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ABSTRACTAn interregional surveillance program was conducted in the northwestern part of France to determine the prevalence of carbapenem-nonsusceptible Enterobacteriaceae(CNSE) isolates and their susceptibility to ceftazidime-avibactam and aztreonam-avibactam combinations. Nonduplicate CNSE clinical isolates were prospectively collected from six hospitals between June 2012 and November 2013. MICs of ceftazidime and aztreonam, alone or combined with a fixed concentration of avibactam (4 μg/ml), and those of carbapenems (comparator agents) were determined. MICs of ertapenem in combination with phenylalanine arginine-naphthylamide dihydrochloride (PAβN) were also determined to assess active efflux. Genes encoding carbapenemases, plasmid-mediated AmpC enzymes, extended-spectrum β-lactamases (ESBLs), and major outer membrane proteins (OMPs) were amplified and sequenced. OMPs were also extracted for SDS-PAGE analysis. Among the 139 CNSE isolates, mainly Enterobacterspp. and Klebsiella pneumoniae, 123 (88.4%) were ertapenem nonsusceptible, 12 (8.6%) exhibited reduced susceptibility to all carbapenems, and 4 Proteeaeisolates (2.9%) were resistant to imipenem. Carbapenemase production was detected in only two isolates (producing OXA-48 and IMI-3). In contrast, OMP deficiency, in association with AmpCs and/or ESBLs (mainly CTX-M-9, SHV-12, and CTX-M-15), was largely identified among CNSE isolates. The ceftazidime-avibactam and aztreonam-avibactam combinations exhibited potent activity against CNSE isolates (MIC50/MIC90, 1/1 μg/ml and 0.5/0.5 μg/ml, respectively) compared to that of ceftazidime and aztreonam alone (MIC50/MIC90, 512/512 μg/ml and 128/512 μg/ml, respectively). This study reveals the in vitroactivity of ceftazidime-avibactam and aztreonam-avibactam combinations against a large collection of porin-deficient enterobacterial isolates that are representative of the CNSE recovered in the northern part of France.

ABSTRACTTigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported in Enterococcus faecium. Using the serial passage method, in vitromutant AusTig and in vitromutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 μg/ml) were obtained from strains E. faeciumAus0004 and HM1070 (MICs, 0.03 μg/ml), respectively. In addition, two vancomycin-resistant E. faeciumclinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 μg/ml, respectively) were studied. Compared to the wild-type strains, the in vitromutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected the rpsJgene (efau004_00094, coding for the S10 protein of the 30S ribosomal subunit), efau004_01228(encoding a cation transporter), efau004_01636(coding for a hypothetical protein), and efau004_02455(encoding the l-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein. In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci.

ABSTRACTRhodococcus equi, the causal agent of rhodococcosis, is a major pathogen of foals and is also responsible for severe infections in immunocompromised humans. Of great concern, strains resistant to currently used antibiotics have emerged. As the number of drugs that are efficient in vivois limited because of the intracellular localization of the bacterium inside macrophages, new active but cell-permeant drugs will be needed in the near future. In the present study, we evaluated, by in vitroand ex vivoexperiments, the ability of the alpha-helical equine antimicrobial peptide eCATH1 to kill intracellular bacterial cells. Moreover, the therapeutic potential of the peptide was assessed in experimental rhodococcosis induced in mice, while the in vivotoxicity was evaluated by behavioral and histopathological analysis. The study revealed that eCATH1 significantly reduced the number of bacteria inside macrophages. Furthermore, the bactericidal potential of the peptide was maintained in vivoat doses that appeared to have no visible deleterious effects for the mice even after 7 days of treatment. Indeed, daily subcutaneous injections of 1 mg/kg body weight of eCATH1 led to a significant reduction of the bacterial load in organs comparable to that obtained after treatment with 10 mg/kg body weight of rifampin. Interestingly, the combination of the peptide with rifampin showed a synergistic interaction in both ex vivoand in vivoexperiments. These results emphasize the therapeutic potential that eCATH1 represents in the treatment of rhodococcosis.

ABSTRACTAs opposed to Enterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A), Enterococcus faeciumis naturally susceptible. Since this phenotype may be selected for in vivoby quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance in E. faecium. Six LSAP-resistant in vitromutants of E. faeciumHM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of an in vitromutant (E. faeciumUCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in all in vitroand in vivoresistant strains but was not present in susceptible strains. The wild-type allele was named eat(A) (for EnterococcusABC transporter), and its mutated allelic variant was named eat(A)v. The introduction of eat(A)vfrom UCN90B into HM1070 conferred the LSAP phenotype, whereas that of eat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance in E. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.

ABSTRACTStreptococcus agalactiaeUCN70, isolated from a vaginal swab obtained in New Zealand, is resistant to lincosamides and streptogramins A (LSAphenotype) and also to tiamulin (a pleuromutilin). By whole-genome sequencing, we identified a 5,224-bp chromosomal extra-element that comprised a 1,479-bp open reading frame coding for an ABC protein (492 amino acids) 45% identical to Lsa(A), a protein related to intrinsic LSAresistance in Enterococcus faecalis. Expression of this novel gene, named lsa(C), in S. agalactiaeBM132 after cloning led to an increase in MICs of lincomycin (0.06 to 4 μg/ml), clindamycin (0.03 to 2 μg/ml), dalfopristin (2 to >32 μg/ml), and tiamulin (0.12 to 32 μg/ml), whereas no change in MICs of erythromycin (0.06 μg/ml), azithromycin (0.03 μg/ml), spiramycin (0.25 μg/ml), telithromycin (0.03 μg/ml), and quinupristin (8 μg/ml) was observed. The phenotype was renamed the LSAP phenotype on the basis of cross-resistance to lincosamides, streptogramins A, and pleuromutilins. This gene was also identified in similar genetic environments in 17 other S. agalactiaeclinical isolates from New Zealand exhibiting the same LSAP phenotype, whereas it was absent in susceptible S. agalactiaestrains. Interestingly, this extra-element was bracketed by a 7-bp duplication of a target site (ATTAGAA), suggesting that this structure was likely a mobile genetic element. In conclusion, we identified a novel gene, lsa(C), responsible for the acquired LSAP resistance phenotype in S. agalactiae. Dissection of the biochemical basis of resistance, as well as demonstration of in vitromobilization of lsa(C), remains to be performed.

ABSTRACTOne hundred twenty-one extended-spectrum β-lactamse-producing enterobacterial clinical isolates were screened for the qepAgene. A single CTX-M-15-positive Escherichia coliisolate (0.8%) that produced the putative pump QepA2 was identified. This qepA2gene was located onto a 90-kb mobilizable plasmid that conferred reduced susceptibility to hydrophilic fluoroquinolones.

ABSTRACTA novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coliclinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19gene.

ABSTRACTCarbapenem resistance results mostly from the expression of acquired carbapenem-hydrolyzing oxacillinases in Acinetobacter baumannii. The blaOXA-23oxacillinase gene is increasingly reported worldwide and may represent an emerging threat. Our goal was to identify the progenitor of that carbapenemase gene. A collection of 50 Acinetobactersp. strains corresponding to several Acinetobacterspecies was screened for blaOXA-23-like genes by PCR and hybridization techniques. Five Acinetobacter radioresistensisolates that were susceptible to carbapenems harbored chromosomally encoded blaOXA-23-like genes. A similar plasmid backbone was identified in several blaOXA-23-positive A. baumanniiand A. radioresistensisolates, further strengthening the vectors of exchanges for these blaOXA-23-like genes. Therefore, A. radioresistens, a commensal bacterial species which is identified on the skin of hospitalized and healthy patients (a property shared with A. baumannii), was identified as the source of the blaOXA-23gene.

ABSTRACTBy studying the β-lactamase content of several Acinetobacterspp. isolates from Argentina, producing the expanded-spectrum β-lactamases (ESBL) VEB-1a or PER-2, a novel Ambler class A β-lactamase gene was identified. It encoded the narrow-spectrum β-lactamase SCO-1, whose activity was inhibited by clavulanic acid. SCO-1 hydrolyzes penicillins at a high level and cephalosporins and carbapenems at a very low level. β-Lactamase SCO-1 was identified from unrelated VEB-1a-positive or PER-2-positive Acinetobacterspp. isolates recovered from three hospitals. The blaSCO-1gene was apparently located on a plasmid of ca. 150 kb from all cases but was not associated with any ESBL-encoding gene. The G+C content of the blaSCOgene was 52%, a value that does not correspond to that of the A. baumanniigenome (39%). β-Lactamase SCO-1 shares 47% amino acid identity with CARB-5 and ca. 40% with the enzymes TEM, SHV, and CTX-M. A gene encoding a putative resolvase was identified downstream of the blaSCO-1gene, but its precise way of acquisition remains to be determined.

The plasmid-mediated quinolone resistance determinant QnrS1 was identified in non-clonally related Enterobacter cloacae isolates in association with a transferable narrow-spectrum {szligbeta}-lactam resistance marker. Cloning experiments allowed the identification of a novel Ambler class A {szligbeta}-lactamase, named LAP-1. It shares 62 and 61% amino acid identity with the most closely related {szligbeta}-lactamases, TEM-1 and SHV-1, respectively. It has a narrow-spectrum hydrolysis of {szligbeta}-lactams and is strongly inhibited by clavulanic acid and sulbactam and, to a lesser extent, by tazobactam. Association of the blaLAP-1gene with the qnrS1 gene was identified in E. cloacae isolates from France and Vietnam. These genes were plasmid located and associated with similar insertion sequences but were not associated with sul1-type class 1 integrons, as opposed to the qnrA genes.

Genome changes are central to the adaptation of bacteria, especially under antibiotic pressure. The aim of this study was to report phenotypic and genomic adaptations undergone by an Enterobacter hormaecheiclinical strain that became highly resistant to key antimicrobials during a 4-month period in a patient hospitalized in an intensive care unit (ICU). All six clinical E. hormaecheistrains isolated in one ICU-hospitalized patient have been studied. MICs regarding 17 antimicrobial molecules have been measured.

The clinical benefit of carbapenems against carbapenemase-producing Enterobacteriaceae(CPE) remains in question. MICs of imipenem (IMP) and ertapenem (ERT) against isogenic derivatives of the wild-type strain Escherichia coliCFT073 producing KPC-3, OXA-48, or NDM-1 were 0.25, 2, 16, and 64 mg/liter for IMP and 0.008, 0.5, 8, and 64 mg/liter for ERT, respectively. Swiss ICR-strain mice with peritonitis were treated for 24 h with IMP or ERT. Despite a limited duration of time during which free antibiotic concentrations were above the MIC (down to 0% for the NDM-1-producing strain), IMP and ERT significantly reduced bacterial counts in spleen and peritoneal fluid at 24 h (P< 0.

Fosfomycin tromethamine activity is well established for oral treatment of uncomplicated lower urinary tract infections, but little is known about its potential efficacy in pyelonephritis. Ascending pyelonephritis was induced in mice infected with 6 strains of Escherichia coli(fosfomycin MICs, 1 μg/ml to 256 μg/ml). The urine pH was 4.5 before infection and 5.5 to 6.0 during infection.

The study evaluated the in vitroactivity of ceftolozane-tazobactam (C/T) against 94 unique clinical isolates of Enterobacter cloacaecomplex (ECC). No difference was observed according to the ECC cluster.