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Temocillin is used for several years in some European countries but, only since 2015 in France. We assessed the susceptibility of Enterobacterales strains isolated from blood culture 1 year before (2014) and 2 years after (2017) its use in France. 1,387 strains were included by 17 clinical laboratories located throughout France: 363 in 2014 and 1,024 in 2017. The rate of resistance to temocillin was 4.6% and 26.7% in 3rd generation cephalosporin (3GC) susceptible and resistant strains respectively. Cephalosporinase-overproducer (COPE) strains were significantly more resistant to temocillin (37.7%) than ESBL-producer (ESBL-PE) (23.5%) (P < 0.01). The rate of temocillin resistance was correlated to the number of inactive beta-lactams. The rate of resistance to temocillin trend to increase from 13.9% in 2014 to 23.9% in 2017 (P < 0.01). Temocillin remains highly active against Enterobacterales but the trend in resistance should be assessed over time., Competing Interests: Competing interest The temocillin Etest strips (bioMérieux, Lyon, France) were provided without charge by Eumedica pharmaceutical., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output., Methods: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins., Results: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance., Conclusions: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: To describe the epidemiological trend of linezolid-resistant enterococci (LRE) collected in France from 2006 to 2016 and to extensively characterize LRE isolates., Methods: The National Reference Center for Enterococci (NRC-Enc) received enterococcal isolates suspected to be VRE and/or LRE from all French hospitals between 2006 and 2016. LRE isolates were phenotypically characterized and their genomes were entirely sequenced by Miseq (Illumina). Transfer of linezolid resistance was attempted by filter mating experiments., Results: Out of 3974 clinical isolates of enterococci received at the NRC-Enc over the period, 9 (0.2%) were LRE (MICs 8 to >32 mg/L), including 6 Enterococcus faecium and 3 Enterococcus faecalis. This overall prevalence significantly increased over the study period, reaching 0.8% in 2016. The five LRE isolated before 2016 were vanA-positive E. faecium whereas strains isolated in 2016 (one E. faecium and three E. faecalis) were susceptible to vancomycin. None of these isolates was part of an outbreak, while E. faecium strains were assigned to four different STs [17 (1), 80 (3), 412 (1) and 650 (1)] and all three E. faecalis belonged to ST480. Except for the strain isolated in 2010, all LRE were positive for optrA, which was located on plasmids (5/8) or in the chromosome (3/8). Plasmid transfer of optrA was successful in three cases., Conclusions: There has been a significant increase in the prevalence of LRE in France over time; this is due to the spread of optrA among E. faecium and E. faecalis human clinical isolates (VRE or not)., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Enterococcus faecium has emerged as a major opportunistic pathogen for 2 decades with the spread of hospital-adapted multidrug-resistant clones. As members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo , with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of this study was to evaluate globally the impact of SICs of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium Transcriptomic analysis was performed by RNA sequencing (RNA-seq) (HiSeq 2500; Illumina) using the vanB -positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/liter, i.e., 1/8 of the MIC). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced, whereas 286 genes were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Among upregulated genes, EFAU004&#95;02294 (fold change, 14.3) encoded a protein (Qnr of E. faecium [EfmQnr]) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive fluoroquinolone (FQ) resistance in E. faecium was experimentally ascertained. Moreover, EFAU004&#95;02292, coding for the collagen adhesin Acm, was also induced by the SIC of ciprofloxacin (fold change, 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both EfmQnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving fluoroquinolone therapy., (Copyright © 2017 American Society for Microbiology.)

Objectives: The aim of the study was to identify the determinant responsible for erythromycin resistance in Helcococcus kunzii clinical isolate UCN99 and to characterize the genetic support and environment of this novel gene., Methods: MICs were determined using the broth microdilution method according to EUCAST guidelines. The entire genome sequence of H. kunzii UCN99 was determined using a 454/Roche GS Junior sequencer. The fragment encompassing the new resistance gene and its own promoter was cloned into the pAT29 shuttle vector and the recombinant plasmid pAT29Ωerm(47) was expressed in both Staphylococcus aureus and Streptococcus agalactiae. The transcription start site (TSS) was experimentally determined by 5' RACE-PCR., Results: UCN99 exhibited a constitutive macrolide/lincosamide/streptogramin B (MLS B ) resistance phenotype, suggesting the presence of an Erm protein. WGS allowed the identification of a novel gene, named erm(47), encoding a protein sharing 44%-48% amino acid identity with known Erm methylases. In both S. aureus and S. agalactiae, the introduction of pAT29Ωerm(47) conferred a significant increase (≥16-fold) in MICs of all macrolides and lincosamides tested, as well as a 4-fold increase in MICs of quinupristin (streptogramin B), confirming the MLS B resistance. The TSS identification revealed the presence of a short leader peptide, potentially implicated in a translational attenuation mechanism. It was also demonstrated that erm(47) was harboured by a 81 kb genomic island integrated into a chromosomal gene., Conclusions: This is the first description of a novel MLS B resistance determinant, named erm(47). The prevalence of this gene among Gram-positive cocci must be further investigated to determine its clinical significance., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Aims of this study were to: (i) evaluate whether the cluster membership could have an impact on hetero-resistance phenotype to colistin in the Enterobacter cloacae complex (ECC); and (ii) determine the genetic mechanism of colistin hetero-resistance in ECC., Methods: A collection of 124 clinical isolates belonging to 13 clusters were used to analyse the hetero-resistance phenotype (MICs were determined using the broth microdilution method, Etest and population analysis profiling). Different mutants (ΔphoP, ΔphoQ, ΔphoPQ, ΔpmrA, ΔpmrB, ΔpmrAB, ΔarnE, ΔarnF and ΔarnBCADTEF) were constructed and tested for their colistin hetero-resistance phenotype., Results: Based on broth microdilution and Etest results, it was shown that the hetero-resistance to colistin depended on the cluster: strains from clusters I, II, IV, VII, IX, X, XI and XII were usually hetero-resistant, whereas those from clusters III, V, VI, VIII and XIII were categorized as susceptible. However, for some cluster V and VIII strains, a small proportion (<10 -7 ) of cells appeared resistant when tested by population analysis profiling. From a mechanistic point of view, analysis of mutants revealed that the mechanism of hetero-resistance was mainly due to the expression of the arn operon and the phoP/phoQ two-component regulatory system., Conclusions: Because the colistin hetero-resistance appeared cluster-dependent in the ECC, it should be advocated to determine the cluster of the strain associated with the infection in parallel with the MIC of colistin. The resistance mechanism may not be similar to other Enterobacteriaceae since only the two-component regulatory system PhoP/PhoQ (and not PmrA/PmrB) seemed to play a role in resistance regulation., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The Gram-negative bacterial outer membrane fortifies the cell against environmental toxins including antibiotics. Unique glycolipids called lipopolysaccharide/lipooligosaccharide (LPS/LOS) are enriched in the cell-surface monolayer of the outer membrane and promote antimicrobial resistance. Colistin, which targets the lipid A domain of LPS/LOS to lyse the cell, is the last-line treatment for multidrug-resistant Gram-negative infections. Lipid A is essential for the survival of most Gram-negative bacteria, but colistin-resistant Acinetobacter baumannii lacking lipid A were isolated after colistin exposure. Previously, strain ATCC 19606 was the only A. baumannii strain demonstrated to subsist without lipid A. Here, we show that other A. baumannii strains can also survive without lipid A, but some cannot, affording a unique model to study endotoxin essentiality. We assessed the capacity of 15 clinical A. baumannii isolates including 9 recent clinical isolates to develop colistin resistance through inactivation of the lipid A biosynthetic pathway, the products of which assemble the LOS precursor. Our investigation determined that expression of the well-conserved penicillin-binding protein (PBP) 1A, prevented LOS-deficient colony isolation. The glycosyltransferase activity of PBP1A, which aids in the polymerization of the peptidoglycan cell wall, was lethal to LOS-deficient A. baumannii Global transcriptomic analysis of a PBP1A-deficient mutant and four LOS-deficient A. baumannii strains showed a concomitant increase in transcription of lipoproteins and their transporters. Examination of the LOS-deficient A. baumannii cell surface demonstrated that specific lipoproteins were overexpressed and decorated the cell surface, potentially compensating for LOS removal. This work expands our knowledge of lipid A essentiality and elucidates a drug resistance mechanism., Competing Interests: The authors declare no conflict of interest.

Objectives: To improve understanding of mechanisms of daptomycin resistance and to dissect the genetic basis of reversion to daptomycin hypersusceptibility in Enterococcus faecium., Methods: Daptomycin-resistant mutants (Mut4, Mut8, Mut16, Mut32, Mut64 and Mut128 with MICs from 4 to 128 mg/L) were obtained in vitro from E. faecium strain Aus0004 (MIC at 2 mg/L). The entire genome sequences of Mut64 and Mut128 were determined as well as those of liaFSR and cls genes for other mutants and corresponding revertants (named Rev4 to Rev128). The study of daptomycin resistance stability was performed without any selective pressure. The expression of liaF, liaS and liaR genes was quantified by quantitative RT-PCR., Results: By comparative genomic analysis, substitutions Asn13Ser in cls and Gly92Asp in liaS were identified in Mut64 and Mut128. Only the liaS mutation was found in Mut16 and Mut32 while Mut4 and Mut8 were devoid of any mutation. After 15 days, all mutants except Mut4 reverted to daptomycin hypersusceptibility (MICs from 0.12 to 0.25 mg/L). In all revertants (except Rev4 and Rev8), an IS was found in the liaFSR operon with a dramatic decrease of its expression: IS66 in the promoter region of liaF (Rev16 and Rev64), IS30 in liaR (Rev32) and IS982 in liaF (Rev128)., Conclusions: We demonstrated the stepwise and sequential acquisition of mutations in liaS and in cls leading to daptomycin resistance in E. faecium, and the instability of daptomycin resistance as well as the role of liaFSR inactivation in reversion to daptomycin hypersusceptibility., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Owing to the emergence of vancomycin-resistant Enterococcus faecium, treatment of enterococcal infections has become challenging. Although spontaneous in vitro resistance frequencies are low, the emergence of resistance is increasingly reported during daptomycin therapy. The mutant selection window (MSW), comprised between the minimum inhibitory concentration (MIC) and the mutant prevention concentration (MPC), corresponds to the concentration range within which resistant mutants may be selected. Since no data are available for enterococci, the aim of this study was to determine MPCs and MSWs for 12 representative E. faecium clinical isolates. MICs and MPCs were determined by broth microdilution and agar dilution methods, respectively. A basic MSW-derived pharmacodynamic analysis was also performed using mean maximum plasma concentration (Cmax) values obtained with dosages from 4 to 12 mg/kg. MICs and MPCs of daptomycin ranged from 0.5 to 4 mg/L and from 2 to 32 mg/L, respectively, with no correlation between them. The wideness of MSWs ranged from 2× to 32× MIC. Mean plasma Cmax values of daptomycin were calculated from 55 to 174.5 mg/L when using a dosage from 4 to 12 mg/kg. All Cmax values were above the MPCs whatever the dosage. Taking into account the protein binding of daptomycin (ca. 90%), the unbound fraction Cmax was just within the MSW in 67-92% of strains at recommended dosages (4-6 mg/kg) and was above the MPC for the majority of strains only with the highest dosage (12 mg/kg). This study shows that free daptomycin Cmax values usually fell into MSWs when using lower dosages (<10 mg/kg)., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

All the 31 isolates of Alloscardovia omnicolens exhibited low MICs for β-lactams, glycopeptides, linezolid, tetracyclines, and cotrimoxazole. One strain showed MICs ≥256μg/mL for both erythromycin and clindamycin with a single point mutation in 23S rRNA. One strain likely had acquired fluoroquinolone resistance associated with a unique mutation in ParC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism's overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD(+) strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.

Objectives: Unique resistance to lincosamides (L phenotype) due to the production of nucleotidyltransferases (Lnu) is uncommon among Gram-positive bacteria. The aim of the study was to characterize the L phenotype in a clinical isolate of the Streptococcus milleri group., Methods: The strain UCN93 was recovered from neonatal specimens and from the mother's vaginal swab. Identification was confirmed by sequencing of the sodA gene. Antimicrobial susceptibility testing was carried out by the disc diffusion method, while MICs were determined using the agar dilution method. Screening for lnu(A), lnu(B), lnu(C) and lnu(D) genes was performed by PCR. Genetic environment and support were determined by thermal asymmetric interlaced PCR and PCR mapping. The transfer of lincomycin resistance was also attempted by conjugation., Results: UCN93 was unambiguously identified as Streptococcus anginosus. It was susceptible to all tested antibiotics, except lincomycin (MIC, 8 mg/L) and tetracycline (2 mg/L). The lnu(C) gene was found to be responsible for the L phenotype. It was shown that lnu(C) was associated with a gene coding for a transposase within a structure similar to the transposon MTnSag1, described once in Streptococcus agalactiae. Since MTnSag1 was found to be mobilized by Tn916 and S. anginosus UCN93 harboured a Tn916 transposon, several attempts at transfer were performed but they all failed. The lnu(C)-containing genetic element was inserted into a chromosomal intergenic sequence of S. anginosus., Conclusions: Since lnu(C) has been detected in only one S. agalactiae clinical isolate so far, this is its second description among clinically relevant streptococci.

To determine if hospital effluent input has an ecological impact on downstream aquatic environment, antibiotic resistance in Enterococcus spp. along a medical center-retirement home-wastewater treatment plant-river continuum in France was determined using a culture-based method. Data on antibiotic consumption among hospitalized and general populations and levels of water contamination by antibiotics were collected. All isolated enterococci were genotypically identified to the species level, tested for in vitro antibiotic susceptibility, and typed by multilocus sequence typing. The erm(B) and mef(A) (macrolide resistance) and tet(M) (tetracycline resistance) genes were detected by PCR. Along the continuum, from 89 to 98% of enterococci, according to the sampled site, were identified as Enterococcus faecium. All E. faecium isolates from hospital and retirement home effluents were multiply resistant to antibiotics, contained erm(B) and mef(A) genes, and belonged to hospital-adapted clonal complex 17 (CC17). Even though this species remained dominant in the downstream continuum, the relative proportion of CC17 isolates progressively decreased in favor of other subpopulations of E. faecium that were more diverse, less resistant to antibiotics, and devoid of the classical macrolide resistance genes and that belonged to various sequence types. Antibiotic concentrations in waters were far below the MICs for susceptible isolates. CC17 E. faecium was probably selected in the gastrointestinal tract of patients under the pressure of administered antibiotics and then excreted together with the resistance genes in waters to progressively decrease along the continuum.

Objectives: To study the impact of qnrA1, qnrB19 and qnrS1 on the ciprofloxacin treatment of urinary tract infection (UTI)., Methods: From a wild-type (wt) Escherichia coli UTI isolate, three isogenic strains were constructed carrying low-level ciprofloxacin resistance genes qnrA1, qnrB19 or qnrS1 (ciprofloxacin MIC range: 0.19-0.38 mg/L). Time-kill studies were performed for all four isogenic strains at the following concentrations: 1×, 2×, 4×, 8× and 16× MIC. Ciprofloxacin serum and urine pharmacokinetics was determined to calculate a murine dose equivalent (AUC(24)) to the standard human dose of 500 mg twice daily, which corresponded to 0.2 mg/mouse four times daily. In the murine UTI model, mice infected with each of the isogenic qnr strains or the wt strain were treated with ciprofloxacin (0.2 mg/mouse) or saline (only the E. coli wt) subcutaneously four times daily for 3 days starting 24 h after bacterial inoculation., Results: In vitro, the strains responded to ciprofloxacin concentrations of 4-16× MIC by several log(10) reductions. In vivo, despite ciprofloxacin reaching urine concentrations far exceeding the MICs for the strains (500 mg/L), ciprofloxacin was significantly less efficient at reducing the urine and bladder bacterial counts of qnrA1-, qnrB19- and qnrS1-positive strains compared with the ciprofloxacin-treated wt strain (P < 0.05). None of the four strains infected the kidneys well, with median cfu counts of <1 log(10)., Conclusions: Although qnr genes only confer low levels of resistance to ciprofloxacin, a reduced bactericidal activity of ciprofloxacin was observed in both urine and bladder in the murine model of UTI.

Objectives: To analyse the in vitro transfer of the qnrA1 gene by a 50 kb (pSZ50) self-transferable plasmid that derives from a 300 kb plasmid (pSZ300) and to determine the complete nucleotide sequence of plasmid pSZ50., Methods: Extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes of an Escherichia coli clinical isolate were analysed. Plasmid analysis included conjugation and selection on seven antibiotics examined by antimicrobial susceptibility testing, RFLP comparison, Southern hybridization, incompatibility group identification and shotgun sequencing., Results: The E. coli 5509 isolate carries the genes encoding the ESBL CTX-M-15 and the quinolone resistance determinants qnrA1, qnrB2 and aac(6')-Ib-cr on a 300 kb plasmid. Seven transfer resistances were analysed by conjugation under two conditions (30 and 37°C), leading to two distinct transconjugant phenotypes with different resistances. Transconjugants of phenotype A harboured a 300 kb plasmid named pSZ300 that conferred resistance to eight antibiotics and harboured the qnrA1, aac(6')-Ib-cr and bla(CTX-M-15) genes. Transconjugants of phenotype B were resistant to three antibiotics and they harboured the qnrA1 gene on an ≈ 50 kb plasmid named pSZ50. Both plasmids were self-transferable at a frequency of 1 × 10(-3). Plasmid pSZ300 was typed to be both an IncF and IncN plasmid, whereas pSZ50 corresponded only to type IncN. Fingerprinting and Southern hybridization showed that plasmid pSZ50 derived from pSZ300. The complete nucleotide sequence of plasmid pSZ50 was determined (51556 bp) and 55 open reading frames were predicted. The qnrA1 gene was identified in a tandem duplicate inside a sul1-type integron structure., Conclusions: The plasmid pSZ300 represented a fusion of two replicons (IncF and IncN), and our observations suggest that the plasmid pSZ50 (IncN) may split and transfer antibiotic resistance determinants. This mechanism could be advantageous in the dissemination of antibiotic resistance genes.

The aim of this study was to evaluate the prevalence of resistance to macrolide-lincosamide-streptogramin (MLS) antibiotics as well as to assess the molecular basis of this resistance amongst 72 Staphylococcus saprophyticus urinary isolates collected from 2005 to 2009 in University Hospital of Caen (France). Of the 72 strains studied, 33 (45.8%) were resistant to at least one MLS antibiotic, including 24 (72.7%) with an M phenotype, 5 (15.2%) with an inducible MLS(B) phenotype, 3 (9.1%) with a combined M+L phenotype and 1 (3.0%) with an L phenotype. All isolates were susceptible to the combination of streptogramins A and B. The resistance genes erm(A), erm(B), erm(C), msr(A) and lnu(A) were detected alone in 0, 0, 5 (15.2%), 24 (72.7%) and 1 (3.0%) of the 33 MLS-resistant isolates, respectively, whereas 2 strains (6.1%) were positive for both msr(A) and lnu(A). All msr(A)-positive isolates exhibited an M phenotype, whereas all five erm(C)-positive and all three lnu(A)-positive strains displayed, respectively, an inducible MLS(B) phenotype and an L phenotype with a positive Hodge test. Plasmid analysis indicated that erm(C) and lnu(A) genes were borne by small-size plasmids (ca. 2.5 kb), whereas larger plasmids (30-90 kb) harboured msr(A). In conclusion, these findings show a high prevalence of MLS resistance in S. saprophyticus, which was mainly associated with the presence of the msr(A) gene. Since S. saprophyticus colonises the gastrointestinal tract, it may constitute an unexpected reservoir for MLS resistance genes, in particular msr(A), amongst coagulase-negative staphylococci., (Copyright © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Objectives: To assess the in vitro susceptibility of Actinobaculum schaalii to 12 antimicrobial agents as well as to dissect the genetic basis of fluoroquinolone resistance., Methods: Forty-eight human clinical isolates of A. schaalii collected in Switzerland and France were studied. Each isolate was identified by 16S rRNA sequencing. MICs of amoxicillin, ceftriaxone, gentamicin, vancomycin, clindamycin, linezolid, ciprofloxacin, levofloxacin, moxifloxacin, co-trimoxazole, nitrofurantoin and metronidazole were determined using the Etest method. Interpretation of results was made according to EUCAST clinical breakpoints. The quinolone-resistance-determining regions (QRDRs) of gyrA and parC genes were also identified and sequence analysis was performed for all 48 strains., Results: All isolates were susceptible to amoxicillin, ceftriaxone, gentamicin, clindamycin (except three), vancomycin, linezolid and nitrofurantoin, whereas 100% and 85% were resistant to ciprofloxacin/metronidazole and co-trimoxazole, respectively. Greater than or equal to 90% of isolates were susceptible to the other tested fluoroquinolones, and only one strain was highly resistant to levofloxacin (MIC ≥32 mg/L) and moxifloxacin (MIC 8 mg/L). All isolates that were susceptible or low-level resistant to levofloxacin/moxifloxacin (n = 47) showed identical GyrA and ParC amino acid QRDR sequences. In contrast, the isolate exhibiting high-level resistance to levofloxacin and moxifloxacin possessed a unique mutation in GyrA, Ala83Val (Escherichia coli numbering), whereas no mutation was present in ParC., Conclusions: When an infection caused by A. schaalii is suspected, there is a risk of clinical failure by treating with ciprofloxacin or co-trimoxazole, and β-lactams should be preferred. In addition, acquired resistance to fluoroquinolones more active against Gram-positive bacteria is possible.

Enterococci are responsible for various community- and hospital-acquired infections. Glycopeptides (vancomycin and teicoplanin) are active against these microorganisms by inhibiting cell wall synthesis through binding to cell wall precursors. Enterococcus faecium has developed multidrug resistance, including resistance to glycopeptides. Resistance to glycopeptides is due to the acquisition of an operon of genes cooperating to synthesize precursors devoid of affinity for the glycopeptides. Outbreaks were recently reported in hospital settings. These outbreaks were due to E. faecium isolates belonging to an hospital-adapted clonal complex (CC17) characterized by high level resistance to ampicillin and fluoroquinolones and frequently containing virulence factors. Outbreaks may be controlled by appropriate measures and new antibiotics are available in therapy. However, spreading of clonal strains adapted to hospitals require close surveillance.

Resistance to quinolones and fluoroquinolones has been increasingly reported among human and veterinary isolates during the last three decades related to their wide clinical use. Until recently, the mechanisms of resistance to quinolones in Enterobacteriaceae were believed to be only chromosome-encoded, i.e. related to modifications of the molecular targets (DNA gyrase and topoisomerase IV), decreased outer-membrane permeability (porin defect) and overexpression of naturally-occurring efflux. However, emergence of plasmid-mediated quinolone resistance (PMQR) has been reported since 1998. Three mechanisms are known to date: Qnr proteins, aminoglycoside acetyltransferase AAC(6')-Ib-cr, and efflux pump QepA. The Qnr proteins protect DNA gyrase and type IV topoisomerase from quinolone inhibition. Four types of Qnr protiens have been reported: QnrA (six variants), QnrB (19 variants), QnrC (one variant), and QnrS (three variants). The AAC(6')-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. The protein AAC(6')-Ib-cr contains two amino acid substitutions as compared to the wild-type enzyme AAC(6')-Ib. Both Qnr and AAC(6')-Ib proteins have been reported worldwide. Lately reported, the plasmid-encoded QepA efflux pump may extrude hydrophilic fluoroquinolones (eg. norfloxacin, ciprofloxacin, and enrofloxacin).

One hundred twenty-one extended-spectrum beta-lactamse-producing enterobacterial clinical isolates were screened for the qepA gene. A single CTX-M-15-positive Escherichia coli isolate (0.8%) that produced the putative pump QepA2 was identified. This qepA2 gene was located onto a 90-kb mobilizable plasmid that conferred reduced susceptibility to hydrophilic fluoroquinolones.

Objectives: The aim of this study was to determine the spread of plasmid-mediated quinolone resistance determinants [qnr-like, aac(6')-Ib-cr and qepA genes] among nalidixic acid-resistant enterobacterial strains isolated from outpatients from Southeast Brazil, their transferability and the genetic structures associated with the qnr genes., Methods: The qnrA, qnrB and qnrS genes were screened by a multiplex PCR-based technique from 257 non-repetitive nalidixic acid-resistant enterobacterial isolates collected from January 2000 to May 2005. Conjugation experiments were performed to determine whether the qnr-carrying plasmids were self-transferable. Genetic structures surrounding the qnr genes were analysed by PCR and cloning. The aac(6')-Ib-cr and qepA genes were screened among qnr-positive strains., Results: Six qnrB-like-positive isolates (2.3%) were detected, whereas no qnrA- or qnrS-positive isolates were detected. Three Escherichia coli and two Klebsiella pneumoniae isolates harboured a qnrB2 gene and a single Citrobacter freundii isolate had the qnrB8 gene. One qnrB2-positive isolate also had the extended-spectrum beta-lactamase bla(CTX-M-2) gene. All these isolates also possessed chromosomal substitutions in gyrase- and topoisomerase-encoding genes, explaining their high-level resistance to quinolones., Conclusions: This study constitutes the first epidemiological survey of the three known Qnr determinants among Brazilian isolates and shows their low prevalence in that country, with the qnrB2 gene being mostly identified.

We searched for plasmid-mediated quinolone resistance determinants of the Qnr type in several water samples collected at diverse locations from the Seine River (Paris, France). The qnrS2 genes were identified from Aeromonas punctata subsp. punctata and A. media. The qnrS2 gene was located on IncU-type plasmids in both isolates, which resulted in increased MIC values of quinolones and fluoroquinolones, once they were transferred into Escherichia coli. The qnrS2 gene identified in A. punctata was part of novel genetic structure corresponding to a mobile insertion cassette element. This identification of plasmid-mediated qnr genes outside Enterobacteriaceae underlines a possible diffusion of those resistance determinants within gram-negative rods.

Objectives: To develop a rapid and reliable single-tube-based PCR technique for detecting simultaneously the plasmid-mediated quinolone resistance qnrA, qnrB and qnrS genes., Methods: After multiple alignments, primers were designed to detect known qnr variants (six for qnrA-, six for qnrB- and two for qnrS-like genes). They were used for screening a collection of 64 expanded-spectrum beta-lactamase (ESBL)-producing enterobacterial isolates from Kuwait, collected from 2002 to 2004, as ESBL genes have been often associated with qnr genes. Sequencing was performed to identify qnr and associated ESBL genes., Results: In optimized conditions, all positive controls (used separately or mixed) confirmed the specificity of the PCR primers. Out of 64 isolates, only 3 isolates were positive for a qnrB-like gene (4.7%), whereas no qnrA-like and qnrS-like gene was detected. A qnrB2 gene was detected in an Enterobacter cloacae K34 (SHV-12+) isolate, whereas qnrB1-like (termed qnrB7) and qnrB6-like (termed qnrB8) genes were identified from E. cloacae K37 (SHV-12+) and Citrobacter freundii K70 (VEB-1b+) isolates, respectively., Conclusions: We report here a fast and reliable technique for rapid screening of qnr-positive strains to be used for epidemiological surveys. A low prevalence of Qnr determinants among ESBL-producing Enterobacteriaceae was identified in the study with Kuwaiti isolates.

The qnr genes are transferable genes that confer low-level quinolone resistance by protection of topoisomerase. The occurrence of mutations in DNA gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes in strains harbouring qnr was investigated in 28 qnrA-positive clinical isolates, among which 7 strains also harboured qnrS. Topoisomerase mutations were found in 25 (89%) of the 28 strains, with at least two mutations (gyrA and parC) in 13 strains and one mutation in 12 strains. Isolates of the Enterobacter cloacae complex were compared with reference strains of the new Enterobacter species. gyrA mutations were found at position 83 (Ser or Thr for Ile, Tyr, Leu or Phe depending on the species), and new gyrB mutations were described (S463A, S464F). qnrA had an additive effect of a 10-fold increase in the minimum inhibitory concentration (MIC) whatever the number of topoisomerase mutations, and qnrS was additive to qnrA with a further 2- to 10-fold increase in the MIC. Comparison of MICs with susceptibility breakpoints showed that strains combining qnrA and topoisomerase mutations were resistant to fluoroquinolones, but the three strains lacking a topoisomerase mutation were susceptible using ciprofloxacin and levofloxacin but not using nalidixic acid or moxifloxacin testing.

Objectives: To detect the qnrA, qnrB and qnrS genes among Salmonella isolates received at the French National Reference Centre for Salmonella in Paris, France., Methods: Antibiotic susceptibility was determined by disc diffusion for 499 Salmonella isolates including 320 Salmonella Typhimurium, 100 Salmonella Enteritidis and 79 Salmonella Hadar collected in 2002. Amplification with specific primers of qnrA, qnrB and qnrS genes was performed for all Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Hadar isolates resistant to quinolones and for 17 additional isolates that produced expanded-spectrum beta-lactamases (ESBLs)., Results: Prevalence of quinolone resistance was 3.75%, 11% and 79.7% for Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Hadar serovars, respectively. A single isolate (0.2%) was qnrA-positive (QnrA1 determinant) being a Salmonella serovar Concord carrying also the ESBL gene bla(CTX-M-15). This strain was probably from East Africa. No qnrB or qnrS genes were identified., Conclusions: Whereas plasmid-mediated quinolone resistance of the Qnr type is emerging in Enterobacteriaceae worldwide, it remains rare in Salmonella in France.

Helicobacter pylori eradication by standard therapy is decreasing due to clarithromycin and metronidazole resistance. Fluoroquinolones are valuable drugs for alternative therapy, but their activity needs to be updated. We determined minimum inhibitory concentrations (MICs) of the newly marketed fluoroquinolones (levofloxacin, moxifloxacin and gatifloxacin) and assessed the prevalence of resistance in 128 H. pylori strains isolated in 2004-2005. The quinolone resistance-determining region (QRDR) of gyrA was sequenced for all strains. Gatifloxacin MICs (MIC(50) = 0.25 mg/L) were two- to four-fold lower than those of the other fluoroquinolones. The prevalence of resistance (ciprofloxacin MIC > 1 mg/L) was 17.2% (22 strains). All resistant strains harboured one gyrA mutation at codons 86, 87 or 91, including three new mutations (Asp86Asn, Thr87Ile and Asn87Tyr). Ciprofloxacin-susceptible strains were devoid of such gyrA mutations, but harboured a polymorphism at codon 87 that distinguished 18 isolates (17%) with a Thr87 like the reference strain J99 from 88 strains with Asn87 like the reference strain 26695. Strains with Thr87 were four-fold more susceptible to nalidixic acid, pefloxacin, ciprofloxacin and levofloxacin and were equally susceptible to moxifloxacin and gatifloxacin. The high rate of quinolone resistance in H. pylori requires the use/implication of a 'test and treat' strategy that can confidently rely on QRDR gyrA sequencing.

Bacteria can resist to antibiotics by active exportation mediated by membrane transporters called efflux pumps. These proteins can be specific of a class of antibiotics or responsible for multidrug resistance (MDR). Energy required by efflux pumps can be provided by transmembrane electrochemical gradient of protons (MFS, RND, SMR families) or sodium ions (MATE family) or by ATP hydrolysis (ABC family). Several physiological functions have been described in prokaryotes, such as protection from environmental toxics and regulation of cell homeostasis, which can indirectly contributes to bacterial virulence. In Gram-negative bacteria, efflux transporters usually are organized as multicomponent systems in which the efflux pump located in the inner membrane works in conjunction with a periplasmic fusion protein and an outer membrane factor. The most frequently encountered pumps are of the RND-type such as AcrB in Escherichia coli or MexB in Pseudomonas aeruginosa. In Gram-positive bacteria, efflux is solely mediated by the pump protein, so described with MFS pumps such as NorA or QacA in Staphylococcus aureus and PmrA in Streptococcus pneumoniae. Efflux transporters have also been described in mycobacteria. Although numerous bacterial pumps have been characterized, the clinical consequences of efflux-mediated resistance are mostly unknown because of variable levels of expression and of the lack of specific markers in laboratory practice. Finally, associating pump-specific inhibitors to efflux-sensitive antibiotics might prove an interesting therapeutic perspective. However, inhibitors that are not toxic to eukaryotic cells remain to be identified.