Extended-spectrum beta-lactamase--producing enterobacteriaceae in the intensive care unit: acquisition does not mean cross-transmission.

BACKGROUND: In intensive care unit (ICU), infection and colonization by resistant Gram-negative bacteria increase costs, length of stay and mortality. Extended-spectrum beta-lactamase--producing Enterobacteriaceae (ESBL-E) is a group of pathogens increasingly encountered in ICU setting. Conditions that promote ESBL-E acquisition are not completely understood. The increasing incidence of infections related to ESBL-E and the unsolved issues related to ESBL-E cross-transmission, prompted us to assess the rates of referred and acquired cases of ESBL-E in ICU and to assess patient-to-patient cross-transmission of ESBL-E using a multimodal microbiological analysis. METHODS: During a 5-month period, all patients admitted to a medical ICU were tested for ESBL-E carriage. A rectal swab was performed at admission and then twice a week until discharge or death. ESBL-E strains were analyzed according to antibiotic susceptibility pattern, rep-PCR (repetitive-element Polymerase chain reaction) chromosomal analysis, and plasmid PCR (Polymerase chain reaction) analysis of ESBL genes. Patient-to-patient transmission was deemed likely when 2 identical strains were found in 2 patients hospitalized simultaneously in the ICU. RESULTS: Among the 309 patients assessed for ESBL-E carriage on admission, 25 were found to carry ESBL-E (importation rate: 8%). During follow-up, acquisition was observed among 19 of them (acquisition rate: 6.5%). Using the multimodal microbiological approach, we found only one case of likely patient-to-patient ESBL-E transmission. CONCLUSIONS: In unselected ICU patients, we found rather low rates of ESBL-E referred and acquired cases. Only 5% of acquisitions appeared to be related to patient-to-patient transmission. These data highlight the importance of jointly analyzing phenotypic profile and molecular data to discriminate strains of ESBL-E.

Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics.

Recent studies revealed that several vibrio species have evolved the capacity to survive inside host cells. However, it is still often ignored if intracellular stages are required for pathogenicity. Virulence of Vibrio tasmaniensis LGP32, a strain pathogenic for Crassostrea gigas oysters, depends on entry into hemocytes, the oyster immune cells. We investigated here the mechanisms of LGP32 intracellular survival and their consequences on the host-pathogen interaction. Entry and survival inside hemocytes were required for LGP32-driven cytolysis of hemocytes, both in vivo and in vitro. LGP32 intracellular stages showed a profound boost in metabolic activity and a major transcription of antioxidant and copper detoxification genes, as revealed by RNA sequencing. LGP32 isogenic mutants showed that resistance to oxidative stress and copper efflux are two main functions required for vibrio intracellular stages and cytotoxicity to hemocytes. Copper efflux was also essential for host colonization and virulence in vivo. Altogether, our results identify copper resistance as a major mechanism to resist killing by phagocytes, induce cytolysis of immune cells and colonize oysters. Selection of such resistance traits could arise from vibrio interactions with copper-rich environmental niches including marine invertebrates, which favour the emergence of pathogenic vibrios resistant to intraphagosomal killing across animal species.

Clostridium difficile: New Insights into the Evolution of the Pathogenicity Locus.

The major virulence factors of Clostridium difficile are toxins A and B. These toxins are encoded by tcdA and tcdB genes, which form a pathogenicity locus (PaLoc) together with three additional genes that have been implicated in regulation (tcdR and tcdC) and secretion (tcdE). To date, the PaLoc has always been found in the same location and is replaced in non-toxigenic strains by a highly conserved 75/115 bp non-coding region. Here, we show new types of C. difficile pathogenicity loci through the genome analysis of three atypical clinical strains and describe for the first time a variant strain producing only toxin A (A(+)B(-)). Importantly, we found that the PaLoc integration sites of these three strains are located in the genome far from the usual single known PaLoc integration site. These findings allowed us to propose a new model of PaLoc evolution in which two "Mono-Toxin PaLoc" sites are merged to generate a single "Bi-Toxin PaLoc".

A single regulatory gene is sufficient to alter Vibrio aestuarianus pathogenicity in oysters.

Oyster diseases caused by pathogenic vibrios pose a major challenge to the sustainability of oyster farming. In France, since 2012 a disease affecting specifically adult oysters has been associated with the presence of Vibrio aestuarianus. Here, by combining genome comparison, phylogenetic analyses and high-throughput infections of strains isolated before or during the recent outbreaks, we show that virulent strains cluster into two V. aestuarianus lineages independently of the sampling dates. The bacterial lethal dose was not different between strains isolated before or after 2012. Hence, the emergence of a new highly virulent clonal strain is unlikely. Each lineage comprises nearly identical strains, the majority of them being virulent, suggesting that within these phylogenetically coherent virulent lineages a few strains have lost their pathogenicity. Comparative genomics allowed the identification of a single frameshift in a non-virulent strain. This mutation affects the varS gene that codes for a signal transduction histidine-protein kinase. Genetic analyses confirmed that varS is necessary for infection of oysters and for a secreted metalloprotease expression. For the first time in a Vibrio species, we show here that VarS is a key factor of pathogenicity.

Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters.

Disease in oysters has been steadily rising over the past decade, threatening the long-term survival of commercial and natural stocks. Our understanding and management of such diseases are of critical importance as aquaculture is an important aspect of dealing with the approaching worldwide food shortage. Although some bacteria of the Vibrio genus isolated from diseased oysters have been demonstrated to be pathogenic by experimental infection, direct causality has not been established. Little is known about the dynamics of how the bacterial population hosted by oysters changes during disease progression. Combining experimental ecology, a high-throughput infection assay and genome sequencing, we show that the onset of disease in oysters is associated with progressive replacement of diverse benign colonizers by members of a phylogenetically coherent virulent population. Although the virulent population is genetically diverse, all members of that population can cause disease. Comparative genomics across virulent and nonvirulent populations identified candidate virulence factors that were clustered in population-specific genomic regions. Genetic analyses revealed that one gene for a candidate virulent factor, a putative outer membrane protein, is necessary for infection of oysters. Finally, analyses of oyster mortality following experimental infection suggest that disease onset can be facilitated by the presence of nonvirulent strains. This is a new form of polymicrobial disease, in which nonpathogenic strains contribute to increase mortality.

Antimicrobial resistance of Listeria monocytogenes isolates from food and the environment in France over a 10-year period.

In order to assess antimicrobial resistance in Listeria monocytogenes, 202 food and environmental isolates from 1996 to 2006 were tested. Only four strains displayed acquired resistance. Resistance to erythromycin, tetracycline-minocycline, and trimethoprim was evidenced, and the genes erm(B), tet(M), and dfrD, already found in L. monocytogenes, were detected.

Comparison of four methods, including semi-automated rep-PCR, for the typing of vancomycin-resistant Enterococcus faecium.

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates.

Association between hand hygiene compliance and methicillin-resistant Staphylococcus aureus prevalence in a French rehabilitation hospital.

We simultaneously investigated the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and compliance with hand hygiene in the clinical wards of a French rehabilitation hospital. We found that the rate of hand hygiene compliance observed at the patient's bedside was a strong predictor of MRSA prevalence.