Novel Qnr Families as Conserved and Intrinsic Quinolone Resistance Determinants in Aeromonas spp.

The environment has been identified as an origin, reservoir, and transmission route of antibiotic resistance genes (ARGs). Among diverse environments, freshwater environments have been recognized as pivotal in the transmission of ARGs between opportunistic pathogens and autochthonous bacteria such as Aeromonas spp. In this study, five environmental strains of Aeromonas spp. exhibiting multidrug resistance (MDR) were selected for whole-genome sequencing to ascertain their taxonomic assignment at the species-level and to delineate their ARG repertoires. Analyses of their genomes revealed the presence of one protein almost identical to AhQnr (A. hydrophila Qnr protein) and four novel proteins similar to AhQnr. To scrutinize the classification and taxonomic distribution of these proteins, all Aeromonas genomes deposited in the NCBI RefSeq genome database (1,222 genomes) were investigated. This revealed that these Aeromonas Qnr (AQnr) proteins are conserved intrinsic resistance determinants of the genus, exhibiting species-specific diversity. Additionally, structure prediction and analysis of contribution to quinolone resistance by AQnr proteins of the isolates, confirmed their functionality as quinolone resistance determinants. Given the origin of mobile qnr genes from aquatic bacteria and the crucial role of Aeromonas spp. in ARG dissemination in aquatic environments, a thorough understanding and strict surveillance of AQnr families prior to the clinical emergence are imperative. In this study, using comparative genome analyses and functional characterization of AQnr proteins in the genus Aeromonas, novel Aeromonas ARGs requiring surveillance has suggested.

Plasmid-Mediated Fluoroquinolone Resistance among Enterobacterales in Africa: Systematic Review.

INTRODUCTION: According to the World Health Organization, antimicrobial resistance (AMR) is a silent global pandemic that plagues everyone. It makes therapy of infectious diseases more difficult and eventually increases morbidity and mortality. AIM: The purpose of this work is to examine existing data on plasmid-mediated quinolone resistance (PMQR), to assess the prevalence of PMQR genes in Enterobacterales, and to determine any knowledge gaps from sub-Saharan Africa. METHODOLOGY: The Preferred Reporting Items of Systematic Reviews and Meta-analyses (PRISMA) standard was followed when conducting this systematic review. The main internet databases examined for pertinent publications were PubMed, Google Scholar, and Ajol. A set of qualifying criteria were used to evaluate the qualified articles. Using the eligibility criteria, 56 full-text articles were chosen for screening. RESULT: Thirty-two (32) articles with the majority originating from West and North Africa and only one article reporting a study carried out in Central Africa were selected for this review. Escherichia coli and Ciprofloxacin were the most reported Enterobacterales and Quinolone respectively. The PMQR genes include qnr (qnrA,qnrB, qnrC, qnrD, and qnrS), aac (6') Ib, aac (6') Ib-cr, oqxAB and qepA gene. The most prevalent PMQR gene is the aac (6') Ib-cr gene (32%) followed by qnrS (26%). CONCLUSION: This study highlighted the requirement for an efficient antimicrobial resistance surveillance system in the continent and revealed a significant incidence of PMQR genes.

INTRODUCTION: Selon l'Organisation mondiale de la santé, la résistance aux antimicrobiens (RAM) est une pandémie mondiale silencieuse qui touche tout le monde. Elle rend le traitement des maladies infectieuses plus difficile et finit par augmenter la morbidité et la mortalité. OBJECTIF: L'objectif de ce travail est d'examiner les données existantes sur la résistance plasmidique aux quinolones (PMQR), d'évaluer la prévalence des gènes PMQR chez les Enterobacterales et de déterminer d'éventuelles lacunes de connaissances en Afrique subsaharienne. MÉTHODOLOGIE: La norme Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) a été suivie lors de la réalisation de cette revue systématique. Les principales bases de données Internet examinées pour des publications pertinentes étaient PubMed, Google Scholar et Ajol. Un ensemble de critères d'admissibilité a été utilisé pour évaluer les articles qualifiés. En utilisant les critères d'éligibilité, 56 articles en texte intégral ont été choisis pour le dépistage. RÉSULTAT: Trente-deux (32) articles, dont la majorité provient d'Afrique de l'Ouest et du Nord, et un seul article rapportant une étude menée en Afrique centrale, ont été sélectionnés pour cette revue. Escherichia coli et la ciprofloxacine étaient les Enterobacterales et les quinolones les plus signalées respectivement. Les gènes PMQR comprennent les gènes qnr (qnrA, qnrB, qnrC, qnrD et qnrS), aac (6 ') Ib, aac (6 ') Ib-cr, oqxAB et qepA. Le gène PMQR le plus prévalent est le gène aac (6 ') Ib-cr (32 %), suivi de qnrS (26 %). CONCLUSION: Cette étude a souligné la nécessité d'un système efficace de surveillance de la résistance aux antimicrobiens sur le continen`t et a révélé une incidence significative des gènes PMQR. MOTS-CLÉS: Enterobacterales, Escherichia coli, Quinolone, Ciprofloxacine, PMQR, "aac(6')-Ib", "aac(6')-Ib-cr", "qnr", "qepA", "oqxAB", "résistance aux antibiotiques".

Coexistence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) genes among clinical Pseudomonas aeruginosa isolates in Egypt.

BACKGROUND: Data about the prevalence of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) production in P. aeruginosa compared to the Enterobacteriaceae family is limited. The availability of limited therapeutic options raises alarming concerns about the treatment of multidrug-resistant P. aeruginosa. This study aimed to assess the presence of PMQR and ESBL genes among P. aeruginosa strains. METHODS: Fifty-six P. aeruginosa strains were isolated from 330 patients with different clinical infections. Phenotypically fluoroquinolone-resistant isolates were tested by PCR for the presence of six PMQR genes. Then, blaTEM, blaSHV, and blaCTX-M type ESBL genes were screened to study the co-existence of different resistance determinants. RESULTS: Overall, 22/56 (39.3%) of the studied P. aeruginosa isolates were phenotypically resistant to fluoroquinolones. PMQR-producing P. aeruginosa isolates were identified in 20 isolates (90.9%). The acc(6')-Ib-cr was the most prevalent PMQR gene (77.3%). The qnr genes occurred in 72.7%, with the predominance of the qnrA gene at 54.5%, followed by the qnrS gene at 27.3%, then qnrB and qnrC at 22.7%. The qepA was not detected in any isolate. The acc(6')-Ib-cr was associated with qnr genes in 65% of positive PMQR isolates. Significant differences between the fluoroquinolone-resistant and fluoroquinolone-susceptible isolates in terms of the antibiotic resistance rates of amikacin, imipenem, and cefepime (P value < 0.0001) were found. The ESBL genes were detected in 52% of cephalosporin-resistant P. aeruginosa isolates. The most frequent ESBL gene was blaCTX-M (76.9%), followed by blaTEM (46.2%). No isolates carried the blaSHV gene. The acc(6')-Ib-cr gene showed the highest association with ESBL genes, followed by the qnrA gene. The correlation matrix of the detected PMQR and ESBL genes indicated overall positive correlations. The strongest and most highly significant correlation was between qnrA and acc(6')-Ib-cr (r = 0.602) and between qnrA and blaCTX-M (r = 0.519). CONCLUSION: A high prevalence of PMQR genes among the phenotypic fluoroquinolone-resistant P. aeruginosa isolates was detected, with the co-carriage of different PMQR genes. The most frequent PMQR was the acc(6')-Ib-cr gene. Co-existence between PMQR and ESBL genes was found, with 75% of PMQR-positive isolates carrying at least one ESBL gene. A high and significant correlation between the ESBL and PMQR genes was detected.

Distribution and genetic characterization of fluoroquinolone resistance gene qnr among Salmonella strains from chicken in China.

The prevalence and dissemination of the plasmid-mediated fluoroquinolone (FQ) resistance gene qnr in Salmonella are considered serious public health concerns worldwide. So far, no comprehensive large-scale studies have focused on the prevalence and genetic characteristics of the qnr gene in Salmonella isolated from chickens. Herein, this study aimed to investigate the prevalence, antimicrobial resistance (AMR) patterns, and molecular characteristics of chicken-originated qnr-positive Salmonella strains from chicken farms, slaughterhouses, and markets in 12 provinces of China in 2020-2021. The overall prevalence of the qnr gene was 21.13% (56/265), with the highest prevalence in markets (36.11%, 26/72), followed in farms (17.95%, 21/117), and slaughterhouses (10.53%, 9/76). Only the qnrS and qnrB genes were detected, and the prevalence rate of the qnrS gene (19.25%, 51/265) was higher than that of the qnrB gene (1.89%, 5/265). Whole genome sequencing identified 37 distinct AMR genes and 15 plasmid replicons, and the most frequent mutation in quinolone resistance determining regions was parC (T57S; 91.49%, 43/47). Meanwhile, four different qnrS and two qnrB genetic environments were discovered among 47 qnr-positive Salmonella strains. In total, 21.28% (10/47) of the strains were capable of conjugative transfer, and all were qnrS1-positive strains, with the majority of transferable plasmids being IncHI2 types (n = 4). Overall, the prevalence of qnr-positive Salmonella strains from chickens in China and their carriage of multiple resistance and virulence genes and transferable plasmids is a major concern, which calls for continuous surveillance of qnr-positive Salmonella and the development of measures to control its prevalence and transmission.IMPORTANCESalmonella is a common foodborne pathogen responsible for 155,000 deaths annually worldwide. Fluoroquinolones (FQs) are used as first-line drugs for the treatment of Salmonella infections in several countries and regions. However, the emergence and increasing prevalence of the FQ-resistant gene qnr in Salmonella isolated from chickens have been widely reported. Gaining insight into the genetic mechanisms of AMR genes in chicken could lead to the development of preventive measures to control and reduce the risk of drug resistance. In this study, we identified qnr-positive Salmonellae isolated from chickens in different regions of China and their AMR patterns and genome-wide characteristics, providing a theoretical basis for further control of their prevalence and transmission.

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

BACKGROUND: Our objective was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in fluoroquinolone-nonsusceptible Klebsiella pneumoniae (FQNSKP) in Taiwan, 1999-2022. METHODS: A total of 938 FQNSKP isolates were identified from 1966 isolates. The presence of PMQR and virulence genes, antimicrobial susceptibility, capsular types, and PMQR-plasmid transferability were determined. RESULTS: An increasing number of PMQR-containing FQNSKP isolates were observed over the study period. Our results showed that 69.0% (647 isolates) of FQNSKP isolates contained at least one PMQR gene, and 40.6%, 37.0%, and 33.9% of FQNSKP carried aac(6')-Ib-cr, qnrB, and qnrS, respectively. None of FQNSKP carried qepA and qnrC. The most common combination of PMQR genes was aac(6')-Ib-cr and qnrB (12.3%). The presence of PMQR genes is strongly related to resistance to aminoglycoside, cephalosporin, tetracycline, and sulfamethoxazole/trimethoprim in FQNSKP. The capsular serotype K64 is the most common serotype we tested in both the non-PMQR and PMQR FQNSKP isolates, while K20 showed a higher prevalence in PMQR isolates. The magA and peg-344 genes showed a significantly higher prevalence rate in non-PMQR isolates than in PMQR isolates. Eleven isolates that carried the PMQR and carbapenemase genes were identified; however, three successful transconjugants showed that the PMQR and carbapenemase genes were not located on the same plasmid. CONCLUSIONS: Our results indicated an increasing prevalence of PMQR genes, especially qnrB and qnrS, in FQNSKP in Taiwan. Moreover, the distribution of PMQR genes was associated with capsular serotypes and antimicrobial resistance gene and virulence gene distribution in FQNSKP.

Quinolone resistance and biofilm formation capability of uropathogenic Escherichia coli isolates from an Iranian inpatients' population.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a major pathogen of the urinary tract infection (UTI), and biofilm formation is crucial as it facilitates the colonization in the urinary tract. We aimed to investigate the antibiotic susceptibility pattern, biofilm formation capability, distribution of quinolone resistance genes, and phylogenetic groups among UPEC isolates from an Iranian inpatients' community. METHODS AND RESULTS: A collection of 126 UPEC obtained from hospitalized patients with symptomatic UTI at 3 teaching hospitals during 2016 were included. Antibiogram of all isolates against quinolone and fluoroquinolones was performed using the disk diffusion method. Phylogenetic groups and qnr A, B, and S genes were assessed by PCR. Susceptibility pattern showed that more than 50% and 81% of the isolates were resistant to fluoroquinolones and quinolones, correspondingly. The frequency of qnrS and qnrB genes was 22% and 13.5%, correspondingly. Our result indicated no significant association between the presence of fluoroquinolone genes and antibiotic resistance to them. The frequent common phylogroup was B2 (84.1%), followed by D (10.3%), A (3.2%) and B1 (2.4%) groups. Indeed, 80.2% of the isolates were biofilm producers, so that 42.1%, 16.7% and 21.4% of them were classified as weak, moderate and strong producers, respectively. CONCLUSIONS: Our results showed considerable fluoroquinolone and quinolone resistance among UPEC along with a remarkable rate of biofilm-producing isolates from symptomatic hospitalized patients, making them a serious health concern in the region. This survey highlights the need for awareness on quinolone resistance and careful prescription of them by physicians.

Serotypes, Antimicrobial Resistance Profiles, and Virulence Factors of Salmonella Isolates in Chinese Edible Frogs (Hoplobatrachus rugulosus) Collected from Wet Markets in Hong Kong.

Salmonella is an important agent of gastrointestinal disease in humans. While livestock, such as cattle, poultry, and pigs, are well-recognised animal reservoirs of Salmonella, there is a lack of data on Salmonella in edible frogs, even though frog meat is a popular food worldwide. In this study, 103 live edible Chinese frogs (Hoplobatrachus rugulosus) were collected from wet markets throughout Hong Kong. After euthanasia, faeces or cloacal swabs were examined for Salmonella. Overall, Salmonella spp. were isolated from 67 (65%, CI: 0.554-0.736) of the samples. The serotypes included S. Saintpaul (33%), S. Newport (24%), S. Bareilly (7%), S. Braenderup (4%), S. Hvittingfoss (4%), S. Stanley (10%), and S. Wandsworth (16%). Many isolates were phylogenetically related. A high number of genes encoding for resistance to clinically relevant antimicrobials, and a high number of virulence determinants, were identified. Antimicrobial susceptibility testing (AST) identified multidrug resistance (MDR) in 21% of the isolates. Resistance to ampicillin, ciprofloxacin, nalidixic acid, and tetracycline was common. These results demonstrate that a high percentage of live frogs sold for human consumption in wet markets are carriers of multidrug-resistant Salmonella. Public health recommendations for handling edible frogs should be considered, to mitigate the risk of Salmonella transmission to humans.

Transferable mechanisms of quinolone resistance are more frequent among enterotoxigenic Escherichia coli isolates displaying low-level quinolone resistance

@#This study analysed the mechanisms of quinolone resistance among enterotoxigenic Escherichia coli (ETEC) in a periurban area of Lima, Peru. The susceptibility to nalidixic acid and ciprofloxacin, the role of Phe-Arg-b-Naphtylamyde inhibitable-(PAbN) efflux pumps, the presence of mutations in gyrA and parC as well as the presence of aac(6’)Ib-cr, qepA, qnrA, qnrB, qnrC, qnrD, qnrVC and oqxAB were determined in 31 ETEC from previous case/control studies of children’s diarrhoea. Discordances between disk diffusion, with all isolates showing intermediate or fully resistance to nalidixic acid, and minimal inhibitory concentration (MIC), with 7 isolates being below considered resistance breakpoint, were observed. Twenty-one isolates possessed gyrA mutations (19 S83L, 2 S83A). AAC(6’) Ib-cr, QnrS, QnrB and QepA were found in 7, 6, 2 and 1 isolates respectively, with 3 isolates presenting 2 transferable mechanisms of quinolone resistance (TMQR) concomitantly. TMQR were more frequent among isolates with MIC to nalidixic acid ranging from 2 to 16 mg/L (p=0.03), while gyrA mutations were more frequent among isolates with nalidixic acid MIC > 128 mg/L (p=0.0002). In summary, the mechanisms of quinolone resistance present in ETEC isolates in Peru have been described. Differences in the prevalence of underlying mechanisms associated with final MIC levels were observed. The results suggest two different evolutive strategies to survive in the presence of quinolones related to specific bacterial genetic background.

Aquatic Environments as Hotspots of Transferable Low-Level Quinolone Resistance and Their Potential Contribution to High-Level Quinolone Resistance.

The disposal of antibiotics in the aquatic environment favors the selection of bacteria exhibiting antibiotic resistance mechanisms. Quinolones are bactericidal antimicrobials extensively used in both human and animal medicine. Some of the quinolone-resistance mechanisms are encoded by different bacterial genes, whereas others are the result of mutations in the enzymes on which those antibiotics act. The worldwide occurrence of quinolone resistance genes in aquatic environments has been widely reported, particularly in areas impacted by urban discharges. The most commonly reported quinolone resistance gene, qnr, encodes for the Qnr proteins that protect DNA gyrase and topoisomerase IV from quinolone activity. It is important to note that low-level resistance usually constitutes the first step in the development of high-level resistance, because bacteria carrying these genes have an adaptive advantage compared to the highly susceptible bacterial population in environments with low concentrations of this antimicrobial group. In addition, these genes can act additively with chromosomal mutations in the sequences of the target proteins of quinolones leading to high-level quinolone resistance. The occurrence of qnr genes in aquatic environments is most probably caused by the release of bacteria carrying these genes through anthropogenic pollution and maintained by the selective activity of antimicrobial residues discharged into these environments. This increase in the levels of quinolone resistance has consequences both in clinical settings and the wider aquatic environment, where there is an increased exposure risk to the general population, representing a significant threat to the efficacy of quinolone-based human and animal therapies. In this review the potential role of aquatic environments as reservoirs of the qnr genes, their activity in reducing the susceptibility to various quinolones, and the possible ways these genes contribute to the acquisition and spread of high-level resistance to quinolones will be discussed.

Characterization of plasmid-mediated quinolone resistance genes and extended-spectrum beta-lactamases in non-typhoidal Salmonella enterica isolated from broiler chickens.

Background and Aim: Antibiotic-resistant Salmonella is a public health concern. Fluoroquinolones and extended-spectrum beta-lactams are widely used for the treatment of Salmonella infections. This study focused on the detection of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamase (ESBL) genes among multidrug-resistant (MDR) Salmonella enterica isolated from broilers. Materials and Methods: A total of 40 non-typhoidal S. enterica isolates were collected from 28 broiler chicken farms in four Iraqi Governorates. These isolates were examined for their susceptibility to 10 antimicrobial agents by disk-diffusion method followed by polymerase chain reaction assay for the detection of PMQR determinants and ESBLs genes. Results: Salmonella strains revealed high levels of resistance to the following antibiotics: Nalidixic acid 100%, levofloxacin (LEV) 97.5%, amoxicillin-clavulanic acid 95.0%, tetracycline 92.5%, and nitrofurantoin 80.0%. Otherwise, all isolates were susceptible to cefotaxime and ceftriaxone. All isolates were MDR, with 15 different profiles observed. Among 38 amoxicillin/clavulanic acid-resistant Salmonella isolates, 20 (52.6%) had the blaTEM gene, while blaSHV, blaCTX-M, and blaOXA genes were not detected. Only 5 (12.8%) out of 39 LEV-resistant isolates were positive for qnrB, three of which had blaTEM. No qnrC or qnrD, qnrS, aac(6`)-Ib-cr, qunA, and oqxAB genes were found in any of the tested isolates. Conclusion: This study demonstrates that broiler chickens may be considered a potential source for spreading MDR non-typhoidal Salmonella and ESBL traits in poultry production. Therefore, it is important to continuously monitor ESBL and PMQR genes to avoid the spread of resistant strains in the food chain and impact public health.

qnrVC occurs in different genetic contexts in Klebsiella and Enterobacter strains isolated from Brazilian coastal waters.

OBJECTIVES: In contrast to other qnr families, qnrVC has been reported mainly in Vibrio spp. and inserted in class 1 integrons. This study aimed to identify the variants of qnrVC genes detected in Klebsiella pneumoniae carbapenemase-2-producing Enterobacter and Klebsiella strains isolated from Brazilian coastal waters and the genetic contexts associated with their occurrence. METHODS: qnrVC variants were identified by Sanger sequencing. Stains were typified by pulsed-field gel electrophoresis. Antimicrobial susceptibility testing, conjugation assays, and whole genome sequencing (WGS) were applied to identify the strains' antimicrobial resistance profile, qnrVC and blaKPC-2 co-transference, and qnrVC genetic context. RESULTS: qnrVC1 was identified in 15 Enterobacter and 3 Klebsiella, and qnrVC4 in 2 Enterobacter strains. Pulsed-field gel electrophoresis revealed 12 clonal profiles of Enterobacter and one of Klebsiella. Strains were resistant to aminoglycosides, beta-lactams, fosfomycin, quinolones, and sulfamethoxazole-trimethoprim. Co-transference of qnrVC and blaKPC-2 were obtained from five representative Enterobacter strains, which showed resistance to ampicillin and amoxicillin-clavulanate, and reduced susceptibility to extended-spectrum cephalosporins, meropenem, and ciprofloxacin. WGS analysis from representative strains revealed one K. quasipneumoniae subsp. similipneumoniae, one E. soli, four E. kobei, and seven isolates belonging to Enterobacter Taxon 3. Long-read WGS showed qnrVC and blaKPC-2 were carried by the same replicon on Klebsiella and Enterobacter strains, and the qnrVC association with not previously described genetic environments composed of insertion sequences and truncated genes. These contexts occurred in small- and high-molecular-weight plasmids belonging to IncFII, IncP6, pKPC-CAV1321, and IncU groups. CONCLUSION: Our results suggest that the dissemination of qnrVC among Enterobacterales in Brazilian coastal waters is associated with several genetic recombination events.

Using Targeted Liquid Chromatography-Tandem Mass Spectrometry to Rapidly Detect ß-Lactam, Aminoglycoside, and Fluoroquinolone Resistance Mechanisms in Blood Cultures Growing E. coli or K. pneumoniae.

New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the detection of ß-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing Escherichia coli or Klebsiella pneumoniae complex. Selected targets were the ß-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, ANT(2 ' ' )-I, and APH(3')-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6')-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the E. coli porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing E. coli or K. pneumoniae complex.

Plasmid-mediated quinolone resistance determinants in fluoroquinolone-nonsusceptible Escherichia coli isolated from patients with urinary tract infections in a university hospital, 2009-2010 and 2020.

OBJECTIVES: This study aimed to characterize the plasmid-mediated quinolone resistance (PMQR) in fluoroquinolone nonsusceptible E. coli (FQNSEC) isolated from patients with urinary tract infections (UTIs) in 2019-2010 and 2020. METHODS: A total of 844 E. coli isolates were collected from UTI patients at National Cheng Kung University Hospital. The antimicrobial susceptibility of E. coli isolates to 21 antibiotics was determined by disk diffusion tests. The distribution of phylogenetic groups, virulence factor, and PMQR genes was determined by PCR. Conjugation assays were performed to investigate the transferability of qnr genes from FQNSEC isolates to E. coli C600. RESULTS: We found 211 (41.9%) and 152 (44.7%) E. coli isolates were FQNSEC in 2009-2010 and 2020, respectively. Phylogenetic group B2 was dominant in FQNSEC isolates (52.34%), followed by group F (10.47%), group B1 (9.64%), and group D (9.64%). FQNSEC isolates were more resistant to 17 of 19 tested antimicrobial agents, compared to the fluoroquinolone susceptible E. coli. PMQR screening results showed 34, 22, and 10 FQNSEC isolates containing aac(6')-Ib-cr, qnr genes, and efflux pump genes (qepA or oqxAB), respectively. PMQR E. coli isolates were more nonsusceptible to gentamicin, amoxicillin, ampicillin/sulbactam, imipenem, cefazolin, cefuroxime, cefmetazole, ceftriaxone, ceftazidime, and cefepime compared to non-PMQR FQNSEC. Moreover, 16 of 22 qnr-carrying plasmids were transferrable to the recipient C600. CONCLUSION: Here, we reported the high prevalence of MDR- and XDR-E. coli in FQNSEC isolates. Moreover, qnr-carrying plasmids were highly transferable and led to the resistance to other classes of antibiotics in the transconjugants.

A qnrD-Plasmid Promotes Biofilm Formation and Class 1 Integron Gene Cassette Rearrangements in Escherichia coli.

Bacteria within biofilms may be exposed to sub-minimum inhibitory concentrations (sub-MICs) of antibiotics. Cell-to-cell contact within biofilms facilitates horizontal gene transfers and favors induction of the SOS response. Altogether, it participates in the emergence of antibiotic resistance. Aminoglycosides at sub-MICs can induce the SOS response through NO accumulation in E. coli carrying the small plasmid with the quinolone resistance qnrD gene (pDIJ09-518a). In this study, we show that in E. coli pDIJ09-518a, the SOS response triggered by sub-MICs of aminoglycosides has important consequences, promoting genetic rearrangement in class 1 integrons and biofilm formation. We found that the integrase expression was increased in E. coli carrying pDIJ09-518a in the presence of tobramycin, which was not observed for the WT isogenic strain that did not carry the qnrD-plasmid. Moreover, we showed that biofilm production was significantly increased in E. coli WT/pDIJ09-518a compared to the WT strain. However, such a higher production was decreased when the Hmp-NO detoxification pathway was fully functional by overexpressing Hmp. Our results showing that a qnrD-plasmid can promote biofilm formation in E. coli and potentiate the acquisition and spread of resistance determinants for other antibiotics complicate the attempts to counteract antibiotic resistance and prevention of biofilm development even further. We anticipate that our findings emphasize the complex challenges that will impact the decisions about antibiotic stewardship, and other decisions related to retaining antibiotics as effective drugs and the development of new drugs.

Genomic analysis of qnr-harbouring IncX plasmids and their transferability within different hosts under induced stress.

BACKGROUND: Conjugative plasmids play a major role in the dissemination of antibiotic resistance genes. Knowledge of the plasmid characteristics and behaviour can allow development of control strategies. Here we focus on the IncX group of plasmids carrying genes conferring quinolone resistance (PMQR), reporting their transfer and persistence within host bacteria of various genotypes under distinct conditions and levels of induced stress in form of temperature change and various concentrations of ciprofloxacin supplementation. METHODS: Complete nucleotide sequences were determined for eight qnr-carrying IncX-type plasmids, of IncX1 (3), IncX2 (3) and a hybrid IncX1-2 (2) types, recovered from Escherichia coli of various origins. This data was compared with further complete sequences of IncX1 and IncX2 plasmids carrying qnr genes (n = 41) retrieved from GenBank and phylogenetic tree was constructed. Representatives of IncX1 (pHP2) and IncX2 (p194) and their qnrS knockout mutants, were studied for influence of induced stress and genetic background on conjugative transfer and maintenance. RESULTS: A high level of IncX core-genome similarity was found in plasmids of animal, environmental and clinical origin. Significant differences were found between the individual IncX plasmids, with IncX1 subgroup plasmids showing higher conjugative transfer rates than IncX2 plasmids. Knockout of qnr modified transfer frequency of both plasmids. Two stresses applied simultaneously were needed to affect transfer rate of wildtype plasmids, whereas a single stress was sufficient to affect the IncX ΔqnrS plasmids. The conjugative transfer was shown to be biased towards the host phylogenetic proximity. A long-term cultivation experiment pointed out the persistence of IncX plasmids in the antibiotic-free environment. CONCLUSIONS: The study indicated the stimulating effect of ciprofloxacin supplementation on the plasmid transfer that can be nullified by the carriage of a single PMQR gene. The findings present the significant properties and behaviour of IncX plasmids carrying antibiotic resistance genes that are likely to play a role in their dissemination and stability in bacterial populations.

Validation of a random Vibrio parahaemolyticus genomic library by selection of quinolone resistance in a heterologous host.

Vibrio parahaemolyticus is a shellfish-borne pathogen that is a highly prevalent causative agent of inflammatory gastroenteritis in humans. Genomic libraries have proven useful for the identification of novel gene functions in many bacterial species. In this study we prepared a library containing 40 kb fragments of randomly sheared V. parahaemolyticus genomic DNA and introduced this into Escherichia coli HB101 using a commercially available low copy cosmid system. In order to estimate coverage and suitability of the library and potentially identify novel antimicrobial resistance determinants, we screened for the acquisition of resistance to the fluoroquinolone norfloxacin - a phenotype exhibited by V. parahaemolyticus but not the heterologous E. coli host. Upon selection on solid medium containing norfloxacin, 0.52% of the library population was resistant, consistent with the selection of a single resistance locus. End-sequencing identified six distinct insert fragments. All clones displayed fourfold increased norfloxacin MIC compared with E. coli HB101 carrying an empty vector. The common locus contained within resistant clones included qnr, a previously described quinolone resistance gene. These results indicate that the library was unbiased, of sufficient coverage and that heterologous expression was possible. While we hope that this library proves useful for identifying the genetic determinants of complex phenotypes such as those related to virulence, not all norfloxacin resistance genes were detected in our screen. As such, we discuss the benefits and limitations of this approach for identifying the genetic basis of uncharacterized bacterial phenotypes.

Characterisation of plasmids harbouring qnrA1, qnrS1, and qnrB4 in E. coli isolated in the Philippines from food-producing animals and their products.

OBJECTIVES: Determinants showing plasmid-mediated quinolone resistance, which usually leads to antimicrobial ineffectiveness, have become an emerging clinical problem. In our previous study in the Philippines, a high prevalence of qnr determinants was found in clinical samples and food-producing animals and their food products. However, no qnr-carrying plasmids have been investigated in animals or animal-derived foods. Hence, in the present, we aimed to characterise qnr-carrying plasmids in Escherichia coli isolated from the food supply chain. METHODS: Plasmids from 44 qnr-positive isolates were assigned to incompatibility groups by Polymerase chain reaction (PCR)-based replicon typing, and the presence of ß-lactamase-encoding genes were investigated by PCR. Localisation of qnr in plasmids was determined by S1-PFGE and Southern blot hybridisation. The transferability of qnr-carrying plasmids was examined by conjugation analysis. RESULTS: Overall, 77.3% (95% confidence interval [CI]: 62.2-88.5) of the isolates harbouring qnr determinants were positive for seven plasmid types, and 56.8% concurrently harboured blaTEM-1. Plasmid IncFrepB was prevalent (65.9% [95% CI: 50.1-79.5]) among qnr determinants. Localisation of qnr determinants in IncFrepB and transferability of plasmids was further confirmed. CONCLUSION: The current study proved that qnr in E. coli isolated from food-producing animals and their food products could spread via plasmid IncFrepB upon selective pressure with quinolones or other antimicrobials. Therefore, to curb the emergence and spread of qnr-harbouring bacteria in the Philippines, prudent use of antimicrobials in animal production and stricter hygiene and food handling are recommended.

Global Distribution of Fluoroquinolone and Colistin Resistance and Associated Resistance Markers in Escherichia coli of Swine Origin - A Systematic Review and Meta-Analysis.

Background: Fluoroquinolones and polymyxins (colistin) are considered as critical drugs for human medicine. Antimicrobials of these classes are also used in swine production worldwide and this usage can contribute to selection of antimicrobial resistance (AMR), which is a threat to both human and animal health. Given the dynamic epidemiology of AMR, updating our knowledge regarding distribution and trends in the proportion of resistant bacteria is of critical importance. Objectives: The aim of this systematic review and meta-analysis was to describe the global prevalence of phenotypic and genotypic resistance to fluoroquinolones and colistin in Escherichia coli collected from swine. Results: Four databases (PubMed, PubAg, Web of Science, and CAB abstracts) and reports of national surveillance programs were scanned and 360 articles were included in the analysis. We identified higher prevalence levels of fluoroquinolone and colistin resistance in isolates from pig populations in Asia compared to Europe. The heterogeneity of pooled estimates was also higher in Asian countries suggesting that prevalence of AMR is still not fully characterized. There was a major knowledge gap about the situation of AMR in South American and African countries. We also identified key deficiencies in how AMR data was reported in the studies. A meta-analysis using 6,167 publicly available genomes of swine E. coli established the prevalence and global distribution of genetic determinants that can lead to fluoroquinolone and colistin resistance. Conclusion: This study provides the most comprehensive information on prevalence of phenotypic and genotypic resistance to key antimicrobials in pig populations globally. There is a need to establish national surveillance programs and effective policies, particularly in certain world regions, to curtail the threat of evolution of resistant isolates in swine production that can potentially contribute to public health detrimentally.

Burden of Fluoroquinolone Resistance in Clinical Isolates of Escherichia coli at the Ho Teaching Hospital, Ghana.

BACKGROUND: The growing burden of antibiotic resistance is a threat to the management of infections. Infections by Escherichia coli are routinely treated with fluoroquinolone antimicrobial agents. Due to their frequent use, there has been increasing resistance to these drugs. We set out to determine the burden of fluoroquinolone resistance among clinical E. coli isolates at the Ho Teaching Hospital, Ghana. METHODS: This was a cross-sectional study conducted from July 2018 to June 2019. One hundred and thirty-five E. coli isolates were cultured from various clinical samples. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method with discs of nalidixic acid (NAL), ciprofloxacin (CIP), norfloxacin (NOR) and levofloxacin (LEV). Deoxyribonucleic acid (DNA) was extracted from the resistant isolates for the detection of fluoroquinolone resistant genes by polymerase chain reaction. RESULTS: Ninety of the 135 isolates (66.7%) were resistant to at least one of the four fluoroquinolone drugs investigated. Resistance to NAL, CIP, NOR, and LEV was 51.0%, 51.1%, 38.8% and 35.7% respectively. Out of the fluoroquinolone resistant isolates, 69 carried one or more fluoroquinolone resistant genes. The predominant resistant genes were aac(6')-Ib-cr (48.9%) and qnrD (25.6%). Seven of the isolates carried both qnrS and aac(6')-Ib-cr genes. Two isolates carried 5 different fluoroquinolone resistant genes. CONCLUSION: High prevalence of resistance to 4 fluoroquinolone drugs was recorded with associated resistant genes. This is a threat to current efforts to control the spread of antibiotic resistance and calls for concerted efforts to curb the spread of these resistant organisms.

The prevalence of plasmid-mediated quinolone resistance genes among Escherichia coli strains isolated from urinary tract infections in southwest Iran.

BACKGROUND: The extensive and inappropriate use of quinolones, which are frequently used as an effective treatment for urinary tract infection (UTI) patients, has led to resistance to these antibiotics. This study was designed to determine the prevalence of quinolones resistance and the presence of plasmid-mediated quinolone resistance (PMQR) genes among extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates. METHODS: One hundred and fourteen E. coli isolates were collected from patients' urine samples. The susceptibility of isolates to selected antibiotics was tested by the Kirby-Bauer disk diffusion method. ESBL-producing isolates were identified phenotypically using a combination disk test. Using specific primers, the frequency of aac (6')-Ib, qnrA, qnrB, qnrC, qnrD, qnrS, and qepA genes was investigated by polymerase chain reaction (PCR). RESULTS: Among 26 ESBL-producing isolates, the highest resistance rate was observed toward nalidixic acid (80.8%) and ciprofloxacin (61.5%), respectively. Ninety-seven (85%) of all isolates harbored at least one PMQR gene, the most frequent one being aac(6')-Ib-cr variant (47.4%). Coexistence of aac(6')-Ib-cr variant and qnrB were the most broadly distributed genotype among quinolone resistance isolates. Notably, none of the isolates contained the qnrC, qnrD, and qepA genes. CONCLUSIONS: Our results highlight the significant prevalence of PMQR genes in ESBL-producing E. coli isolates in this region. Also, the aac (6')-Ib-cr variant was the most frequent gene, particularly in ESBL positive isolates. A regular periodic monitoring program is needed to control and hinder the more spread of antibiotic resistance phenomenon and contributed genes among UTI-causing E. coli isolates.