Biomedical subjects

Jean-Christophe Giard

Publications and source records attributed to Jean-Christophe Giard.

11 recordsLinked to original sources

PubMed2026

In-host adaptation of Staphylococcus aureus during recurrent prosthetic joint infections: a retrospective longitudinal study.

UNLABELLED: The aim of this study was to characterize the in vivo evolution of Staphylococcus aureus strains involved in recurrent prosthetic joint infections (PJIs) both phenotypically and genomically. We conducted a monocentric retrospective study in a 1,437-bed French teaching hospital between 2013 and 2021. All patients presenting a recurrent S. aureus-related PJI-defined as at least two strains isolated from distinct clinical samples more than 90 days apart-of the knee, hip, or shoulder were included. Clinical data were reviewed, and all isolates underwent phenotypic characterization, including antimicrobial susceptibility testing, growth rate determination, biofilm production assays, metabolic profiling (API 50 CH), and virulence evaluation using the Galleria mellonella infection model. Whole-genome sequencing (WGS) was performed for all strains, followed by analyses of core-genome multilocus sequence typing (cgMLST), resistome, virulome, and mobilome composition, and single-nucleotide polymorphisms (SNPs). Thirteen patients met inclusion criteria, yielding 55 S. aureus isolates. Eight patients experienced recurrent infections caused by genetically closely related strains throughout the clinical course (median: three strains per patient; range: 2-6), whereas five patients were infected by genetically distinct strains. At baseline, isolates were genetically diverse and susceptible to methicillin and rifampicin; two showed fluoroquinolone resistance due to grlA and/or gyrA mutations. In one patient (patient C), a recurrent isolate acquired an rpoB S486L mutation, conferring rifampicin resistance after rifampicin exposure. Due to the limited sample size, it is difficult to draw definitive conclusions from the phenotypic analyses. This study highlights the adaptive evolution of S. aureus during chronic PJIs and underscores the need for further research to better understand intra-host dynamics in long-standing infections. IMPORTANCE: This study conducted in a 1,437-bed French teaching hospital analyzed the genomic and phenotypic evolution of 55 Staphylococcus aureus strains recovered in recurrent PJIs from 13 patients. The first strains showed high genotypic diversity across 12 different sequence types. Among the 13 patients, only eight experienced a true recurrence with the same strain, while five were contaminated with a different strain of S. aureus, indicating a new infection. Moreover, this study underscores the complex within-host evolution of S. aureus and highlights the phenotypical and genotypical adaptation during chronic infection.

Staphylococcus aureus↗

PubMed2006

Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages.

The gene encoding the manganese-containing superoxide dismutase (MnSOD) of Enterococcus faecalis was characterized. It is transcribed monocistronically from an upstream promoter identified by rapid amplification of cDNA ends (RACE)-PCR. A sodA mutant was constructed and characterized. Growth of the mutant strain was not significantly different from that of its wild-type counterpart in standing and aerated cultures. However, the mutant was more sensitive towards menadione and hydroperoxide stresses. The response to H(2)O(2) stress was analysed in more detail, and the mode of killing of this oxidant was different under anaerobic and aerobic conditions. Cultures grown and challenged under anaerobic conditions were highly sensitive to treatment with 35 mM H(2)O(2). They were largely protected by the iron chelator deferoxamine, which suggested that killing was mainly due to an enhanced Fenton reaction. In contrast, neither strain was protected by the iron chelators deferoxamine and diethylenetriaminepentaacteic acid when grown and challenged under aerobic conditions, which suggested that inactivation of the cells by H(2)O(2) was due to another killing mode. The sodA mutant was more sensitive under these conditions, showing that MnSOD is also important for protecting the cells from damage under aerobic conditions. Finally, the MnSOD of Ent. faecalis may be considered to be a virulence factor, since survival of the corresponding mutant strain was highly affected inside mouse peritoneal macrophages.

Animals↗

PubMed2006

Characterization of Ers, a PrfA-like regulator of Enterococcus faecalis.

We have identified a transcriptional regulator, named Ers (for enterococcal regulator of survival), of Enterococcus faecalis, an important opportunistic bacterium commonly recovered from hospitalized patients. Ers is a member of the Crp/Fnr family and is 69% similar to Srv, a PrfA-like regulator of Streptococcus pyogenes implicated in virulence, and is the E. faecalis protein most closely related to PrfA, a positive regulator of virulence genes in Listeria monocytogenes. In an in vivo-in vitro macrophage infection model, the survival of an ers mutant was highly significantly decreased compared with that of the parental strain JH2-2. This mutant was more than 10-fold more sensitive to oxidative challenge by hydrogen peroxide. In order to identify genes whose expression was under Ers control, the RNA levels of 31 likely candidates were measured by real-time quantitative PCR. The results indicate that ers may be autoregulated and that the locus ef0082 appears to be positively regulated by Ers. Nevertheless, mutation of ef0082 did not result in any detectable changes in the survival of the bacterium within murine macrophages.

Amino Acid Sequence↗

PubMed2005

Implication of hypR in the virulence and oxidative stress response of Enterococcus faecalis.

HypR has recently been described as the first transcriptional regulator involved in the oxidative stress response and in the intracellular survival of Enterococcus faecalis within macrophages. In order to characterize the HypR regulon, real-time quantitative RT-PCR experiments were performed. The expression of four genes involved in the oxidative stress response encoding catalase, glutathione reductase, and the two subunits of alkyl hydroperoxide reductase were down regulated in the hypR background under H(2)O(2) condition. These findings show that HypR acts as a transcriptional activator, especially during oxidative stress. In addition, DNAse I footprinting assays allowed us to identify the HypR-protected DNA regions corresponding to the "HypR box" in the hypR promoter. Moreover, the effect of the hypR mutation on the virulence of E. faecalis was evaluated in comparison with the wild-type JH2-2 strain using a mouse peritonitis model. Our results revealed that HypR appears to be an important virulence factor in E. faecalis.

Animals↗

PubMed2005

Contribution of a PerR-like regulator to the oxidative-stress response and virulence of Enterococcus faecalis.

PerR is one of the most important transcriptional regulators involved in the oxidative-stress response in Bacillus subtilis. Here, the homologous gene in Enterococcus faecalis, ranked among the leading causes of nosocomial infection, was characterized and analysed. Phenotype analysis showed that the perR mutant was significantly more resistant to H2O2 challenge (P < 0.05). Expression of eight genes with potential roles in the oxidative-stress response was determined in the wild-type and perR-mutant strains by real-time quantitative PCR. Surprisingly, low quantitative differences in the transcriptional activity of these genes in the mutant versus wild-type were observed. Likewise, this locus was not involved in survival within murine macrophages, but in the mouse peritonitis model, the perR mutant appeared less lethal than the JH2-2 wild-type strain. The combined results show that PerR affects E. faecalis virulence and that its implication in the transcriptional regulation in this bacterium deviates from the B. subtilis model.

Bacterial Proteins↗

PubMed2005

The Enterococcus faecalis sigV protein is an extracytoplasmic function sigma factor contributing to survival following heat, acid, and ethanol treatments.

Analysis of the genome sequence of Enterococcus faecalis allowed the identification of two genes whose protein products showed 33 and 34% identity with those of sigV and yrhM of Bacillus subtilis, respectively. These genes, named sigV and rsiV, are predicted to encode members of the extracytoplasmic function subfamily of eubacterial RNA polymerase sigma and anti-sigma factors, respectively. This group of sigma factors has been shown to regulate gene expression in response to stress conditions. sigV and rsiV were shown to be under the control of the same promoter. The transcriptional start site was determined, and the 1.5-kb mRNA transcript was shown to be overexpressed under glucose and complete starvation, as well as under physicochemical treatments. Three mutants, affected in sigV, rsiV, and both genes, were constructed by double-crossover recombination within the genome of E. faecalis strain JH2-2. Compared with the wild type and the rsiV mutant, the sigV mutants were more susceptible to heat shock, acid, and ethanol treatments and displayed decreased survival during long-term starvation. A nisin-inducible sigV gene construction used in complementation assays restored the wild phenotype of the sigV mutants, confirming the involvement of SigV in the heat shock, ethanol, and acid stress responses. Northern blot analysis carried out with the three mutant strains revealed the inhibition of sigV expression by the related anti-sigma factor gene rsiV. In addition, putative candidates of the sigV regulon determined by computer search for the sigV promoter sequence were analyzed.

Amino Acid Sequence↗

PubMed2004

Is 2-phosphoglycerate-dependent automodification of bacterial enolases implicated in their export?

We observed that in vivo and in vitro a small fraction of the glycolytic enzyme enolase became covalently modified by its substrate 2-phosphoglycerate (2-PG). In modified Escherichia coli enolase, 2-PG was bound to Lys341, which is located in the active site. An identical reversible modification was observed with other bacterial enolases, but also with enolase from Saccharomyces cerevisiae and rabbit muscle. An equivalent of Lys341, which plays an important role in catalysis, is present in enolase of all organisms. Covalent binding of 2-PG to this amino acid rendered the enzyme inactive. Replacement of Lys341 of E.coli enolase with other amino acids prevented the automodification and in most cases strongly reduced the activity. As reported for other bacteria, a significant fraction of E.coli enolase was found to be exported into the medium. Interestingly, all Lys341 substitutions prevented not only the automodification, but also the export of enolase. The K341E mutant enolase was almost as active as the wild-type enzyme and therefore allowed us to establish that the loss of enolase export correlates with the loss of modification and not the loss of glycolytic activity.

Animals↗

PubMed2004

Effects of the Enterococcus faecalis hypR gene encoding a new transcriptional regulator on oxidative stress response and intracellular survival within macrophages.

In order to identify regulators of the oxidative stress response in Enterococcus faecalis, an important human pathogen, several genes annotated as coding for transcriptional regulators were inactivated by insertional mutagenesis. One mutant, affected in the ef2958 locus (designated hypR [hydrogen peroxide regulator]), appeared to be highly sensitive to oxidative challenge caused by hydrogen peroxide. Moreover, testing of the hypR mutant by using an in vivo-in vitro macrophage infection model resulted in a highly significant reduction in survival compared to the survival of parent strain JH2-2. Northern blot analyses were carried out with probes specific for genes encoding known antioxidant enzymes, and they showed that the ahpCF (alkyl hydroperoxide reductase) transcript was expressed less in mutant cells. Mobility shift protein-DNA binding assays revealed that HypR regulated directly the expression of hypR itself and the ahpCF operon. Our combined results showed that HypR appeared to be directly involved in the expression of ahpCF genes under oxidative stress conditions and suggested that this regulator could contribute to the virulence of E. faecalis.

Animals↗

PubMed2003

Physiological and molecular aspects of bile salt response in Enterococcus faecalis.

Analysis of the susceptibility and the acquisition of tolerance in Enterococus faecalis towards bile salts showed a nearly instantaneous killing effect and yielded evidence for homologous tolerance as well as for cross-protections. Two-dimensional (2-D) electrophoresis revealed 45 proteins which are amplified in response to the bile salt treatment. These include a set of seven proteins, the synthesis of which is increased not only with the bile salts but also with multiple sublethal stresses of various nature. Characterisation of the latter (called general stress proteins) showed that at least five of them are related to resistance to bile salts, heat, ethanol, oxidative and alkaline pH stresses and are probably involved in cross-protection development. On the other hand, random mutagenesis of E. faecalis allowed the isolation of 10 bile salt-sensitive mutants. Their characterisation revealed that the mutation loci corresponded to genes related to DNA repair, oxidative response, transcriptional regulation, dGTP hydrolysis, membrane composition or cell wall synthesis. Further characterisation of one mutant revealed that the insertion within the E. faecalis sagA gene led to morphology changes, to perturbations of cell division and to a decrease of the resistance towards several independent physicochemical stresses.

Bacterial Proteins↗

PubMed2002

Characterization of genes homologous to the general stress-inducible gene gls24 in Enterococcus faecalis and Lactococcus lactis.

Three genes (Ef0604, ymgG, and ytgH) were identified as homologous to the gls24 gene (encoding the general stress protein Gls24) in Enterococcus faecalis V583 and Lactococcus lactis IL1403 genomes sequences, and therefore are part of the 'gls24 family'. The other proteins encoded by the different surrounding genes in each of their respective operons are also highly similar. Our transcriptional analysis showed that Ef0604 and ymgG are not induced under the stress conditions tested. On the other hand, ytgH is induced at the onset of glucose starvation and by heat, ethanolic, osmotic, and CdCl(2) stresses. The transcriptional start site of this operon was identified and the promoter region appears to contain an 'extended -10 Box'. Moreover, the over-expression under several stress conditions of the YtgH protein observed on 2D gel electrophoresis confirms that it corresponds to a general stress protein in L. lactis.

Bacterial Proteins↗

PubMed2002

The Enterococcus faecalis gene encoding the novel general stress protein Gsp62.

The Enterococcus faecalis general stress protein Gsp62 was purified using two-dimensional gel electrophoresis and its 25 N-terminal amino acid sequence determined. Analysis of the corresponding gene revealed that the gsp62 product is a 172 aa protein. Transcriptional analysis of gsp62 gave evidence for a monocistronic mRNA, the synthesis of which was induced at the onset of stationary phase and in response to heat shock, acid pH, detergents (i.e. SDS or bile salts), ethanol, tert-butyl hydroperoxide, sodium chloride and, to a lesser extent, hydrogen peroxide. 5' rapid amplification of cDNA ends by PCR experiments showed that gsp62 transcription initiates 30 nt upstream of the ATG start codon. Although gsp62 expression was induced in response to various stresses, its disruption had no significant effect on the cell survival after each individual stress. Two-dimensional protein gels from wild-type and mutant cells revealed no pleiotropic effect of the mutation on protein synthesis. Transcriptional fusions with the lacL lacM beta-galactosidase genes showed that an inverted repeat located upstream of the promoter is required for transcriptional induction by environmental stresses but not by entrance into stationary phase. Two distinct mechanisms responding to different signals are thus involved in gsp62 induction.

Amino Acid Sequence↗