Detection of gyrA and parC Mutations and Prevalence of Plasmid-Mediated Quinolone Resistance Genes in Klebsiella pneumoniae.

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.

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.

One of the treatment options of Escherichia coli and Klebsiella spp. infections which are the most common opportunistic pathogens of gram-negative sepsis is quinolones. Resistance to quinolones which act by disrupting DNA synthesis has been increasing. Horizontal transfer of plasmid-mediated quinolone resistance (PMQR) genes play an important role in the spread of resistance. The data about the prevalence of PMQR genes in our country is quite limited. The aim of this study was to investigate the presence of known PMQR genes namely qnrA, qnrB, qnrC, qnrS, qnrD, aac(6')-Ib-cr, qepA and oqxAB amongst quinolone-resistant E. coli and Klebsiella spp. strains isolated from blood cultures. One hundred twenty seven E.coli and 66 Klebsiella isolates detected as nalidixic acid- and/or ciprofloxacin-resistant by phenotypical methods, from 193 blood samples of 187 patients admitted to Karadeniz Technical University, Faculty of Medicine, Department of Medical Microbiology, Bacteriology Unit of Patient Service Laboratory between January 2012 to August 2013 were included in the study. The presence of PMQR genes were investigated by polymerase chain reaction (PCR) and for the detection of aac(6')-Ib-cr variants PCR-restriction fragment length polymorphism (PCR-RFLP) method was used. The positive bands were sequenced using the same primers, and aligned with formerly defined resistance gene sequences, and confirmed. In the study, 56.7% (72/127) of E.coli and 19.7% (13/66) of Klebsiella spp. isolates, with a total of 44% (85/193) of all the isolates were found to be phenotypically resistant to quinolones. Of the 13 resistant Klebsiella isolates, 11 were K.pneumoniae, and two were K.oxytoca. Extended-spectrum beta-lactamase (ESBL)-producing isolates showed higher resistance (50/80, 62.5%) to quinolones than the negative ones (35/113, 30.9%). The prevalence of quinolone resistance genes among resistant E. coli and Klebsiella spp. isolates was determined as qnrA, 1.4% and 15.4%; qnrB, 4.2% and 61.5%; qnrS, 1.4% and 7.7%; qepA, 1.4% (only E.coli); aac(6')-1b-cr, 38.9% and 92.3%; and oqxAB, 1.4% and 84.6%, respectively. qnrC and qnrD genes were not detected. Carriage of multiple resistance genes were observed more frequently among resistant Klebsiella isolates than E.coli strains. As a result, in this study investigating the contribution of transferrable genes to quinolone resistance, prevalence of PMQR genes in quinolone-resistant and blood isolates of E.coli and Klebsiella in our university hospital serving the region were found to be higher than the current data reported from the other studies in our country. Furthermore, this study presented the initial data for the first time in our country on the prevalence of qnrD which was undetected, and the frequency of oqxAB gene in clinical samples. However, location of oqxAB gene needs to be confirmed by conjugation or hybridization methods.

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.

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

This first nationwide study was conducted to analyze the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in phenotypically unselected (consecutive) clinical enterobacteria. We studied 1,058 isolates that had been consecutively collected in 66 hospitals of the WHONET-Argentina Resistance Surveillance Network. Overall, 26% of isolates were nonsusceptible to at least one of the three quinolones tested (nalidixic acid, ciprofloxacin, and levofloxacin). The overall prevalence of PMQR genes was 8.1% (4.6% for aac(6')-Ib-cr; 3.9% for qnr genes; and 0.4% for oqxA and oqxB, which were not previously reported in enterobacteria other than Klebsiella spp. from Argentina). The PMQR prevalence was highly variable among the enterobacterial species or when the different genes were considered. The prevalent PMQR genes were located in class 1 integrons [qnrB2, qnrB10, and aac(6')-Ib-cr]; in the ColE1-type plasmid pPAB19-1 or Tn2012-like transposons (qnrB19); and in Tn6238 or bracketed by IS26 and blaOXA-1 [aac(6')-Ib-cr]. The mutations associated with quinolone resistance that were located in the quinolone resistance-determining region (QRDR mutations) of gyrA, parC, and gyrB were also investigated. The occurrence of QRDR mutations was significantly associated with the presence of PMQR genes: At least one QRDR mutation was present in 82% of the PMQR-harboring isolates but in only 23% of those without PMQR genes (p < 0.0001, Fisher's Test). To the best of our knowledge, this is the first report on the prevalence of PMQR genes in consecutive clinical enterobacteria where all the genes currently known have been screened.

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.

The determination of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in carbapenem resistant Klebsiella pneumonia ST11 and ST76 strains isolated from patients in Heilongjiang Province, China

This work describes the characterization of plasmid-mediated quinolone-resistance (PMQR) genes from a multicenter study of ESBL-producing Enterobacteriaceae pediatric clinical isolates in Mexico. The PMQR gene-positive isolates were characterized with respect to ESBLs, and mutations in the GyrA and ParC proteins were determined. The phylogenetic relationship was established by PFGE and the transfer of PMQR genes was determined by mating assays. The prevalence of the PMQR genes was 32.1%, and the rate of qnr-positive isolates was 15.1%; 93.3% of the latter were qnrB and 6.4% were qnrA1. The distribution of isolates in terms of bacterial species was as follows: 23.5% (4/17) corresponded to E. cloacae, 13.7% (7/51) to K. pneumoniae, and 13.6% (6/44) to E. coli. In addition, the prevalence of aac(6’)-Ib-cr and qepA was 15.1% and 1.7%, respectively. The molecular characteristics of qnr- and qepA-positive isolates pointed to extended-spectrum β-lactamase (ESBL) CTX-M-15 as the most prevalent one (70.5%), and to SHV-12 in the case of aac(6’)-Ib-cr-positive isolates. GyrA mutations at codons Ser-83 and Asp-87, and ParC mutations at codons Ser-80 were observed in 41.1% and 35.2% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a co-transmission of blaCTX-M-15 with the qnrB alleles. In general, the prevalence of PMQR genes (qnr and aac(6’)-Ib-cr) presented in this work was much lower in the pediatric isolates, in comparison to the adult isolates in Mexico. Also, ESBL CTX-M-15 was the main ESBL identified in the pediatric isolates, whereas in the adult ones, ESBLs corresponded to the CTX-M and the SHV families. In comparison with other studies, among the PMQR-genes identified in this study, the qnrB-alleles and the aac(6’)-Ib-cr gene were the most prevalent, whereas the qnrS1, qnrA1 and qnrB-like alleles were the most prevalent in China and Uruguay.

Escherichia coli is the major causative agent for urinary tract infections (UTIs), and fluoroquinolones (FQ) are commonly used in the treatment of patients with UTIs. The surge in FQ-resistant E. coli is an important public health threat worldwide. We investigated the prevalence and mechanisms of FQ resistance among FQ-resistant E. coli isolated from UTI patients. A total of 131 FQ-resistant E. coli strains were characterized and broth microdilution assay showed that 108 strains (82.4%) were highly resistant to ciprofloxacin (MIC ≥ 32 μg/mL). All strains were analyzed for plasmid-mediated quinolone resistance (PMQR) genes, with 37 (28.2%) testing positive. Among the PMQR genes detected, aac(6')-Ib-cr was the most frequent, found in 30 strains (22.9%), followed by qnrS in 10 strains (7.6%) and qnrB in 1 strain (0.8%). PCR assay showed that all carried acrA, acrB, and tolC genes, but 33 strains (25.2%) revealed at least 4-fold reduction in ciprofloxacin MIC when using PAβN and CCCP as efflux pump inhibitors, indicating the role of the AcrAB efflux pump in ciprofloxacin resistance in these strains. The 19 strains of high-level ciprofloxacin-resistant E. coli were selected to determine the target enzyme alteration by PCR assay and DNA sequencing. Genetic analysis revealed that 16 strains (84.2%) had double mutations in gyrA (S83L and D87 to N or Y) with single mutation in parC (S80I), while 3 strains (15.8%) had double mutations in gyrA (S83L and D87 to N or Y) and parC (S80I and E84 to G or V). The positive efflux activity was linked to increased MIC values of ciprofloxacin (P < 0.001). Overall, the carriage of PMQR genes, efflux activity, and target mutations across E. coli strains contribute to ciprofloxacin resistance, a result that may necessitate a reassessment of the antibiotics in use for empirical UTIs therapy.

Background and Aim:Riemerella anatipestifer is a Gram-negative bacterium causing systemic infections in ducks, often treated with quinolones. However, increasing resistance to quinolones poses a threat to effective treatment, and the molecular mechanisms underlying this resistance remain inadequately understood in Thailand. This study aimed to determine the minimum inhibitory concentrations (MICs) of nalidixic acid, ciprofloxacin, and enrofloxacin; identify mutations in the quinolone resistance-determining regions of gyrA and parC; and detect plasmid-mediated quinolone resistance (PMQR) genes in R. anatipestifer isolates from Thai ducks.Materials and Methods:A total of 37 clinical isolates of R. anatipestifer were collected from diseased ducks between 2021 and 2023. MICs were determined using the agar dilution method, following the guidelines of the Clinical and Laboratory Standards Institute. Polymerase chain reaction and Sanger sequencing were employed to detect mutations in gyrA and parC and to screen for PMQR genes (qnrA, qnrB, and qnrS). Phylogenetic analysis of the gyrA gene was performed to assess the relatedness among isolates.Results:Nalidixic acid MICs ranged from 16 μg/mL to ≥128 μg/mL; ciprofloxacin from 1 μg/mL to 8 μg/mL; and enrofloxacin from 0.25 μg/mL to 4 μg/mL. All isolates had a single point mutation at codon 83 of gyrA, either C248T (Ser83Ile, n = 35) or C248G (Ser83Arg, n = 2). No mutations were observed in parC, and none of the PMQR genes were detected. Phylogenetic analysis grouped most Thai isolates into one major cluster, with a few aligning with Chinese strains and the American Type Culture Collection reference strain.Conclusion:This study provides the first molecular evidence of quinolone resistance mechanisms in R. anatipestifer from ducks in Thailand. Resistance appears primarily associated with a single mutation at codon 83 of gyrA, while no parC mutations or PMQR genes were detected. These findings highlight the importance of ongoing resistance surveillance and prudent antimicrobial use. Despite limitations in sample size and gene scope, this study provides essential baseline data to inform treatment guidelines and supports the inclusion of R. anatipestifer monitoring in Thailand’s national antimicrobial resistance action plan. Future research should explore additional resistance genes using advanced genomic tools.

A longitudinal epidemiology study of fluoroquinolone-nonsusceptible Klebsiella pneumoniae reveals an increasing prevalence of qnrB and qnrS in Taiwan

Investigation of mutation distribution in DNA gyrase and topoisomerase IV genes in ciprofloxacin-non-susceptible Enterobacteriaceae isolated from blood cultures in a tertiary care university hospital in South Korea, 2005–2010

Ninety-three Malaysian extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6′)-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs 4– ≥ 32 μg/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone (n = 1) or in both gyrA and parC regions (n = 32). aac(6′)-Ib-cr was the most common PMQR gene detected in this study (n = 61), followed by qnrB and qnrS (n = 55 and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μg/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6′)-Ib-cr (n = 34) or a single gyrA 83 mutation (n = 6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital.

BackgroundThe purpose of this study was to look into the presence of plasmid-mediated quinolone resistance (PMQR) genes and biofilm formation in several species of clinical Shigella isolates that were resistant to quinolones.MethodsThe stool samples of 150 patients (younger than 10 years) with diarrhea were collected in this cross-sectional study (November 2020 to December 2021). After cultivation of samples on Hektoen Enteric agar and xylose lysine deoxycholate agar, standard microbiology tests, VITEK 2 system, and polymerase chain reaction (PCR) were utilized to identify Shigella isolates. The broth microdilution method was used to determine antibiotic susceptibility. PMQR genes including qnrA, qnrB, qnrC, qnrD, qnrE, qnrS, qnrVC, qepA, oqxAB, aac(6′)-Ib-cr, and crpP and biofilm formation were investigated in quinolone-resistant isolates by PCR and microtiter plate method, respectively. An enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used to determine the clonal relatedness of quinolone-resistant isolates.ResultsA total of 95 Shigella isolates including S. sonnei (53, 55.8%), S. flexneri (39, 41.1%), and S. boydii (3, 3.2%) were identified. The highest resistance rates of the isolates were against ampicillin (92.6%, n = 88/95). Overall, 42 of 95 (44.2%) isolates were simultaneously resistant against two or more quinolones including 26 (61.9%) S. sonnei and 16 (38.1%) S. flexneri. All isolates were multidrug-resistant (resistance to more than 3 antibiotics). The occurrence of PMQR genes was as follows: qnrS (52.4%), qnrA and aac(6′)-Ib-cr (33.3%), and qnrB (19.0%). The prevalence in species was as follows: 61.5% and 37.5% (qnrS), 19.2% and 56.3% (qnrA), 38.5% and 25.0 (aac(6′)-Ib-cr), and 19.2% and 18.8% (qnrB) for S. sonnei and S. flexneri, respectively. The other PMQR genes were not detected. In total, 52.8% (28/53) of quinolone-susceptible and 64.3% (27/42) of quinolone-resistant isolates were biofilm producers. Biofilm formation was not significantly different between quinolone-resistant and quinolone-susceptible isolates (P-value = 0.299). Quinolone-resistant isolates showed a high genetic diversity according to the ERIC-PCR.ConclusionIt seems that qnrS, qnrA, and aac(6′)-Ib-cr play a significant role in the quinolone resistance among Shigella isolates in our region. Also the quinolone-resistant S. flexneri and S. sonnei isolates had a high genetic diversity. Hence, antibiotic therapy needs to be routinely revised based on the surveillance findings.