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.

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.

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.

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.

Impact of AAC(6')-Ib-cr in combination with chromosomal-mediated mechanisms on clinical quinolone resistance in Escherichia coli.

OBJECTIVES: aac(6')-Ib-cr is the most prevalent plasmid-mediated fluoroquinolone (FQ) resistance mechanism in Enterobacteriaceae. We aimed to analyse the interplay between this plasmid-mediated gene and chromosomal-mediated quinolone resistance mechanisms on both FQ resistance and bacterial fitness in Escherichia coli. METHODS: E. coli ATCC 25922 and derived isogenic strains carrying chromosomal-mediated quinolone resistance modifications (Ser83Leu-Asp87Asn in GyrA, Ser80Arg in ParC and/or a marR gene deletion) were electroporated with a pBK-CMV vector encoding AAC(6')-Ib-cr. The MICs of FQs were determined by microdilution and bactericidal activity was determined using time-kill curves. A peritoneal sepsis murine model was used to evaluate the in vivo impact. Bacterial fitness was analysed using growth curves and competition assays. RESULTS: The presence of the aac(6')-Ib-cr gene increased the MICs of ciprofloxacin and norfloxacin 4-8-fold for all E. coli genotypes, independently of the initial resistance level. Combination of the aac(6')-Ib-cr gene with three or four chromosomal mechanisms was necessary to reach MIC values above the susceptible category. Killing curve assays showed a clear selective advantage for survival in strains harbouring the aac(6')-Ib-cr gene (up to 7 log10 cfu/mL after 24 h). AAC(6')-Ib-cr significantly reduced the ciprofloxacin efficacy in vivo. In terms of bacterial fitness cost, maximal OD was significantly lower for all strains harbouring the aac(6')-Ib-cr gene, independently of chromosomal mutations associated. CONCLUSIONS: The aac(6')-Ib-cr gene, in spite of producing low-level resistance by itself, plays a relevant role in acquisition of a clinical level of ciprofloxacin and norfloxacin resistance, when combined with three or four chromosomal mutations, both in vitro and in vivo.

Comparison of clinical categories for Escherichia coli harboring specific qnr and chromosomal-mediated fluoroquinolone resistance determinants according to CLSI and EUCAST.

EUCAST breakpoints are more restrictive than those defined by CLSI. This study highlights the discrepancies between CLSI and EUCAST in a well characterized isogenic Escherichia coli collection and their correlations with specific quinolone resistance mechanisms. The greatest number of discrepancies was observed in strains containing 2-4 resistance mechanisms (MIC values on the borderline of clinical resistance). Bearing in mind that quinolones are concentration dependent antimicrobial agents, small changes in MIC may have relevant consequences for treatment outcomes.

Effect of the efflux pump QepA2 combined with chromosomally mediated mechanisms on quinolone resistance and bacterial fitness in Escherichia coli.

OBJECTIVES: The aim of the study was to determine the interplay between the plasmid-mediated qepA2 gene and multiple chromosomally mediated fluoroquinolone resistance determinants in the development of fluoroquinolone resistance in Escherichia coli and its influence on bacterial fitness. METHODS: E. coli ATCC 25922 and derived isogenic strains harbouring different chromosomally mediated fluoroquinolone resistance determinants were electroporated with pBK-CMV vector encoding QepA2. The MICs of fluoroquinolones were determined by standardized microdilution. The mutant prevention concentration (MPC) was evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays. RESULTS: The ciprofloxacin MIC for strains harbouring the qepA2 gene was 4- to 8-fold higher compared with strains without the qepA2 gene. The qepA2 gene also increased the MPC of ciprofloxacin 4- to 16-fold. Combination of the qepA2 gene plus two to three additional mechanisms conferred a clinically relevant resistance level. The presence of the qepA2 gene was associated with fitness costs in strains with mutations in the gyrA and/or parC genes, although the presence of an additional deletion of the marR gene compensated for this fitness cost by increasing bacterial fitness by 5%-23%. CONCLUSIONS: The additive effect of chromosomally mediated fluoroquinolone resistance mechanisms and the qepA2 gene led to clinical levels of fluoroquinolone resistance. Under competitive conditions, the qepA2 gene had a biological cost in E. coli that was compensated for by the presence of an additional deletion in the marR gene.

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.

Interplay between plasmid-mediated and chromosomal-mediated fluoroquinolone resistance and bacterial fitness in Escherichia coli.

OBJECTIVES: The aim of this study was to analyse the interplay among plasmid-mediated qnr genes, alone or in combination with multiple chromosomal-mediated fluoroquinolone (FQ) resistance determinants, susceptibility to FQs and bacterial fitness in an isogenic Escherichia coli collection. METHODS: E. coli ATCC 25922 was used to modify or delete chromosomal genes. qnr genes were cloned into the pBK-CMV vector. The MICs of FQs were determined by microdilution. Mutant prevention concentration and frequency of mutants were evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays using in vitro and in vivo models. RESULTS: The relationships between the number of resistance mutations and bacterial fitness were complex. With specific combinations of resistance mechanisms the addition of a new resistance mutation was shown to improve bacterial fitness. qnrA1 caused a decrease in fitness (7%-21%) while qnrS1 caused an increase in fitness (9%-21%) when combined with chromosomal mutations. We identified susceptible triple mutants in which the acquisition of a fourth resistance mutation significantly increased fitness and at the same time reached the clinical resistance level (the acquisition of qnrS1 in a S83L + D87N + ΔmarR genetic background). A strong correlation with the production of reactive oxygen species, as well as changes in susceptibility, was observed following treatment with ciprofloxacin. CONCLUSIONS: Our data indicate that there may be critical stages (depending on the genotype) in resistance development, including chromosomal- and plasmid-mediated mechanisms, at which some low-fitness mutants below the resistance breakpoint are able to evolve clinical resistance with just one or two mutations, and show increased fitness.

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.

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.

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.

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.

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.

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.

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.

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.