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International audience; β-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to β-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of β-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained

International audience; No abstract available

International audience; To address the problem of resistance to paclitaxel treatment, we have investigated to which extent is possible to predict Breast Cancer (BC) patient response to this drug. We carried out a large-scale tumor-based prediction analysis using data from the US National Cancer Institute's Genomic Data Commons. These data sets comprise the responses of BC patients to paclitaxel along with six molecular profiles of their tumors. We assessed 10 Machine Learning (ML) algorithms on each of these profiles and evaluated the resulting 60 classifiers on the same BC patients. DNA methylation and miRNA profiles were the most informative overall. In combination with these two profiles, ML algorithms selecting the smallest subset of molecular features generated the most predictive classifiers: a complexity-optimized XGBoost classifier based on CpG island methylation extracted a subset of molecular factors relevant to predict paclitaxel response (AUC = 0.74). A CpG site methylation-based Decision Tree (DT) combining only 2 of the 22,941 considered CpG sites (AUC = 0.89) and a miRNA expression-based DT employing just 4 of the 337 analyzed mature miRNAs (AUC = 0.72) reveal the molecular types associated to paclitaxel-sensitive and resistant BC tumors. A literature review shows that features selected by these three classifiers have been individually linked to the cytotoxic-drug sensitivities and prognosis of BC patients. Our work leads to several molecular signatures, unearthed from methylome and miRNome, able to anticipate to some extent which BC tumors respond or not to paclitaxel. These results may provide insights to optimize paclitaxel-therapies in clinical practice.

Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D β-lactamases and 65 new Acinetobacter -derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D β-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of bla OXA genes by Acinetobacter species. The class D β-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii , Acinetobacter pittii , Acinetobacter calcoaceticus , and Acinetobacter lwoffii . Neither bla OXA-40/143 - nor bla OXA-58 -like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from bla ADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of bla OXA or bla ADC transfer was observed between distantly related species, except for bla OXA-279 , possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus . Analysis of β-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of β-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.

International audience; To investigate the possible role of the hospital environment in the dissemination of extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli isolates, 300 samples were taken during 2013 from abiotic surfaces (n = 250), healthcare worker hands (n = 27), and hands of patients (n = 23) in a Tunisian Hospital. ESBL-producing E. coli isolates were recovered in 3.7% of analyzed samples (4% abiotic surfaces; 4.3% hands of patients; 0% in healthcare worker hands), and one isolate/sample was further studied. The characterization of beta-lactamase genes, as well as the genetic environment of bla CTX-M gene, was performed by PCR and sequencing. The ESBL genes found were as follows: bla CTX-M-15 (eight isolates), bla CTX-M-15 +bla SHV-12 (two isolates), and bla SHV-12 (one isolate). The bla TEM-1b gene was detected in seven ESBL-positive isolates. The orf477 was found downstream of bla CTX-M-15 gene in 10 strains, whereas the ISEcp1 sequence was identified upstream of this gene in two isolates. The analysis of class 1 integrons by PCR and sequencing revealed five positive isolates with the following gene cassette arrangements: dfrA1-aadA1 (two isolates), aadA1 (two isolates), and aadA2 (one isolate). The virulence-encoding genes aer, eae, bfp, and hly were detected by PCR in six, four, four, and three isolates, respectively. The following sequence types and associated phylogroups were detected among ESBL-producing strains: ST167-phylogroup-A (six isolates) and ST131-phylogroup-B2 (two isolates). In conclusion, the hospital environment could be a reservoir of multiresistant bacteria, including ESBL-positive E. coli isolates, which could be acquired by the patient population, and strict control measures should be established to minimize this problem.

International audience; Comparative genomics with Staphylococcus aureus suggested the existence of a regulatory system governing beta-lactamase (BlaC) production in Mycobacterium tuberculosis. The crystal structure of Rv1846c, a winged helix regulator of previously unknown function, was solved thus revealing strong similarity to the BlaI and MecI repressors of S. aureus, which both respond to beta-lactam treatment. Using chromatin immunoprecipitation and hybridization to microarrays (ChIP-on-chip), the Rv1846c regulon was shown to comprise five separate genomic loci. Two of these mediate responses and resistance to beta-lactam antibiotics (rv1845c, rv1846c-rv1847; blaC-sigC); two encode membrane proteins of unknown function (rv1456c, rv3921c) while the last codes for ATP synthase (rv1303-atpBEFHAGDC-rv1312). The ChIP-on-chip findings were confirmed independently using electrophoretic mobility shift assays, DNAse footprinting and transcript analysis leading to Rv1846c being renamed BlaI. When cells were treated with beta-lactams, BlaI was released from its operator sites causing derepression of the regulon and upregulation of ATP synthase transcription. The existence of a potential regulatory loop between cell wall integrity and ATP production was previously unknown.

OBJECTIVE AND DESIGN: The aim of this study was to compare the use of a late (CD63) and an early (IgE) marker of basophil activation in the flow cytometric diagnosis of beta-lactam induced allergic hypersensitivity reactions. SUBJECTS: Twelve patients who had had a clear cut betalactam induced immediate reaction and 16 controls were selected, as well as 11 patients who had had an immediate reaction to bee or wasp stings. METHODS: Leukocyte suspensions were incubated with allergen dilutions as well as 2 positive controls (anti-IgE and NFormyl- Methionyl-Leucyl-Phenylalanine (fMLP)). Basophils were labelled with an anti-IgE FITC (fluorescein isothiocyanate) and an anti-CD63 PE (phycoerythrin). Results were expressed as percentage CD63 expression and index calculated according to a specific algorithm including the two activation markers. RESULTS: Significant CD63 expression (>5 %) was observed in 3/12 cases for the beta-lactam sensitized population, in 0/16 cases for the controls and in 11/11 cases for the venom sensitized population. A significant index (determined by a ROC analysis) was observed in 11/12 beta-lactam sensitized patients and in 0/16 controls. CONCLUSION: These results show that IgE (an early activation marker) is more sensitive than CD63 (a later activation marker) in the diagnosis of beta-lactam allergy.

We report here a rare case of chronic lumbar discitis caused by Clostridium perfringens in an elderly patient that was treated with a combination of β-lactams and clindamycin. Molecular analysis performed on the strain revealed an unusual toxin gene pattern.

International audience; Understanding the molecular basis of bacterial cell wall assembly is of paramount importance in addressing the threat of increasing antibiotic resistance worldwide. Streptococcus pneumoniae presents a particularly acute problem in this respect, as it is capable of rapid evolution by homologous recombination with related species. Resistant strains selected by treatment with β-lactams express variants of the target enzymes that do not recognize the drugs but retain their activity in cell wall building, despite the antibiotics being mimics of the natural substrate. Until now, the crucial transpeptidase activity that is inhibited by β-lactams was not amenable to in vitro investigation with enzymes from Gram-positive organisms, including streptococci, staphylococci, or enterococci pathogens. We report here for the first time the in vitro assembly of peptidoglycan using recombinant penicillin-binding proteins from pneumococcus and the precursor lipid II. The two required enzymatic activities, glycosyl transferase for elongating glycan chains and transpeptidase for cross-linking stem-peptides, were observed. Most importantly, the transpeptidase activity was dependent on the chemical nature of the stem-peptide. Amidation of the second residue glutamate into iso-glutamine by the recently discovered amido-transferase MurT/GatD is required for efficient cross-linking of the peptidoglycan.

International audience; OBJECTIVES: To investigate the resistance mechanisms of β-lactam-resistant Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients in France. METHODS: Two-hundred-and-four P. aeruginosa CF isolates were collected in 10 French university hospitals in 2007. Their susceptibility to 14 antibiotics and their resistance mechanisms to β-lactams were investigated. Their β-lactamase contents were characterized by isoelectric focusing, PCR and enzymatic assays. Expression levels of efflux pumps and the intrinsic β-lactamase AmpC were quantified by reverse transcription real-time quantitative PCR. Genotyping was performed using multiple-locus variable number of tandem repeats analysis (MLVA). The oprD genes were sequenced and compared with those of reference P. aeruginosa strains. To assess deficient OprD production, western blotting experiments were carried out on outer membrane preparations. RESULTS: MLVA typing discriminated 131 genotypes and 47 clusters. One-hundred-and-twenty-four isolates (60.8%) displayed a susceptible phenotype to β-lactams according to EUCAST breakpoints. In the 80 remaining isolates, resistance to β-lactams resulted from derepression of intrinsic cephalosporinase AmpC (61.3%) and/or acquisition of secondary β-lactamases (13.8%). Efflux pumps were up-regulated in 88.8% of isolates and porin OprD was lost in 53.8% of isolates due to frameshifting or nonsense mutations in the oprD gene. CONCLUSIONS: β-Lactam resistance rates are quite high in CF strains of P. aeruginosa isolated in France and not really different from those reported for nosocomial strains. Development of β-lactam resistance is correlated with patient age. It results from intrinsic mechanisms sequentially accumulated by bacteria isolated from patients who have undergone repeated courses of chemotherapy.

Antimicrobials: mismatch excels when ampicillin runs low. [Nat Rev Microbiol. 2013]; International audience; Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.

We report the complete nucleotide sequence of the pKpS90 plasmid, carrying the bla KPC-2 and bla SHV-12 genes. This plasmid was isolated from a sequence type 258 (ST258) Klebsiella pneumoniae strain responsible for an outbreak in a French university hospital in 2009. pKpS90 is a 53,286-bp plasmid that belongs to the IncX incompatibility group. pKpS90 consists of a backbone from IncX plasmids, in which the KPC-2-encoding Tn 4401 transposon and a bla SHV-12 -encoding region have been inserted.

International audience; The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a biopolymer highly cross-linked through d,d-transpeptidation. Peptidoglycan cross-linking is catalyzed by penicillin-binding proteins (PBPs) that are the essential target of β-lactam antibiotics. PBPs are functionally replaced by l,d-transpeptidases (Ldts) in ampicillin-resistant mutants of Enterococcus faecium and in wild-type Mycobacterium tuberculosis. Ldts are inhibited in vivo by a single class of β-lactams, the carbapenems, which act as a suicide substrate. We present here the first structure of a carbapenem-acylated l,d-transpeptidase, E. faecium Ldtfm acylated by ertapenem, which revealed key contacts between the carbapenem core and residues of the catalytic cavity of the enzyme. Significant reorganization of the antibiotic conformation occurs upon enzyme acylation. These results, together with the analysis of protein-to-carbapenem proton transfers, provide new insights into the mechanism of Ldt acylation by carbapenems.

The purpose of this work was to study the genetic determinants responsible for extended-spectrum cephalosporin (ESC) resistance of Salmonella collected during the period of 1995-2008 at the Hussein Dey hospital in Algiers (Algeria). Fourteen ESC-resistant Salmonella isolates were tested towards 22 antimicrobial agents. Polymerase chain reaction (PCR) and sequencing were used to determine the underlying genetic determinants responsible for the extended-spectrum beta-lactamase (ESBL) phenotypes. Enterobacterial Repetitive Intergenic Consensus PCR was employed to type the isolates. All tested isolates were resistant to ticarcillin, ticarcillin-clavulanate, piperacillin, cefuroxime, aztreonam, ceftazidime, cefotaxime (except two isolates), cefepime, and cefpirome. PCR and DNA sequencing identified these ESBLs as TEM-48 (n=6), TEM-4 (n=3), CTX-M-15 (n=4), and one new TEM, designated TEM-188. Thus, continued surveillance for the presence of ESBL-producing (non-typhoidal) salmonellae in Algeria is essential.

A rapid and specific high-performance liquid chromatography method with UV detection (HPLC-UV) for the simultaneous determination of 12 beta-lactam antibiotics (amoxicillin, cefepime, cefotaxime, ceftazidime, ceftriaxone, cloxacillin, imipenem, meropenem, oxacillin, penicillin G, piperacillin, and ticarcillin) in small samples of human plasma is described. Extraction consisted of protein precipitation by acetonitrile. An Atlantis T3 analytical column with a linear gradient of acetonitrile and a pH 2 phosphoric acid solution was used for separation. Wavelength photodiode array detection was set either at 210 nm, 230 nm, or 298 nm according to the compound. This method is accurate and reproducible (coefficient of variation [CV] < 8%), allowing quantification of beta-lactam plasma levels from 5 to 250 μg/ml without interference with other common drugs. This technique is easy to use in routine therapeutic drug monitoring of beta-lactam antibiotics.

International audience

The prevalence of class D extended-spectrum oxacillinases (ES-OXAs) in ceftazidime-resistant strains of Pseudomonas aeruginosa is often underestimated by double-disk synergy tests (DDST) using clavulanate. A DDST with a customized distance between a disk of ceftazidime or cefepime and inhibitors (clavulanate and imipenem) detected 14 out of 15 different ES-OXAs.

International audience; OBJECTIVES: To investigate the high prevalence of extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia coli (4%, 10/250 consecutive isolates) recovered during a 5 month period in the maternity ward of the University Hospital of Bordeaux, France. METHODS: beta-Lactam resistance transfer was analysed by conjugation and transformation. ESBLs were characterized by isoelectric focusing, PCR amplification and sequencing. The relatedness of the strains was examined by PFGE and phylogenetic group determination. Plasmids were characterized by incompatibility group and restriction analysis. RESULTS: Ten ESBL-producing E. coli were isolated from urinary or genital samples of eight mothers and from gastric fluids of two newborns of carrier mothers. The patients were hospitalized in five different units of the maternity ward. Transconjugants, obtained for 7 of the 10 strains, and wild-type strains exhibited various antibiotypes. Different CTX-M enzymes were characterized: CTX-M-1 (n = 4); CTX-M-14 (n = 3); CTX-M-32 (n = 2); and CTX-M-28 (n = 1). The strains recovered from two mothers and their respective babies were identical. All the other strains were epidemiologically unrelated. Furthermore, various plasmids were identified. Environmental samples from the common echographic and sampling rooms did not reveal the presence of ESBL-producing enterobacteria. CONCLUSIONS: The data argue against the occurrence of a nosocomial outbreak and support the hypothesis of an importation of community-acquired ESBL-producing strains into the hospital through colonized/infected patients. At present, not only patients transferred from other hospitals or long-term care facilities are at risk of carrying ESBL-producing enterobacteria on hospital admission, but also community patients.

International audience; BACKGROUND: Beta-lactamase production and porin decrease are the well-recognized mechanisms of acquired beta-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and susceptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this beta-lactam phenotype was the aim of this study. METHODOLOGY/FINDINGS: MICs of 9 beta-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other beta-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also 16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other beta-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of beta-lactams. CONCLUSION: This is the first study demonstrating that efflux mechanism plays a key role in the beta-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in beta-lactam resistance is specially underestimated in clinical isolates.

International audience; A new liquid chromatography assay with isocratic elution and tandem mass spectrometry detection (LC-MS/MS) using an electrospray ionization interface in the multiple reaction monitoring mode was developed and validated for ertapenem determination in microdialysate samples. Linearity was demonstrated between 10ngmL(-1) (lower limit of quantification, LLoQ) and 160ngmL(-1). The precision (CV%) and accuracy (bias%) in microdialysates at the LLoQ were respectively 2.2% and 17.3% within-day and 10.6% and 2.7% between-days. Ertapenem was stable for 1 month at -20 degrees C and -80 degrees C but unstable at +4 degrees C. This new LC-MS/MS assay is simple, rapid and more sensitive than previously described assays.

We describe the first IMP metallo-β-lactamase in Aeromonas caviae : IMP-19, which differed from IMP-2 by a single amino acid change (Arg to Ala at position 38). bla IMP-19 was found within a class 1 integron located on a 35-kb plasmid. This is also the first description of an IMP producer in France.

In this study, we systematically investigated the resistance mechanisms to β-lactams, aminoglycosides, and fluoroquinolones of 120 bacteremic strains of Pseudomonas aeruginosa . Pulsed-field gel electrophoresis genotyping showed that 97 of these strains were represented by a single isolate, 10 by 2 and 1 by 3 clonally related isolates, respectively. Seventy-five percent (90 out of 120) of the bacteremic P. aeruginosa strains displayed a significant resistance to one or more of the tested antimicrobials (up to 11 for 1 strain). These strains were found to harbor a great diversity of resistance mechanisms (up to 7 in 1 strain), leading to various levels of drug resistance. Interestingly, 11 and 36% of the isolates appeared to overproduce the MexAB-OprM and MexXY-OprM efflux systems, respectively. Altogether, our results show that P. aeruginosa may accumulate intrinsic (overproduction of cephalosporinase AmpC, increased drug efflux, fluoroquinolone target mutations, and deficient production of porin OprD) and exogenous (production of secondary β-lactamases and aminoglycoside-modifying enzymes) resistance mechanisms without losing its ability to generate severe bloodstream infections. Consequently, clinicians should be aware that multidrug-resistant P. aeruginosa may remain fully pathogenic.

Received 19 December 2006; returned 29 January 2007; revised 14 February 2007; accepted 22 February 2007Objectives: Pseudomonas aeruginosa is a major causative agent of hospital infections. The purposeof this study was to determine the antibiotic susceptibility of P. aeruginosa in a French multicentrestudy and to investigate the mechanisms of b-lactam resistance.Methods: Four hundred and fifty non-repetitive strains of P. aeruginosa were collected in 15 Frenchuniversity hospitals in 2004. MICs of antibiotics were measured by agar dilution methods. For all thestrains with MICs of ticarcillin >16 mg/L, detection and identification of the b-lactamases, quantitativedetermination of cephalosporinase and overproduction of the MexAB-OprM efflux pump wereevaluated.Results: The percentages of susceptible isolates were as follows: ticarcillin, 62%; ticarcillin1clavulanicacid, 61%; piperacillin, 78%; piperacillin1tazobactam, 80% (MICs 16 mg/L); aztreonam, 50%; ceftazi-dime, 78%; cefepime, 64%; imipenem, 83%; tobramycin, 80% (MICs 4 mg/L); amikacin, 86%(MIC 8 mg/L); ciprofloxacin, 68%; and levofloxacin, 57% (MICs 1 mg/L). Decreased susceptibility toimipenem was linked in two cases to VIM-type carbapenemase production. Overexpression of theAmpC cephalosporinase, production of acquired b-lactamases including SHV2a extended-spectrumb-lactamase and overproduction of the MexAB-OprM efflux pump were present in 16.9%, 6.5% and22.3% of the strains, respectively.Conclusions: In the last decade, the overall susceptibility of P. aeruginosa hospital isolates to anti-biotics has remained quite stable in France. However, the emergence of extended-spectrum b-lacta-mases and carbapenemases in different locations is a matter of concern.Keywords: Pseudomonas spp., mechanisms of resistance, antimicrobial resistance surveillance, b-lactams

To the Editor: A 38-year-old French man with a history of chronic juvenile arthritis was referred to the Necker-Enfants Malades University hospital (Paris, France) with a dysenteric syndrome. The patient had returned the day before from a 1-month stay in Port-au-Prince, Haiti, where he spent most of his time in close contact with young children from an orphanage, most of whom had diarrhea. Clinical examination at admission showed fever (39°C), chills, diffuse abdominal pain, bloody diarrhea, and vomiting. The patient received ceftriaxone, which was stopped on day 4 because initial blood and stool cultures were negative for pathogens and clinical signs had completely resolved. Ten days later, he reported the recurrence of diarrhea without fever. A novel stool culture grew Shigella sonnei. An extended-spectrum β-lactamase (ESBL) was detected by double-disk synergy test; the isolate was also resistant to aminoglycosides (except amikacin), tetracycline, and cotrimoxazole. The strain was susceptible to fluoroquinolones and fosfomycin. It also appeared susceptible to azithromycin (MIC 4 μg/mL), although azithromycin MIC for Shigella spp. should be interpreted with caution (1). The patient was successfully treated with azithromycin at a dose of 500 mg/d for 5 days. Azithromycin was preferred to fluoroquinolones to avoid the risk for tendinopathy because of the patient’s history of chronic juvenile arthritis and because this antimicrobial agent was shown to be effective in the treatment of shigellosis caused by multidrug-resistant strains (2). To identify the molecular basis of this ESBL, a series of PCR primers were used for detection of TEM-, SHV-, or CTX-M–type ESBL (3). Only the TEM PCR showed positive results. Sequencing of 2 independent PCR products showed a new allele (www.lahey.org/studies/temtable.asp). Analysis of the deduced amino acid sequence allowed characterization of TEM-137, derived from TEM-1 with 2 substitutions, Arg-16→Ser and Glu-240→Arg. This ESBL (and resistance to aminoglycosides and tetracyclines) was easily transferred to Escherichia coli J53-2 by conjugation. MICs of β-lactams alone or in association with clavulanic acid, were determined by E-test, according to manufacturer’s instructions (AB Biodisk, Solna, Sweden). High-level resistance to ceftazidime (MIC 32 μg/mL) and intermediate resistance to cefotaxime (MIC 8 μg/mL) were observed; the strain remained susceptible to cefepime and imipenem (MIC 0.5 and 0.25 μg/mL, respectively). Clavulanic acid did not restore susceptibility to ceftazidime (MIC 4 μg/mL) but did restore susceptibility to cefotaxime (MIC 0.5 μg/mL). With clavulanic acid, the MIC of cefepime was 0.06 μg/mL. ESBL in S. sonnei is rare worldwide. In Argentina, a CTX-M-2 was found in an isolate of S. sonnei resistant to cefotaxime but not to ceftazidime (4). In South Korea, TEM-15, TEM-17, TEM-19, TEM-20, TEM-52, and CTX-M-14 were characterized in S. sonnei (5); TEM-52 and CTX-M-14 were also widely distributed, particularly in Salmonella spp. (6,7). In Turkey, an isolate of S. sonnei producing CTX-M-3 was reported (8). In Hong Kong, sequencing of 2 S. sonnei isolates showed the presence of CTX-M-14 and CTX-M-15 (9). Finally, in Bangladesh, 2 isolates of S. sonnei with a class A ESBL were reported; they were not characterized at the molecular level, but the resistance phenotypes suggested a CTX-M type (10). In our case, little information on antimicrobial drug resistance could be obtained from Haiti because no systematic investigation on resistance in Enterobacteriaceae is performed. Nevertheless, the emergence of TEM-137 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM286274","term_id":"110350793","term_text":"AM286274"}}AM286274) harbored by this imported S. sonnei isolate clearly demonstrates that ESBL-associated shigellosis has emerged in Haiti and that potentially large and severe shigellosis outbreaks could occur, for which the use of azithromycin could be beneficial, as illustrated in our patient. Because treating shigellosis is becoming problematic, it is essential to focus on prevention measures such as simple rules of personal hygiene that might drastically decrease the risk of transmission.

International audience; Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.

International audience; Mycobacterium fallax (M. fallax) is naturally sensitive to many beta-lactam antibiotics (MIC < 2 microg/ml) and devoid of beta-lactamase activity. In this paper, we show that the production of the beta-lactamase of Mycobacterium fortuitum by M. fallax significantly increased the MIC values for good substrates of the enzyme, whereas the potency of poor substrates or transient inactivators was not modified. The rates of diffusion of beta-lactams through the mycolic acid layer were low, but for all studied compounds the half-equilibration times were such that they would only marginally affect the MIC values in the absence of beta-lactamase production. These results emphasize the importance of enzymatic degradation as a major factor in the resistance of mycobacteria to penicillins.