Mutations in the efflux regulator gene oqxR provide a simple genetic switch for antimicrobial resistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.

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".

Transferable Mechanisms of Quinolone Resistance from 1998 Onward.

While the description of resistance to quinolones is almost as old as these antimicrobial agents themselves, transferable mechanisms of quinolone resistance (TMQR) remained absent from the scenario for more than 36 years, appearing first as sporadic events and afterward as epidemics. In 1998, the first TMQR was soundly described, that is, QnrA. The presence of QnrA was almost anecdotal for years, but in the middle of the first decade of the 21st century, there was an explosion of TMQR descriptions, which definitively changed the epidemiology of quinolone resistance. Currently, 3 different clinically relevant mechanisms of quinolone resistance are encoded within mobile elements: (i) target protection, which is mediated by 7 different families of Qnr (QnrA, QnrB, QnrC, QnrD, QnrE, QnrS, and QnrVC), which overall account for more than 100 recognized alleles; (ii) antibiotic efflux, which is mediated by 2 main transferable efflux pumps (QepA and OqxAB), which together account for more than 30 alleles, and a series of other efflux pumps (e.g., QacBIII), which at present have been sporadically described; and (iii) antibiotic modification, which is mediated by the enzymes AAC(6')Ib-cr, from which different alleles have been claimed, as well as CrpP, a newly described phosphorylase.

Plasmid-mediated quinolone resistance (PMQR) among Enterobacteriales in Latin America: a systematic review.

The plasmid-mediated quinolone resistance (PMQR) genes have changed the resistance pattern to quinolones, especially among Enterobacteriales. The dissemination of these genes in Latin American countries, where the prescription of fluoroquinolones is high, has been described in several studies; however, no review of the impact of PMQR in this continent has been conducted. This review summarized current knowledge about the circulation of PMQR among Enterobacteriales in Latin American. After the search and selection, 61 articles were included in the study. Most of studies reported PMQR genes among Enterobacteriales from human (47/61, 77%) and animal (18/61, 29.5%) samples, recovered mainly in Brazil (23/61, 37.7%), Mexico (11/61, 18%), and Uruguay (7/61, 11.5%). Nine different PMQR genes (qnrA, qnrB, qnrS, qnrD, qnrE, aac-(6')-Ib-cr, oqxA, oqxB, and qepA) were found in Latin America, with aac (6')-Ib-cr (37/61, 60.6%) and qnrB (26/61, 42.6%) being the most frequently reported. Escherichia coli (40/61, 65.6%) was the species most frequently reported to carry a PMQR gene, followed by Klebsiella pneumoniae (24/61, 39.3%), Enterobacter cloacae (15/61, 24.6%), and Salmonella spp. (14/61, 22.9%). Thus, this review provides important information which might help in designing measures to control the spread of quinolone resistance determinants on this continent.

Co-occurrence of biofilm formation and quinolone resistance in Salmonella enterica serotype typhimurium carrying an IncHI2-type oqxAB-positive plasmid.

The objective of this study was to investigate the co-occurrence of biofilms and quinolone resistance in Salmonella enterica serotype Typhimurium mediated by IncHI2-type oqxAB-positive plasmids. Among the 40 Salmonella strains, we found that 27 isolates formed biofilms and displayed identical multidrug-resistance profiles to ciprofloxacin, doxycycline, sulfamethoxazole-trimethoprim, ampicillin and streptomycin, based on biofilm formation assays and antimicrobial susceptibility testing. In particular, a single S. Typhimurium isolate named SC523 produced the thickest biofilms and exhibited the highest-level resistance (MIC = 8 µg/mL) to ciprofloxacin compared to those of the other isolates. The detection of known plasmid-mediated quinolone resistance (PMQR) genes and point mutations in the quinolone resistance-determining region (QRDR) by PCR assay showed that oqxAB genes were present in 27 biofilm-positive isolates. Conjugation experiments, S1-pulse-field gel electrophoresis and biofilm formation assays demonstrated that the conjugative plasmid that encoded biofilms and quinolone resistance in Salmonella SC523 could be transferred to a recipient with a frequency of 4.7 × 10-3 per recipient cell. The results of PCR-based replicon typing (PBRT) showed that the IncHI2-type plasmids accounted for 100% of the biofilm-oqxAB-positive isolates and transconjugants. The sequence analysis of Salmonella SC523 confirmed that the oqxAB cassette and fourteen DNA transfer genes in the IncHI2-type oqxAB-positive conjugative plasmid were genetically responsible for the phenotypic quinolone resistance and biofilm formation. The conclusion is that the IncHI2-type plasmid in S. Typhimurium isolate from chicken farm was identified and sequenced, which contained oqxAB and tra/trh and encoded quinolone resistance and biofilms, and could be transferred to recipients through conjugation. Notably, the prevalence of IncHI2-type biofilm-oqxAB-positive plasmids in animal-origin Salmonella poses a threat to public health, as these Salmonella from poultry farms show a decreased susceptibility to quinolones and could spread to humans.

Plasmid-mediated quinolone resistance: Two decades on.

After two decades of the discovery of plasmid-mediated quinolone resistance (PMQR), three different mechanisms have been associated to this phenomenon: target protection (Qnr proteins, including several families with multiple alleles), active efflux pumps (mainly QepA and OqxAB pumps) and drug modification [AAC(6')-Ib-cr acetyltransferase]. PMQR genes are usually associated with mobile or transposable elements on plasmids, and, in the case of qnr genes, are often incorporated into sul1-type integrons. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. Although the three PMQR mechanisms alone cause only low-level resistance to quinolones, they can complement other mechanisms of chromosomal resistance to reach clinical resistance level and facilitate the selection of higher-level resistance, raising a threat to the treatment of infections by microorganisms that host these mechanisms.

First survey on antibiotic resistance markers in Enterobacteriaceae in Cochabamba, Bolivia.

A molecular survey was conducted in Cochabamba, Bolivia, to characterize the mechanism involved in the resistance to clinically relevant antibiotics. Extended Spectrum ß-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) markers were investigated in a total of 101 oxyimino-cephalosporin-resistant enterobacteria recovered from different health centers during four months (2012-2013). CTX-M enzymes were detected in all isolates, being the CTX-M-1 group the most prevalent (88.1%). The presence of blaOXA-1 was detected in 76.4% of these isolates. A high quinolone resistance rate was observed among the included isolates. The aac(6')-Ib-cr gene was the most frequent PMQR identified (83.0%). Furthermore, 6 isolates harbored the qnrB gene. Interestingly, qepA1 (6) and oqxAB (1), were detected in 7 Escherichia coli, being the latter the first to be reported in Bolivia. This study constitutes the first molecular survey on resistance markers in clinical enterobacterial isolates in Cochabamba, Bolivia, contributing to the regional knowledge of the epidemiological situation. The molecular epidemiology observed herein resembles the scene reported in South America.

Challenges to accurate susceptibility testing and interpretation of quinolone resistance in Enterobacteriaceae: results of a Spanish multicentre study.

OBJECTIVES: The objective of this study was to evaluate the proficiency of Spanish laboratories with respect to accurate susceptibility testing and the detection and interpretation of quinolone resistance phenotypes in Enterobacteriaceae. METHODS: Thirteen strains of Enterobacteriaceae were sent to 62 participating centres throughout Spain; strains harboured GyrA/ParC modifications, reduced permeability and/or plasmid-mediated quinolone resistance genes. The centres were requested to evaluate nalidixic acid and five quinolones, provide raw/interpreted clinical categories and to detect/infer resistance mechanisms. Consensus results from reference centres were used to assign minor, major and very major errors (mEs, MEs and VMEs, respectively). RESULTS: Susceptibility testing in the participating centres was frequently performed using the MicroScan WalkAway, Vitek 2 and Wider systems (48%, 30% and 8%, respectively). CLSI/EUCAST breakpoints were used in 71%/29% of the determinations. The percentage of VMEs for all quinolones was well below 2%. Only ofloxacin and moxifloxacin showed higher values for raw VMEs (6.6%), which decreased to 0% and 2.9%, respectively, in the interpreted VMEs. These errors were particularly associated with the CC-03 strain [qnrS2 + aac(6')-Ib-cr]. For MEs, percentages were always <10%, except in the case of ofloxacin and nalidixic acid. There was a significantly higher percentage of all types of errors for strains whose MICs were at the border of clinical breakpoints. CONCLUSIONS: The use of different breakpoints and methods, the complexity of mutation-driven and transferable resistance mechanisms and the absence of specific tests for detecting low-level resistance lead to high variability and represent a challenge to accuracy in susceptibility testing, particularly in strains with MICs on the border of clinical breakpoints.

Prevalence of plasmid-mediated quinolone resistance genes among ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from blood cultures in Korea.

OBJECTIVES: To analyze the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from patients at a tertiary care hospital in Korea. METHODS: A total of 102 nonduplicate isolates of ciprofloxacin-intermediate or ciprofloxacin-resistant E coli (n=80) and K pneumoniae (n=22) from blood cultures were obtained. The qnr (qnrA, qnrB, qnrS), aac(6')-Ib-cr, qepA and oqxAB genes were detected using polymerase chain reaction (PCR) and confirmed using direct sequencing. To determine whether the PMQR-positive plasmid was horizontally transferable, conjugation experiments were performed. RESULTS: Of the 102 isolates, 81 (79.4%) had one or more PMQR genes; these consisted of 59 (73.8%) E coli and 22 (100%) K pneumoniae isolates. The qnr genes were present in 15 isolates (14.7%): qnrB4 was detected in 10.8% and qnrS1 was detected in 3.9%. The aac(6')-Ib-cr, qepA and oqxAB genes were detected in 77.5%, 3.9% and 10.8%, respectively. In conjugation experiments, PMQR genes were successfully transferred from seven (8.6%) isolates. The range of minimum inhibitory concentrations of ciprofloxacin for these seven transconjugants increased to 0.5 mg/L to 1 mg/L, which was 16- to 33-fold that of the recipient E coli J53 bacteria. CONCLUSIONS: PMQR genes were highly prevalent among ciprofloxacin-nonsusceptible E coli and K pneumoniae from blood cultures in the authors' hospital. Therefore, it is necessary to monitor for the spread of PMQR genes of clinical isolates and to ensure careful antibiotic use in a hospital setting.

OBJECTIFS: Analyser la prévalence des déterminants de la résistance à la quinolone à médiation plasmidique (RQMP) en cas d'Escherichia coli et de Klebsiella pneumoniae non susceptibles à la ciprofloxacine, isolés chez des patients d'un hôpital de soins tertiaires de la Corée. MÉTHODOLOGIE: Au total, les chercheurs ont obtenu 102 isolats non dupliqués d'E coli (n=80) et de K pneumoniae (n=22) moyennement résistants ou résistants à la ciprofloxacine dans les hémocultures. Ils ont décelé les gènes qnr (qnrA, qnrB, qnrS), aac(6')-Ib-cr, qepA et oqxAB au moyen de la réaction en chaîne de la polymérase (PCR) et les ont confirmés par séquençage direct. Pour déterminer si les plasmides ayant une RQMP pouvaient opérer un transfert horizontal, les chercheurs ont effectué des expériences de conjugaison. RÉSULTATS: Sur les 102 isolats, 81 (79,4 %) avaient au moins un gène de RQMP. De ce nombre, 59 (73,8 %) étaient des isolats d'E coli et 22 (100 %), de K pneumoniae. Les gènes qnr étaient présents dans 15 isolats (14,7 %), soit 10,8 % de gène qnrB4 et 3,9 % de gène qnrS1. Les gènes aac(6')-Ib-cr, qepA et oqxAB ont été décelés dans 77,5 %, 3,9 % et 10,8 % des isolats, respectivement. Dans les expériences de conjugaison, sept isolats (8,6 %) ont entraîné un transfert des gènes de RQMP. La plage de concentrations inhibitrices minimales de la ciprofloxacine de ces sept produits de transconjugaison est passée de 0,5 mg/L à 1 mg/L, soit 16 fois à 33 fois plus que celles des bactéries d'E coli J53 des receveurs. CONCLUSIONS: Les gènes de RQMP étaient hautement prévalents dans les hémocultures d'E coli et de K pneumoniae non susceptibles à la ciprofloxacine à l'hôpital des auteurs. Par conséquent, il faut surveiller la propagation des gènes de RQMP dans les isolats cliniques et vérifier attentivement l'utilisation des antibiotiques en milieu hospitalier.

First Emergence of NDM-5 and OqxAB Efflux Pumps Among Multidrug-Resistant Klebsiella pneumoniae Isolated from Pediatric Patients in Assiut, Egypt.

Introduction: New Delhi metallo-ß-lactamase (NDM)-producing K. pneumoniae poses a high risk, especially among Egyptian pediatric patients who consume carbapenems antibiotics very widely and without adequate diagnostic sources. In addition, presence of efflux pump genes such as OqxAB increases resistance against many groups of antimicrobials which exacerbates the problem faced for human health. This study aimed to determine NDM variants among K. pneumoniae strains isolated from pediatric patients in Egypt, analyze the presence of OqxAB genes, and molecular characterization of blaNDM-5-positive K. pneumoniae. Methods: Fifty-six K. pneumoniae isolates were recovered from pediatric patients, and tested for carbapenemase by modified carbapenem inactivation methods (mCIM) test. Minimum inhibitory concentrations of meropenem and colistin were determined by meropenem E-test strips and broth microdilution, respectively. PCR was used for the detection of the resistant genes (ESBL gene (blaCTX-M), carbapenemase genes (blaNDM, blaKPC) colistin resistant (mcr1, mcr2)) and genes for efflux pump (oqxA and oqxB). BlaNDM was sequenced. The effect of efflux pump in NDM-5-producing isolates was assessed by measuring MIC of ciprofloxacin and meropenem before and after exposure to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The horizontal gene transfer ability of blaNDM-5 was determined using liquid mating assay and PCR-based replicon typing (PBRT) was done to determine the major plasmid incompatibility group. Results: Twenty-nine isolates were positive for blaNDM-1, nine isolates were positive for blaNDM-5, and 15 isolates were positive for blaKPC. There is a significant increase of meropenem MIC of NDM-5-positive isolates compared with NDM-1-positive isolates. In addition, 38 isolates were positive for CTX-M, and 15 isolates were positive for mcr1. Both OqxA and OqxB were detected in 26 isolates and 13 isolates were positive for OqxA while 11 isolates were positive for OqxB only. All NDM-5-producing isolates except one isolate could transfer their plasmids by conjugation to their corresponding transconjugants (E. coli J53). Plasmid replicon typing showed that FII was predominant in NDM-5-producing K. pneumoniae. Similar strains were found between the three isolates and similarity was also detected between the two isolates. Conclusion: The highly resistant K. pneumoniae producing blaNDM-5 type was firstly isolated from pediatric patients. The association of efflux pump genes such as OqxAB is involved in resistance to ciprofloxacin. This highlighted the severity risk of blaNDM-5-positive K. pneumonia as it could transfer blaNDM-5 to other bacteria and has more resistance against carbapenems. This underlines the importance of continuous monitoring of infection control guidelines, and the urgent need for a national antimicrobial stewardship plan in Egyptian hospitals.

Transcriptional Regulation and Functional Characterization of the Plasmid-Borne oqxAB Genes in Salmonella Typhimurium.

Coexistence of oqxAB and aac(6')-Ib-cr is often associated with the expression of fluoroquinolone resistance in Salmonella. The actual role of the plasmid-borne oqxAB gene and its regulatory mechanism compared to its chromosomally encoded counterpart in Klebsiella pneumoniae remain unclear We found that cloning of oqxAB gene only or chromosomally encoded oqxABR (ABRc) locus did not lead to an increase of ciprofloxacin (CIP) minimum inhibitory concentration (MIC) in S. Typhimurium, while cloning of the plasmid-encoded oqxABR (ABRp) locus led to a 4-fold increase in CIP MIC, reaching 0.0065 µg/mL. The co-carriage of these constructs with aac(6')-Ib-cr further increased the CIP MIC to 0.25 µg/mL in S. Typhimurium carrying aac(6')-Ib-cr and ABRp. Analysis of the transcription start site sequences showed that the expression level of suppressor protein gene, oqxR, in strains carrying ABRp was lower than that of its chromosomal counterpart due to the truncated promoter region in ABRp. The lower expression of OqxR in ABRp led to the overexpression of OqxAB, which elevated CIP MIC and exhibited a synergistic antimicrobial effect with the aac(6')-Ib-cr gene product to confer intermediate CIP (MIC = 0.25 µg/mL) in S. Typhimurium. Global transcriptional regulators in S. Typhimurium did not seem to play a role in regulating the plasmid-borne oqxAB genes. In conclusion, findings in this work showed that neither aac(6')-Ib-cr nor oqxABRp, but the combination of both genes, could mediate intermediated resistance to fluoroquinolone in Salmonella. The truncated promoter region in the oqxR gene of the plasmid-encoded locus led to the constituted expression of oqxAB genes. IMPORTANCE The transferable mechanisms of quinolone resistance (TMQR) gene, oqxAB, has been widely detected in Salmonella and is commonly associated with aac(6')-Ib-cr. It is thought to be associated with fluoroquinolone resistance, while its ancestor gene from K. pneumoniae is not. This study evaluated the actual role of the plasmid-borne oqxAB genes in Salmonella and showed that it was not able to mediate intermediated resistance to fluoroquinolone and only did so when it coexisted with aac(6')-Ib-cr. Chromosomally encoded oqxABRc from K. pneumoniae was not able to mediate enhanced CIP MIC due to tight regulation by the suppressor oqxR. However, plasmid-encoded oqxABRp enabled oqxAB to be expressed constitutionally due to the truncated promoter region of oqxR, leading to lower expression of the suppressor oqxR. This study clarified the roles of oqxAB and aac(6')-Ib-cr in mediating fluoroquinolone resistance in Salmonella and provides insights into the regulation of plasmid-encoded TMQR determinant, oqxAB.

RamA upregulates multidrug resistance efflux pumps AcrAB and OqxAB in Klebsiella pneumoniae.

Overexpression of the acrAB genes regulated by RamA and overexpression of oqxAB regulated by RarA have been reported to mediate multidrug resistance in Gram-negative bacilli. In this study, regulation of acrAB and oqxAB simultaneously by the global regulator RamA was investigated in a multidrug-resistant Klebsiella pneumoniae clinical isolate (KP22) resistant to tigecycline and other antimicrobials. KP22 overexpressed ramA due to a ramR mutation, along with an unexpected overexpression of oqxB. Deletion of ramA led to a 16-fold decrease in the tigecycline minimum inhibitory concentration (MIC) with decreased expression of acrB (4.3-fold) and oqxB (7.1-fold) compared with KP22. Transcomplementation of KP22ΔramA with the wild-type ramA gene restored the tigecycline MIC and upregulation of the acrB (3.9-fold) and oqxB (4.0-fold) genes compared with KP22. When oqxB was knocked out, MICs of ciprofloxacin, olaquindox and nitrofurantoin were considerably decreased, while deletion of acrB led to MIC decreases for cefepime, piperacillin/tazobactam and tigecycline in addition to the above three antimicrobials. The results of electrophoretic mobility shift assay showed that RamA could bind the promoter regions of both the acrAB and oqxAB operons. This study demonstrates for the first time that RamA can directly regulate multidrug resistance efflux pumps AcrAB and OqxAB in K. pneumoniae.

Molecular epidemiology and nitrofurantoin resistance determinants of nitrofurantoin-non-susceptible Escherichia coli isolated from urinary tract infections.

OBJECTIVES: The worldwide emergence of multidrug-resistant uropathogens has resulted in the revival of old antibiotics such as nitrofurantoin (NIT) for the treatment of uncomplicated urinary tract infections (UTIs). This study aimed to identify determinants of NIT resistance and to investigate the genetic diversity of NIT-resistant (NIT-R) Escherichia coli isolates. METHODS: Six NIT-R and three NIT-susceptible clinical E. coli isolates from patients with UTI were studied. The susceptibility of the isolates to various classes of antibiotics was evaluated by disk diffusion. The presence of plasmid-encoded efflux pump genes (oqxA and oqxB) was investigated by PCR. Nucleotide sequences of the nfsA, nfsB and ribE genes were determined. The genetic relatedness of the NIT-R isolates was evaluated by multilocus sequence typing (MLST). RESULTS: All six NIT-R isolates were characterised with high-level NIT resistance (MIC ≥ 512 mg/L) and they belonged to five distinct STs including ST131 (n = 2), ST73, ST405, ST10 and ST354 (n = 1 each). Amikacin, carbapenems, minocycline, tigecycline and fosfomycin were the most active agents against the studied uropathogens. The oqxA and oqxB genes were not detected in any isolate. All NIT-R isolates harboured inactivating genetic alterations in nfsA and nfsB [NfsA H11Y, S33N, S38Y, W212R substitutions, Δg638 (frameshift), Δa64-g73 (frameshift) and NfsB F84S, P45S, W94Stop, E197Stop substitutions, ΔnfsB locus]. The ribE gene of most isolates was unaffected, except for one isolate co-harbouring a deleterious RibE G85C substitution and NfsA/B alterations. CONCLUSION: NIT resistance in the studied E. coli isolates was mainly mediated by nfsA and nfsB alterations.

oqxAB-Positive IncHI2 Plasmid pHXY0908 Increase Salmonella enterica Serotype Typhimurium Strains Tolerance to Ciprofloxacin.

Salmonella enterica serotype Typhimurium is a major global food-borne pathogen and causes life-threatening infections. Although the resistance mechanisms to fluoroquinolones in S. Typhimurium had been well-defined, tolerance to fluoroquinolones and the associated mechanism for this are obscure. In the current work, we investigated an oqxAB-positive plasmid pHXY0908 and analyzed its role in S. Typhimurium tolerance to ciprofloxacin using time-kill, transcriptome sequencing and real-time PCR. S. Typhimurium ATCC14028 could survive under lethal concentrations of ciprofloxacin after acquiring plasmid pHXY0908. Transcriptome sequence analysis showed the chromosomal genes were systematically regulated after acquiring this plasmid suggesting an interaction between chromosome and plasmid. Additionally, the chromosomal efflux pump genes acrB, acrA, tolC, and yceE were up-regulated after acquiring plasmid pHXY0908 suggesting that these efflux pumps may contribute to the survival of ATCC14028 exposed to the lethal concentrations of ciprofloxacin. In conclusion, this is the first known report demonstrating that an IncHI2 type plasmid harboring oqxAB could assist S. Typhimurium survival under lethal concentrations of ciprofloxacin.

The nature and epidemiology of OqxAB, a multidrug efflux pump.

Background: OqxAB efflux pump has been found to mediate multidrug resistance (MDR) in various bacteria over the past decades. The updates on the nature and epidemiology of OqxAB efflux pump need to be fully reviewed to broaden our understanding of this MDR determinant. Methods: A literature search using the keyword of "oqxAB" was conducted in the online databases of Pubmed and ISI Web of Science with no restriction on the date of publication. The 87 publications were included into this review as references due to their close relevance to the nature and/or epidemiology of OqxAB efflux pump. Results: The oqxAB gene generally locates on chromosome and/or plasmids flanked by IS26-like elements in clinical isolates of Enterobacteriaceae and Klebsiella pneumoniae, conferring low to intermediated resistance to quinoxalines, quinolones tigecycline, nitrofurantoin, several detergents and disinfectants (benzalkonium chloride, triclosan and SDS). It could co-spread with other antimicrobial resistance genes (blaCTX-M, rmtB and aac(6')-Ib etc.), virulence genes and heavy metal resistance genes (pco and sil operons). Both RarA (activator) and OqxR (repressor) play important roles on regulation of the expression of OqxAB. Conclusions: The dissemination of oqxAB gene may pose a great risk on food safety and public health. Further investigation and understanding of the natural functions, horizontal transfer, and regulation mechanism of the OqxAB efflux pump will aid in future strategies of antimicrobial usage.

Prevalence and molecular characterization of oqxAB in clinical Escherichia coli isolates from companion animals and humans in Henan Province, China.

Background: The plasmid-encoded multidrug efflux pump oqxAB confers bacterial resistance primarily to olaquindox, quinolones, and chloramphenicol. The aims of this study were to investigate the prevalence of oqxAB among Escherichia coli isolates from dogs, cats, and humans in Henan, China and the susceptibilities of E. coli isolates to common antibiotics. Methods: From 2012 to 2014, a total of 600 samples which included 400 rectal samples and 200 clinical human specimens were tested for the presence of E. coli. All isolates were screened for oqxAB genes by PCR and sequencing. The MICs of 11 antimicrobial agents were determined by the broth microdilution method. A total of 30 representative oqxAB-positive isolates were subjected to ERIC-PCR and MLST. Additionally, conjugation experiments and southern hybridizations were performed. Results: Of 270 isolates, 58.5% (62/106) of the isolates from dogs, 56.25% (36/64) of the isolates from cats, and 42.0% (42/100) of the isolates from humans were positive for the oqxAB. Olaquindox resistance was found for 85.7%-100% of oqxAB-positive isolates. Of oqxAB-positive isolates from dogs, cats, and humans, ciprofloxacin resistance was inspected for 85.8%, 59.1%, and 93.8%, respectively. Several oqxAB-positive isolates were demonstrated by ERIC-PCR and MLST, and have high similarity. Phylogenetic analysis showed that oqxAB-positive isolates could be divided into 7 major clusters. OqxAB-positive conjugants were obtained, southern hybridization verified that the oqxAB gene complex was primarily located on plasmids. Conclusion: In conclusion, oqxAB-positive isolates were widespread in animals and humans in Henan, China. Carriage of oqxAB on plasmids of E. coli isolates may facilitate the emergence of multidrug resistant and its transmission via horizontal transfer, and might pose a potential threat to public health.

Identification and prevalence of RND family multidrug efflux pump oqxAB genes in Enterococci isolates from swine manure in China.

PURPOSE: The resistance/nodulation/cell division (RND) family multidrug efflux pump, OqxAB, has been identified as one of the leading mechanisms of plasmid-mediated quinolone resistance and has become increasingly prevalent among Enterobacteriaceae in recent years. However, oqxAB genes have not yet been reported in Enterococcus isolates. The aim of the present study was to identify the oqxAB genes and investigate their prevalence among Enterococcus from swine manure in China. METHODOLOGY: The oqxAB genes were screened in 87 Enterococcus isolates by PCR. The transferability of the oqxAB genes in Enterococcus was determined by conjugation experiments. The genetic environment of oqxAB genes was investigated by cloning experiments, PCR mapping and sequencing. RESULTS: A high prevalence (86.2 %) of olaquindox resistance was observed in Enterococcus and 98.9 % isolates exhibited multidrug-resistance phenotypes. The occurrence of oqxA and oqxB in Enterococcus was also high (79.3 and 65.5 %, respectively). Sequence analysis of the cloned fragment indicated that the oqxAB cassette was linked to an incomplete Tn5 transposon containing aph(3')-IIa and flanked by IS26 [IS26-oqxAB-IS26-aph(3')-IIa]. The oqxAB-aph(3')-IIa-positive transconjugant or transformant showed resistance or reduced susceptibility to enrofloxacin, ciprofloxacin, olaquindox, mequindox, florfenicol, neomycin and kanamycin. CONCLUSION: This is the first time that the oqxAB genes have been identified in Enterococcus faecalis from swine manure. The genetic linkage of oqxAB-aph(3')-IIa in Enterococcus has not been described before. The high prevalence of oqxAB genes in Enterococcus suggests that it may constitute a reservoir for oqxAB genes and pose a potential threat to public health.

Characterization of quinolone resistance mechanisms in Enterobacteriaceae isolated from companion animals in Europe (ComPath II study).

ComPath is an ongoing European programme dedicated to monitor antibiotic susceptibility of bacterial pathogens from diseased dogs and cats. The objective was to characterize determinants associated with quinolone resistance among 160 enrofloxacin non-wild type strains (100 Escherichia coli, 45 Proteus mirabilis, 14 Klebsiella pneumoniae, 1 Enterobacter cloacae) selected among 843 non-duplicate Enterobacteriaceae isolates collected in 12 European countries (2013-2014). These strains with non-wild type MICs of ≥0.25 mg/L, for P. mirabilis ≥0.5 mg/L, were screened for PMQR determinants (qnr, oqxAB, qepA and aac(6')-Ib-cr), and for QRDR mutations in gyrA, gyrB, parC and parE genes. Among them, 20% (32/160) carried at least one PMQR (18/32 qnrB, qnrS or qnrD, 10/32 aac(6')-Ib-cr and 13/32 oqxAB), and 80% (128/160) no PMQR. qnrB was detected in 3 E. coli, 2 K. pneumoniae and 1 E. cloacae strains; qnrS in 6 E. coli and 1 P. mirabilis and aac(6')-Ib-cr in 4 E. coli, 5 K. pneumoniae and 1 E. cloacae strains. All qnrD1 were detected in P. mirabilis. oqxAB was detected in 12/14 K. pneumoniae and 1 E. cloacae. No qepA genes were detected. From the 32 PMQR-positive strains, 10 showed enrofloxacin MICs ≤2 mg/L and 22 MICs ≥8 mg/L, the latter carrying 1-4 mutations in QRDR. For the 128 non-PMQR strains, 37 showed enrofloxacin MICs ≤2 mg/L with 0-2 QRDR mutations, and 91 MICs ≥4 mg/L carrying 1-4 QRDR mutations. In conclusion, qnr was the major PMQR and qnrD only detected in Proteeae. Mutations in QRDR play a markedly greater role in mediating fluoroquinolone resistance than PMQR.

Molecular characterization of antimicrobial susceptibility of Salmonella isolates: First identification of a plasmid carrying qnrD or oqxAB in Taiwan.

BACKGROUND/PURPOSE: The aim of this study is to characterize antibiotic-nonsusceptible Salmonella isolates in Taiwan. METHODS: A total of 76 Salmonella isolates showing lower susceptibility to cephalosporins or quinolones were identified from 1416 clinical isolates from 1999 to 2008. Minimal inhibitory concentrations for selected antimicrobial agents were tested by the agar dilution method. Antibiotic resistance-related genes were determined by polymerase chain reaction (PCR) combined with sequencing. Southern blotting, conjugation tests, and transformation tests were used to characterize plasmid-mediated quinolone resistance (PMQR) determinants. RESULTS: The observed nonsusceptible phenotypes of 76 isolates were against cefoxitin (57.9%), cefotaxime (43.4%), ceftazidime (40.8%), ceftriaxone (42.1%), cefepime (5.3%), ciprofloxacin (80.3%), and levofloxacin (81.6%). Among 44 cephalosporin-resistant isolates, TEM-1, CMY-2, CMY-14, CTX-M-3-like and CTX-M-15-like determinants were present in 31 (70.5%), 32 (72.7%), 1 (2.3%), 1 (2.3%), and 1 (2.3%) of isolates, respectively. PCR screening for PMQR genes of 62 quinolone-nonsusceptible isolates revealed the presence of qnrS, qnrD, aac(6')-Ib-cr, and oqxAB in 3 (4.8%), 2 (3.2%), 1 (1.6%), and 10 (16.1%) isolates, respectively. Among 36 isolates showing high resistance to quinolones, S83F/D87N and S83F/D87G amino acid substitutions of GyrA were found in 29 (80.6%) and 6 (16.7%) isolates, respectively. Moreover, among quinolone highly resistant isolates, eight (22.2%) of isolates showed over-expression of the PAßN-sensitive efflux pump. Transformants and transconjugants harboring qnrD- or oqxAB-plasmids showed decreased susceptibility to quinolones. CONCLUSION: GyrA mutations are the major mechanisms associated with quinolone-resistant Salmonella isolates in Taiwan. Overproduction of efflux pump genes and the presence of qnr and oqxAB play additional roles in reduced susceptibility to quinolones.

Impact of enrofloxacin and florfenicol therapy on the spread of OqxAB gene and intestinal microbiota in chickens.

Horizontal transfer of plasmid-encoded multidrug-resistant determinants is a major health problem and has attracted much public attention. We studied the dissemination of the efflux pump gene oqxAB located on transferable plasmid pHXY0908 between Salmonella Typhimurium and Escherichia coli in the gut of chickens. After an inoculation with Salmonella Typhimurium harboring oqxAB-bearing plasmid pHXY0908, chickens were treated with enrofloxacin and florfenicol. Inoculated, but non-treated chickens were included as a control group. Our results revealed that commensal E. coli isolates from the gut of chickens acquired the oqxAB-bearing plasmid in both treated and non-treated groups. Additionally, in the florfenicol treatment group, the average isolation rate of oqxAB-positive E. coli was significantly higher than that in the non-treated group. PFGE analysis showed that oqxAB-positive E. coli strains belonged to different patterns with one predominating. Moreover, multilocus sequence typing analysis revealed that E. coli ST533 was closely associated with the spread of oqxAB gene. qPCR analysis indicated that antibiotic administration provided selective advantages for sustaining a significantly high level of oqxAB gene from the DNA extracted from the feces. There was also a fluctuation in the intestinal microbiota with antibiotic therapy. In conclusion, the present study indicates that the oqxAB gene could be readily spread within the intestinal microflora. This could be enhanced by administrated with clinical doses of florfenicol and enrofloxacin, resulting in the enlargement of resistance gene reservoirs. In addition, ST533 E. coli isolates were found to contribute to transfer of the oqxAB gene.