Edouard Bingen

Background: The increasing incidence of community acquired infection due to Extended-Spectrum Beta-Lactamase (ESBL) -Producing Enterobacteriaceae represent a great concern because there are few therapeutic alternatives. The fecal flora of children in the community can represent a reservoir for ESBLs genes which are located on highly transmissible plasmids and the spread of these genes among bacterial pathogens is concerning. Because intestinal carriage is a key factor in the epidemiology of ESBL-producing Enterobacteriaceae, the study of the prevalence of these resistant bacteria and risk factors in young children is of particular interest. Methods: We assessed the prevalence and risk factors of community-acquired faecal carriage of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae in children aged from 6 to 24 months, by means of rectal swabbing in community pediatric practices. Child’s lifestyle and risk factors for carriage of resistant bacteria were noted. Results: Among the 411 children enrolled, 4.6% carried ESBL-producing Enterobacteriaceae. CTX-M-1, CTX-M-15 and CTX-M-14 were the predominant ESBLs. The 18 E. coli isolates were genetically heterogeneous. Recent third-generation oral-cephalosporin exposure was associated with a higher risk of ESBL carriage (AOR=3.52, 95% CI[1.06-11.66], p=0.04). Conclusions: The carriage rate of ESBL-producing Enterobacteriacae in young children in the French community setting is noteworthy, underlining the importance of this population as a reservoir. Exposure to third-generation oral cephalosporins was associated with a significant risk of ESBL carriage in our study. Because of the significant public health implications including the treatment of community-acquired urinary tract infections, the spread of organisms producing ESBLs in the community merits close monitoring with enhanced efforts for surveillance.

Background: The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. Results: Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. Conclusion: We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine.

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Nosocomial outbreaks of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae are an increasing concern in neonatal intensive care units (NICUs). We describe an outbreak of ESBL-producing K. pneumoniae that lasted 5 months and affected 23 neonates in our NICU. Proton pump inhibitor and extended-spectrum cephalosporin exposure were significantly associated with the risk of ESBL-producing K. pneumoniae colonisation and/or infection. Thirty isolates recovered from clinical, screening and environmental samples in the NICU were studied by means of Raman spectroscopy, pulsed-field gel electrophoresis and repetitive extragenic palindromic polymerase chain reaction (rep-PCR). The Raman clustering was in good agreement with the results of the other two molecular methods. Fourteen isolates belonged to the Raman clone 1 and 16 to the Raman clone 3. Molecular analysis showed that all the strains expressed SHV-1 chromosomal resistance, plasmid-encoded TEM-1 and CTX-M-15 β-lactamases. Incompatibility groups of plasmid content identified by PCR-based replicon typing indicated that resistance dissemination was due to the clonal spread of K. pneumoniae and horizontal CTX-M-15 gene transfer between the two clones.

M/emm typing, based either on serotyping of the M protein or on sequencing of the emm gene, is a major tool for epidemiological studies of group A streptococci (GAS). In order to simplify M/emm typing, we designed two multiplex polymerase chain reaction (PCR) formats capable of identifying the most frequent GAS M/emm types involved in invasive infections and antimicrobial resistance. A heptaplex PCR procedure was first developed in a conventional format coupled with gel electrophoresis to identify emm types 1, 3, 4, 6, 12, 28, and 89, based on the size of the amplification products. The other method, designed to identify the same seven emm types, together with emm11, was based on a real-time PCR format coupled with high-resolution melting (HRM) analysis, allowing the rapid typing of large strain collections.

Dissemination of carbapenem resistance among Enterobacteriaceae poses a considerable threat to public health. Carbapenemase gene detection by molecular methods is the gold standard but is available in only a few laboratories. The aim of this study was to test phenotypic methods for the detection of metallo-β-lactamase (MBL)- or Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and associated mechanisms of β-lactam resistance against a panel of 30 genotypically characterized carbapenem-resistant Enterobacteriaceae : 9 MBL, 7 KPC, 6 OXA-48, and 8 extended-spectrum β-lactamase (ESBL) or AmpC β-lactamases associated with decreased permeability. We used carbapenemase inhibitor-impregnated agar to test for carbapenem-resistant strains. Differences in the inhibition zone sizes of the meropenem, imipenem, ertapenem, and doripenem disks were measured between control and inhibitor (EDTA or phenylboronic acid [PBA] with or without cloxacillin)-impregnated Mueller-Hinton agar with a cutoff of 10 mm. All 9 MBL- and 7 KPC-producing Enterobacteriaceae were identified from the differences in zone size in the presence and absence of specific inhibitors, regardless of the carbapenem MICs and including isolates with low-level resistance to carbapenems. We also detected their associated β-lactam resistance mechanisms (11 ESBL-type and 5 class A β-lactamase 2b). No differences in zone size were observed for OXA-48-producing strains or other carbapenem resistance mechanisms such as ESBL and decreased permeability. We propose a new strategy to detect carbapenemases (MBL- and KPC-type) and associated mechanisms of β-lactam resistance (ESBL or class A β-lactamase 2b) by the use of inhibitor-impregnated agar. A rapid phenotypic detection of resistance mechanisms is important for epidemiological purposes and for limiting the spread of resistant strains by implementing specific infection control measures.

Several studies have investigated the impact of 7-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal (Sp) and staphylococcal (Sa) nasopharyngeal (NP) carriage. Few have investigated the impact on Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mc) carriage. We aimed to compare the NP carriage rates in young children with acute otitis media (AOM) before and after PCV7 implementation in France.

Rapid identification of metallo-β-lactamase-producing Gram-negative species is crucial for the timely implementation of infection control measures. We describe two pediatric cases in which colonization by VIM-1- and New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae was rapidly detected by phenotypic and genotypic methods. Phenotypic methods can be useful for routine detection of carbapenemase production.