fluoroquinolone resistance. Resistance to quinolones in Escherichia coli appears to be caused mainly by alterations in the gyrA gene of the DNA gyrase and in the parC gene of the topoisomerase. The mutations are located at the N-terminus of the GyrA protein (residues 67–106 in E. coli) in the quinolone resistance-determining region (QRDR). A similar QRDR has also been reported in parC. We have reported as part of a national surveillance programme in Taiwan 526 E. coli isolates that were classified as fluoroquinolone susceptible according to the NCCLS guidelines. However, the distribution of zone diameters of ciprofloxacin indicated that these isolates consisted of two sub-populations with susceptibility and reduced susceptibility. From five of 44 hospitals surveyed, 193 E. coli isolates were selected for further study. Of these 193 isolates, 20 (10.4%) were with resistance, 44 (22.8%) with reduced susceptibility and 129 (66.8%) with susceptibility with zone diameters of ciprofloxacin 15, 16–29 and 30 mm, respectively, The 20 isolates with resistance, 44 isolates with reduced susceptibility and a random sample consisting of 17 E. coli isolates with susceptibility were assessed for point mutations in the QRDRs of gyrA and parC to determine the genetic bases of fluoroquinolone susceptibility. Based on their mutations in gyrA and parC, the 81 isolates were divided into 13 classes (Table). All resistant isolates in our study had two mutations in gyrA and at least one additional mutation in parC (Table, classes 1–5). A major type of combined mutations, Ser-83 Leu, Asp-87 Asn in gyrA and Ser-80 Ile in parC, was found in 17 resistant isolates (85%). Although single mutations in gyrA have previously been associated with reduced susceptibility to fluoroquinolones in E. coli, the molecular epidemiology of mutations in both gyrA and parC and their associations with reduced susceptibility have not been delineated. All isolates with reduced susceptibility in our study had one mutation in gyrA (Table, classes 6–12). Ser-83 Leu was a major mutation found in the isolates with reduced susceptibility, detected in 39 strains (87%). Isolates with double mutations in gyrA and parC (classes 6–8) had higher MIC90s of fluoroquinolones (0.75 and 2 mg/L of ciprofloxacin and ofloxacin, respectively) than isolates with only one mutation in gyrA (class 9, 0.25 and 1 mg/L of ciprofloxacin and ofloxacin, respectively). These data indicate that additional mutations in parC result in slight increases in MICs, consistent with the results reported for Neisseria gonorrhoeae. No strain possessed a parC mutation without the simultaneous presence of a gyrA mutation. These findings are consistent with fluoroquinolone resistance arising in a multi-step fashion with mutations in gyrA occurring as the first step. Isolates with zone diameters of ciprofloxacin of 15, 16–20 and 21 mm are considered as resistant, intermediate and susceptible, respectively, to ciprofloxacin according to current NCCLS resistance breakpoints. A major concern related to finding a large number of E. coli (22.8%) with reduced susceptibility, especially those with zone diameters of ciprofloxacin between 21 and 29 mm, is the fact that these isolates are unlikely to be detected using current NCCLS guidelines. Increased clinical failures have been reported when human infections caused by Salmonella spp. with reduced susceptibility are treated with fluoroquinolones. Owing to similar concerns over treatment efficacy, the breakpoint for the definition of resistance to fluoroquinolones in N. gonorrhoeae was recently adjusted as follow: isolates with zone diameters of ciprofloxacin 27, 28–40 and 41 mm are interpreted as resistant, intermediate and susceptible, respectively, to ciprofloxacin. Antimicrobial susceptibility test results should not only guide clinicians in determining the most appropriate therapy for their patients, but also assist efforts to monitor and control the spread of resistance. We therefore recommend that current NCCLS breakpoints for fluoroquinolone resistance in Enterobacteriaceae, such as E. coli and Salmonella spp., be modified according to our findings: Correspondence Journal of Antimicrobial Chemotherapy (2001) 48, 931–942

Sir, Acinetobacter baumannii is an emerging Gram-negative multidrug-resistant (MDR) nosocomial pathogen responsible for causing lung, bloodstream, urinary tract and skin infections. Additionally, A. baumannii has emerged as a significant threat in injured US military personnel returning for medical treatment from Afghanistan and Iraq. Because an increasing number of A. baumannii isolates are resistant to the most commonly clinically utilized antibiotics, it is imperative to understand the development of antibiotic resistance in A. baumannii at the molecular level. Fluoroquinolones are broad-spectrum bactericidal agents used to treat diverse bacterial infections. Fluoroquinolones are emerging as viable alternatives for treating A. baumannii infections, but the clinical incidence of fluoroquinolone resistance continues to increase. The most extensively described mechanism of resistance to fluoroquinolones is mediated by mutations in the quinolone resistance-determining regions (QRDRs) of gyrA that encodes the DNA gyrase A subunit and parC that encodes the topoisomerase IV subunit. Additionally, resistance can also be mediated by efflux pumps such as AdeABC and AdeM. Recently, plasmid-mediated quinolone resistance (PMQR) genes have been reported in Gram-negative bacteria, including Escherichia coli and Pseudomonas spp., and include the qnr, qep and oqx systems. The qnr system encodes a pentapeptide repeat protein that blocks the action of ciprofloxacin on bacterial DNA gyrase and topoisomerase IV and was first described in Klebsiella pneumoniae. In contrast, the qep and oqx systems encode plasmid-mediated efflux pumps. However, the PMQR genes have not been demonstrated in A. baumannii. The prevalent theory is that the fluoroquinolone resistance is chiefly mediated by chromosomal mutation, whereas the acquisition of the PMQR genes results in enhancement of the resistance because the PMQR genes by themselves result in a low level of fluoroquinolone resistance. To address the progression of antibiotic resistance in A. baumannii ATCC 17978, we attempted to isolate A. baumannii ATCC 17978 ciprofloxacin-resistant mutants by plating 10– 10 cfu on Mueller–Hinton agar plates containing 2×–16× the MIC of ciprofloxacin. Among the mutants analysed, we isolated a mutant named AB-7, which exhibited a ciprofloxacin MIC of 16 mg/L (Table 1). The CLSI breakpoints for ciprofloxacin are ≤1 mg/L, susceptible; and ≥4 mg/L, resistant. Additionally, the AB-7 strain also exhibited resistance to other fluoroquinolones tested, thus demonstrating class-wide resistance; however, AB-7 was not resistant to the unrelated antibiotic polymyxin B (Table 1). Surprisingly, however, DNA sequencing of the QRDRs did not exhibit any mutations in gyrA, gyrB, parC and parE when compared with the parental strain (Table 1). A. baumannii ATCC 17978 has been whole-genome sequenced and the data have been deposited at the National Center for Biotechnology Information (NCBI; GenBank accession number CP000521.1). The DNA sequence was then analysed for existence of the PMQR genes, namely qnr, qep and oqx. None of the PMQR genes was identified, as has been reported by multiple investigators. – 9 It has been speculated that a number of clinical bacterial isolates that express phenotypic resistance do not exhibit co-relatable genotypic mutations. This phenomenon has recently been reported in the HUE1 E. coli clinical isolate from Japan. In comparison with the HUE1 E. coli isolate, no mutation was detected in the QRDRs as well as no PMQR genes in AB-7. However, other mechanisms may be involved in fluoroquinolone resistance and they are being investigated further. This finding demonstrates a clear exception to the well-accepted mechanism of resistance for fluoroquinolones. The results also underscore the importance of undertaking systems-level studies to re-learn mechanisms of action and evolution of resistance for conventional antibiotics. In conclusion, we present the first report of fluoroquinolone resistance in A. baumannii that is independent of mutations in QRDRs and in the absence of PMQR.

Fluoroquinolones have been used for prophylaxis against infections in cancer patients but their impact on the resistance mechanisms still require further investigation. To elucidate mechanisms underlying ciprofloxacin (CIP) resistance in Gram-negative pathogens causing infections to cancer patients, 169 isolates were investigated. Broth microdilution assays showed high-level CIP resistance in 89.3% of the isolates. Target site mutations were analyzed using PCR and DNA sequencing in 15 selected isolates. Of them, all had gyrA mutations (codons 83 and 87) with parC mutations (codons 80 and 84) in 93.3%. All isolates were screened for plasmid-mediated quinolone resistance (PMQR) genes and 56.8% of them were positive in this respect. Among PMQR genes, aac(6′)-Ib-cr predominated (42.6%) while qnr genes were harbored by 32.5%. This comprised qnrS in 26.6% and qnrB in 6.5%. Clonality of the qnr-positive isolates using ERIC-PCR revealed that most of them were not clonal. CIP MIC reduction by CCCP, an efflux pump inhibitor, was studied and the results revealed that contribution of efflux activity was observed in 18.3% of the isolates. Furthermore, most fluoroquinolone resistance mechanisms were detected among Gram-negative isolates recovered from cancer patients. Target site mutations had the highest impact on CIP resistance as compared to PMQRs and efflux activity.

Frequency of DNA gyrase and topoisomerase IV mutations and plasmid-mediated quinolone resistance genes among Escherichia coli and Klebsiella pneumoniae isolated from urinary tract infections in Azerbaijan, Iran.

Mutations in gyrase and topoisomerase genes associated with fluoroquinolone resistance in Salmonella serovars from retail meats