Molecular characterisation of an outbreak of NDM-7-producing Klebsiella pneumoniae reveals ST11 clone expansion combined with interclonal plasmid dissemination.

The aim of this study was to characterise a hospital outbreak of NDM-7-producing Klebsiella pneumoniae associated with the successful multidrug-resistant (MDR) high-risk clone ST11 between 2017 and 2019 in southern Spain. A total of 46 NDM-7-producing isolates were recovered during the outbreak, including 16 from clinical samples, 27 from surveillance samples and 3 from environmental samples. All isolates were MDR, including carbapenem-resistant. Pulsed-field gel electrophoresis using XbaI restriction enzyme (XbaI-PFGE) showed three pulsotypes belonging to three different clones by multilocus sequence typing (MLST): ST307 (1 isolate); ST152 (1 isolate); and ST11 (44 isolates). Representative isolates were selected for characterisation of blaNDM-7-carrying plasmids using PCR-based replicon typing and whole-genome sequencing analysis. IncX3 plasmids containing NDM-7 were identified in the three clones. The blaNDM-7-carrying plasmids from the ST307 and ST11 clones were identical and were very similar to the IncX3 NDM-7 plasmid previously described. The NDM-7 carbapenemase was introduced into the hospital by means of the ST307 clone, while the ST11 high-risk clone was responsible for NDM-7 dissemination. It is essential to develop and implement strategies to control the introduction and spread of successful MDR clones in hospitals that include active surveillance programmes to detect colonised patients.

Carbapenemase-Producing Gram-Negative Bacteria in Andalusia, Spain, 2014-2018.

The emergence and spread of carbapenemase-producing gram-negative bacteria is a major public health concern. We used data collected from microbiology laboratories as part of the PIRASOA program during 2014-2018 to study the epidemiology of carbapenemase-producing bacteria in Andalusia, Spain. Our findings highlight the importance of ongoing surveillance and epidemiologic studies for these bacteria.

Co-Occurrence of mcr-1 and qnrS1 on an IncHI2 Plasmid in Clinical Isolates of Salmonella Typhimurium in Spain.

Salmonella enterica is a well-adapted zoonotic bacterium associated to cases of gastroenteritis and bacteremia with increased morbidity and mortality. In this study, three isolates of Salmonella Typhimurium obtained from human clinical samples, showing colistin resistance and low-level resistance to quinolones, have been genetically characterized. We detected the co-occurrence of mcr-1 and qnrS1 on a single IncHI2 plasmid in isolates of Salmonella Typhimurium obtained from Spanish children without a travel history. The multiresistant region contained numerous resistance genes. Isolates were clonally related, which suggests the presence of these clones in the community and the potential to cause outbreaks affecting the most susceptible population. It is necessary to monitor the presence of these plasmid-mediated resistance genes in human European strains of Salmonella spp. because of the risk of producing outbreaks of community-acquired infections.

Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates / Colonización por Staphylococcus aureus en pacientes institucionalizados en residencias geriátricas: prevalencia y caracterización molecular de los aislados resistentes a meticilina

Introduction: The epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. Methods: A cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton-Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. Results: A total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. Conclusions: A low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA

Introducción: La epidemiología de S. aureus depende de las condiciones particulares de cada población. En España se han realizado pocos estudios sobre la colonización por S. aureus en la población geriátrica. El objetivo de este estudio es determinar la prevalencia de colonización por S. aureus resistente a meticilina (SARM) y sus características epidemiológicas moleculares en población institucionalizada en residencias de ancianos en Cádiz, España. Métodos: Se realizó un estudio epidemiológico transversal en 3 residencias de ancianos. Se estudiaron muestras de las fosas nasales y axilas. La identificación y las pruebas de sensibilidad se realizaron utilizando MALDI-TOF y paneles MicroScan(R). En los aislados de SARM se determinó el tipo de SCCmec y se tiparon mediante Multilocus Sequence Typing (MLST) y Pulsed-field Gel Electrophoresis (PFGE). La presencia de genes de la leucocidina de Panton-Valentine (LPV) se determinó mediante PCR en todas las cepas de S. aureus. Resultados: Se incluyeron un total de 293 residentes. Cincuenta y un residentes (17,4%) estaban colonizados por S. aureus sensible a la meticilina (SASM) y 11 (3,8%) por SARM. Se observó resistencia frente al menos 2 aminoglucósidos en el 25,4 y 90,9% y resistencia a levofloxacino en el 80,3 y 100% de los aislamientos de SASM y SARM, respectivamente. Se detectó SCCmecIV en todos los aislados, y todos, excepto uno (ST-125) correspondían al ST-8. Ninguno de los aislados de S. aureus fue positivo para LPV. Conclusiones: Se detectó una baja tasa de portadores de S. aureus, siendo el porcentaje de SARM muy bajo. ST8-MRSA-IVc fue el clon predominante, y solo una cepa pertenecía a ST125-MRSA-IVc. Se objetivó transmisión de SARM intracentro. Se observó una tasa muy alta de resistencia a quinolonas en SASM y SARM

OXA-48-Like-Producing Klebsiella pneumoniae in Southern Spain in 2014-2015.

The aim of this study was to characterize the population structure of 56 OXA-48-like-producing Klebsiella pneumoniae isolates, as well as extended-spectrum ß-lactamase (ESBL) and carbapenemase genes, recovered in 2014 and 2015 from 16 hospitals in southern Spain. XbaI pulsed-field gel electrophoresis and multilocus sequence typing were performed to assess clonal relatedness. Representative isolates belonging to OXA-48-like-producing and CTX-M-15-coproducing pulsotypes were selected for characterization of blaOXA-48-like- and blaCTX-M-15-carrying plasmids by PCR-based replicon typing, IncF subtyping, whole-genome sequencing analysis, and typing of Tn1999 structures. Forty-three OXA-48-producing isolates (77%) were recovered from clinical samples and 13 from rectal swabs. All isolates showed ertapenem MIC values of ≥1 mg/liter, although 70% remained susceptible to imipenem and meropenem. Forty-nine isolates (88%) produced OXA-48, 5 produced OXA-245, and 2 produced OXA-181. Twenty-eight different pulsotypes (5 detected in more than 1 hospital) and 16 sequence types (STs) were found. The most prevalent clones were ST15 (29 isolates [52%]) and ST11 (7 isolates [13%]). Forty-five (80%) isolates were also blaCTX-M-15 carriers. The blaCTX-M-15 gene was mostly (82%) located on IncR plasmids, although ST15 and ST11 isolates also carried this gene on IncF plasmids. The composite transposon variant Tn1999.2-like was the most frequent. Among ST15 and ST11 isolates, different transposon variants were observed. The blaOXA-48 gene was mainly located on IncL plasmids, although IncM plasmids were also observed. The spread of OXA-48-like-producing K. pneumoniae in southern Spain is mainly due to ST15 and ST11 clones. Variation within clonal lineages could indicate different acquisition events for both ESBL and carbapenemase traits.

Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates.

INTRODUCTION: The epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. METHODS: A cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton-Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. RESULTS: A total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. CONCLUSIONS: A low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA.

Prevalencia en España de mecanismos de resistencia a quinolonas en enterobacterias productoras de betalactamasas de clase C adquiridas y/o carbapenemasas / Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC Beta-lactamases and/or carbapenemases in Spain

Introducción: En los últimos años se ha observado un incremento de la resistencia a fluoroquinolonas en enterobacterias, estando asociado significativamente a la resistencia a betalactámicos. Nuestro objetivo fue conocer la prevalencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas en aislados productores de betalactamasas de claseC adquiridas y/o carbapenemasas. Métodos: Se evaluó la presencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas [mutaciones en la región determinante de resistencia a quinolonas de gyrA y parCy genes qnr, aac(6')-Ib-cr y qepA] en 289 aislados de enterobacterias productoras de betalactamasas de claseC adquiridas y/o carbapenemasas recogidos entre febrero y julio de 2009 en 35 hospitales españoles. Resultados: Se detectaron determinantes plasmídicos en 92 aislados (31,8%); en 83 aislados (28,7%) se detectó algún gen qnr, y en 20 (7%), la variante aac(6')-Ib-cr. El gen qnr más prevalente fue qnrB4 (20%), asociado en la mayoría de los casos a DHA-1. El 14,6% de los aislados con una CMI de ciprofloxacino superior a 0,25mg/l no presentaban mutaciones en gyrA ni parC, detectándose en el 90% de los mismos algún determinante plasmídico de resistencia a quinolonas. Conclusión: qnrB4 fue el determinante plasmídico más prevalente, claramente asociado a DHA-1. Los mecanismos plasmídicos en asociación con mecanismos cromosómicos diferentes a las mutaciones en los genes de las topoisomerasas (sobreexpresión de bombas de expulsión, alteración del lipopolisacárido o disminución de porinas) pueden dar lugar a valores de CMI de ciprofloxacino que superan los puntos de corte establecidos por los principales comités internacionales de definición de puntos de corte para interpretación de datos de sensibilidad (AU)

Background: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC Beta-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. Methods: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC Beta -lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. Results: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. Conclusion: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC Beta -lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints (AU)

Cellular Response to Ciprofloxacin in Low-Level Quinolone-Resistant Escherichia coli.

Bactericidal activity of quinolones has been related to a combination of DNA fragmentation, reactive oxygen species (ROS) production and programmed cell death (PCD) systems. The underlying molecular systems responsible for reducing bactericidal effect during antimicrobial therapy in low-level quinolone resistance (LLQR) phenotypes need to be clarified. To do this and also define possible new antimicrobial targets, the transcriptome profile of isogenic Escherichia coli harboring quinolone resistance mechanisms in the presence of a clinical relevant concentration of ciprofloxacin was evaluated. A marked differential response to ciprofloxacin of either up- or downregulation was observed in LLQR strains. Multiple genes implicated in ROS modulation (related to the TCA cycle, aerobic respiration and detoxification systems) were upregulated (sdhC up to 63.5-fold) in mutants with LLQR. SOS system components were downregulated (recA up to 30.7-fold). yihE, a protective kinase coding for PCD, was also upregulated (up to 5.2-fold). SdhC inhibition sensitized LLQR phenotypes (up to ΔLog = 2.3 after 24 h). At clinically relevant concentrations of ciprofloxacin, gene expression patterns in critical systems to bacterial survival and mutant development were significantly modified in LLQR phenotypes. Chemical inhibition of SdhC (succinate dehydrogenase) validated modulation of ROS as an interesting target for bacterial sensitization.

Molecular topology: A new strategy for antimicrobial resistance control.

The control of antimicrobial resistance (AMR) seems to have come to an impasse. The use and abuse of antibacterial drugs has had major consequences on the genetic mutability of both pathogenic and nonpathogenic microorganisms, leading to the development of new highly resistant strains. Because of the complexity of this situation, an in silico strategy based on QSAR molecular topology was devised to identify synthetic molecules as antimicrobial agents not susceptible to one or several mechanisms of resistance such as: biofilms formation (BF), ionophore (IA) activity, epimerase (EI) activity or SOS system (RecA inhibition). After selecting a group of 19 compounds, five of them showed significant antimicrobial activity against several strains of Staphylococcus (2 S. aureus, including 1 methicillin resistant, and 1 S. epidermidis), with MIC values between 16 and 32 mg/L. Among the compounds active on RecA, one showed a marked activity in decreasing RecA gene expression in Escherichia coli.

Molecular Characterization of Fluoroquinolone-Resistant Haemophilus parainfluenzae Clinical Isolates in Spain.

OBJECTIVE: The objective was to characterize a group of clinical isolates of fluoroquinolone-resistant Haemophilus parainfluenzae collected in Northern Spain (March-December 2014). METHODS: Twelve clinical isolates of H. parainfluenzae were studied by performing antimicrobial susceptibility testing and PCR amplification and nucleotide sequencing of the QRDR (quinolone resistance-determining region) of gyrA, parC, gyrB, and parE genes. Screening for plasmid-mediated quinolone resistance (PMQR) was also studied. Pulsed-field gel electrophoresis (PFGE) was used for molecular typing. RESULTS: Antimicrobial susceptibility testing showed that all the isolates were resistant to the fluoroquinolones tested (ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin). Analysis of the QRDR demonstrated that all the isolates presented mutations in gyrA and parC. A Glu88Lys substitution in ParC is reported for the first time in H. parainfluenzae. No PMQR gene was detected. PFGE results showed that isolates were not clonally related. CONCLUSION: Multiple H. parainfluenzae fluoroquinolone-resistant isolates grouped in the same area in a short period of time showed diverse substitutions in QRDR of gyrA/parC and were not clonally related, indicating individual emergence. In addition, we described the first report of Glu88Lys substitution in ParC.

Cytotoxic Effect Associated with Overexpression of QNR Proteins in Escherichia coli.

OBJECTIVE: The objective was to evaluate the cytotoxic effect associated with overexpression of multiple Qnr-like plasmid-mediated quinolone resistance (PMQR) mechanisms in Escherichia coli. METHODS: Coding regions of different PMQR genes (qnrA1, qnrB1, qnrC, qnrD1, qnrS1, and qepA2) and efsqnr were cloned into pET29a(+) vector and overexpressed in E. coli BL21. E. coli BL21 with and without an empty pET29a(+) vector were used as controls. The cytotoxic effect associated with PMQR mechanism overexpression was determined by transmission electron microscopy and viability assays. RESULTS: Overexpressed qnr genes produced loss of bacterial viability in the range of 77-97% compared with the controls, comparable with loss of viability associated with EfsQnr overexpression (97%). No loss of viability was observed in E. coli overexpressing QepA2. In transmission electron microscopy assays, signs of cytotoxicity were observed in E. coli cells overexpressing EfsQnr and Qnr proteins (30-45% of the bacterial population showed morphological changes). Morphological changes were observed in less than 5% of bacterial populations from the control strains and E. coli overexpressing QepA2. CONCLUSIONS: Overexpression of qnr genes produces a cytotoxic cellular and structural effect in E. coli, the magnitude of which varies depending on the family of Qnr proteins.

Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC ß-lactamases and/or carbapenemases in Spain. / Prevalencia en España de mecanismos de resistencia a quinolonas en enterobacterias productoras de betalactamasas de clase C adquiridas y/o carbapenemasas.

BACKGROUND: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC ß-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. METHODS: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC ß-lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. RESULTS: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. CONCLUSION: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC ß-lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints.

Characterization of Plasmid-Mediated Quinolone Resistance Determinants in High-Level Quinolone-Resistant Enterobacteriaceae Isolates from the Community: First Report of qnrD Gene in Algeria.

OBJECTIVE: The objective was to assess the prevalence of plasmid-mediated quinolone resistance (PMQR)-producing isolates in a collection of quinolone-resistant Enterobacteriaceae of community origin isolated in Bejaia, Algeria. METHODS: A total of 141 nalidixic acid-resistant Enterobacteriaceae community isolates were collected in Bejaia (Northern Algeria) and screened for PMQR genes using polymerase chain reaction (PCR). For PMQR-positive strains, antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion. Mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected with a PCR-based method and sequencing. Southern blotting, conjugation and transformation assays and molecular typing by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing were also performed. RESULTS: The prevalence of PMQR-producing Enterobacteriaceae isolates was 13.5% (19/141); 11 of these isolates produced Aac(6')-Ib-cr and 8 were qnr-positive (4 qnrB1-like, 2 qnrS1-like, and 2 qnrD1-like), including the association with aac(6')-Ib-cr gene in three cases. PMQR gene transfer by conjugation was successful in 6 of 19 isolates tested. PFGE revealed that most of the PMQR-positive Escherichia coli isolates were unrelated, except for two groups comprising two and four isolates, respectively, including the virulent multidrug-resistant clone E. coli ST131 that were clonally related. CONCLUSION: Our findings indicate that PMQR determinants are prevalent in Enterobacteriaceae isolates from the community studied. We describe the first report of the qnrD gene in Algeria.

Occurrence of the Plasmid-Mediated Fluoroquinolone Resistance qepA1 Gene in Two Clonal Clinical Isolates of CTX-M-15-Producing Escherichia coli from Algeria.

QepA is a plasmid-mediated quinolone resistance determinant of low prevalence described worldwide, mainly in Enterobacteriaceae. This study describes, for the first time in Algeria, two clonally related, QepA-producing Escherichia coli clinical isolates positive for CTX-M-15. The clonal spread of these multidrug-resistant isolates is a major public health concern.

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.