Virulence of Klebsiella pneumoniae isolates harboring bla KPC-2 carbapenemase gene in a Caenorhabditis elegans model.

Klebsiella pneumoniae carbapenemase (KPC) is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i) five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii) seven transformants obtained after electroporation of either these natural KPC plasmids or a recombinant plasmid harboring only the bla KPC-2 gene into reference strains K. pneumoniae ATCC10031/CIP53153; and (iii) five clinical strains cured of plasmids. The virulence of K. pneumoniae isolates was evaluated in the Caenorhabditis elegans model. The clinical KPC producers and transformants were significantly less virulent (LT50: 5.5 days) than K. pneumoniae reference strain (LT50: 4.3 days) (p<0.01). However, the worldwide spread KPC-2 positive K. pneumoniae ST258 strains and reference strains containing plasmids extracted from K. pneumoniae ST258 strains had a higher virulence than KPC-2 strains belonging to other ST types (LT50: 5 days vs. 6 days, p<0.01). The increased virulence observed in cured strains confirmed this trend. The bla KPC-2 gene itself was not associated to increased virulence.

A single amino acid change in the transmembrane domain of the VirB8 protein affects dimerization, interaction with VirB10 and Brucella suis virulence.

VirB8 is a critical component of the Brucella suis type IV secretion system (T4SS). We previously showed that the transmembrane (TM) domain plays an essential role in interactions of this protein with itself and the other proteins of the T4SS. We report that a point mutation in this TM domain stabilizes homodimers of VirB8 and heterodimers with VirB10. A similar variant of Agrobacterium tumefaciens VirB8 showed the same phenotype. The B. suis VirB8 variant was unable to complement a virB8 mutant and displayed a dominant negative phenotype when expressed in wild type B. suis. We suggest that interaction of VirB8 with VirB10 could play a major role in the correct function of the B. suis VirB T4SS.

Interactions between Brucella suis VirB8 and its homolog TraJ from the plasmid pSB102 underline the dynamic nature of type IV secretion systems.

The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.

Virulent synergistic effect between Enterococcus faecalis and Escherichia coli assayed by using the Caenorhabditis elegans model.

BACKGROUND: The role of enterococci in the pathogenesis of polymicrobial infections is still debated. The purpose of this study was to evaluate the effect of virulent enterococci in the presence or absence of Escherichia coli strains in the in vivo Caenorhabditis elegans model. PRINCIPAL FINDINGS: This study demonstrated that there was a synergistic effect on virulence when an association of enterococci and E. coli (LT50 = 1.6 days+/-0.1 according to the tested strains and death of nematodes in 4 days+/-0.5) was tested in comparison with enterococci alone (LT50 = 4.6 days+/-0.1 and death in 10.4 days+/-0.6) or E. coli alone (LT50 = 2.1+/-0.9 and deaths 6.6+/-0.6) (p<0.001). In addition, there was a relation between the virulence of E. faecalis strains alone and the virulence potential of the association with E. coli strains. Finally, in the presence of avirulent E. coli strains, enterococci have no effect (LT50 = 4.3+/-0.5 and deaths in 10.8+/-0.8), independently of the level of their own virulence, demonstrating that the 'enterococci effect' only occurred in the presence of virulent E. coli strains. CONCLUSION: This study allows a better understanding of a bacterial cooperation. Moreover, it could help to optimize the antibiotic regimen during polymicrobial infections.

Virulence potential of Staphylococcus aureus strains isolated from diabetic foot ulcers: a new paradigm.

OBJECTIVE: The purpose of this study was to assess the virulence potential of Staphylococcus aureus strains isolated from diabetic foot ulcers and to discriminate noninfected from infected ulcers. RESEARCH DESIGN AND METHODS: Diabetic patients hospitalized in a diabetic foot department with a foot ulcer were prospectively enrolled if they had been free of antibiotic treatment over the previous 6 months. At admission, ulcers were classified as infected or noninfected on the basis of clinical examination, according to the International Working Group on the Diabetic Foot system. Only patients carrying S. aureus as the sole pathogen were included. In individuals with a grade 1 ulcer, a second bacterial specimen was obtained 1 month later. Using virulence genotyping markers, clonality tools, and an in vivo Caenorhabditis elegans model, we correlated the virulence of 132 S. aureus strains with grade, time of collection, and ulcer outcome. RESULTS: Among virulence genes, the most relevant combination derived from the logistic regression was the association of cap8, sea, sei, lukE, and hlgv (area under the curve 0.958). These markers were useful to distinguish noninfected (grade 1) from infected (grades 2-4) ulcers and to predict wound status at the follow-up. With use of the nematode model, S. aureus strains isolated from grade 1 ulcers were found to be significantly less virulent than strains from ulcers at or above grade 2 (P < 0.001). CONCLUSIONS: This study highlights the coexistence of two S. aureus populations on diabetic foot ulcers. A combination of five genes that may help distinguish colonized grade 1 from infected grade >or=2 wounds, predict ulcer outcome, and contribute to more appropriate use of antibiotics was discovered.

The genomic structure of Brucella strains isolated from marine mammals gives clues to evolutionary history within the genus.

The genomic structure and the restriction maps were studied in 24 Brucella strains isolated from marine mammals. From SpeI restriction profiles, the strains could be ascribed to three clonal groups, each corresponding to a specific host. Cross contamination between exclusively terrestrial and exclusively marine hosts is unlikely suggesting the divergence of the different species of the genus Brucella which may have taken place 60 million years ago, concomitant with the radiation of their mammalian hosts (Artiodactyla) from other mammalian orders.

Swapping of periplasmic domains between Brucella suis VirB8 and a pSB102 VirB8 homologue allows heterologous complementation.

A Brucella suis mutant with a nonpolar deletion in the virB8 gene was attenuated in a macrophage infection model. Complementation with the B. suis VirB8 protein expressed from the virB promoter restored virulence. Expression of TraJ, a VirB8 homologue from plasmid pSB102, did not restore virulence; however, virulence was partially restored by a chimeric protein containing the N terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ.

The IncP island in the genome of Brucella suis 1330 was acquired by site-specific integration.

An 18,228-bp region containing open reading frames predicted to be derived from the IncP plasmid or phage ancestors is present in the genomes of Brucella suis biovars 1 to 4, B. canis, B. neotomae, and strains isolated from marine mammals, but not in B. melitensis, B. abortus, B. ovis, and B. suis biovar 5. The presence of circular excision intermediates and the results of an analysis of sequenced bacterial genomes suggest that the region downstream of the guaA gene is a hotspot for site-specific integration of foreign DNA mediated by a CP4-like integrase.

Identification of a new virulence factor, BvfA, in Brucella suis.

We report the identification of BvfA (for Brucella virulence factor A), a small periplasmic protein unique to the genus Brucella, which is essential for the virulence of Brucella suis. A BvfA knockout mutant was highly attenuated both in in vitro macrophage infection assays and in vivo in the murine model of brucellosis. Fluorescence-activated cell sorting analysis with green fluorescent protein fusions showed that the expression of bvfA is induced within macrophages by phagosome acidification and coregulated with the B. suis virB operon, suggesting that it too may play a role in the establishment of the intracellular replication niche.

Type IV secretion and Brucella virulence.

The type IV secretion system, encoded by the virB region, is a key virulence factor for Brucella. The 12 genes of the region form an operon that is specifically induced by phagosome acidification in cells after phagocytosis. We speculate that the system serves to secrete unknown effector molecules, which allow Brucella to pervert the host cell endosomal pathways and to create a novel intracellular compartment in which it can replicate.

The Brucella genome at the beginning of the post-genomic era.

The year 2002 began with the publication of the first complete genome sequence for a Brucella species, that of the two replicons of B. melitensis 16M. Hopefully in 2002, the complete genome of B. suis 1330, and, perhaps, a B. abortus strain will be published. This is the culmination of over 30 years investigation of the composition, structure, organisation and evolution of the Brucella genome. Brucella research must now adapt to the new challenges of the post-genomic era.

The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell.

The pathogen Brucella suis resides and multiplies within a phagocytic vacuole of its host cell, the macrophage. The resulting complex relationship has been investigated by the analysis of the set of genes required for virulence, which we call intramacrophagic virulome. Ten thousand two hundred and seventy-two miniTn5 mutants of B. suis constitutively expressing gfp were screened by fluorescence microscopy for lack of intracellular multiplication in human macrophages. One hundred thirty-one such mutants affected in 59 different genes could be isolated, and a function was ascribed to 53 of them. We identified genes involved in (i) global adaptation to the intracellular environment, (ii) amino acid, and (iii) nucleotide synthesis, (iv) sugar metabolism, (v) oxidoreduction, (vi) nitrogen metabolism, (vii) regulation, (viii) disulphide bond formation, and (ix) lipopolysaccharide biosynthesis. Results led to the conclusion that the replicative compartment of B. suis is poor in nutrients and characterized by low oxygen tension, and that nitrate may be used for anaerobic respiration. Intramacrophagic virulome analysis hence allowed the description of the nature of the replicative vacuole of the pathogen in the macrophage and extended our understanding of the niche in which B. suis resides. We propose calling this specific compartment "brucellosome."

The Brucella suis virB operon is induced intracellularly in macrophages.

A type IV secretion system similar to the VirB system of the phytopathogen Agrobacterium tumefaciens is essential for the intracellular survival and multiplication of the mammalian pathogen Brucella. Reverse transcriptase-PCR showed that the 12 genes encoding the Brucella suis VirB system form an operon. Semiquantitative measurements of virB mRNA levels by slot blotting showed that transcription of the virB operon, but not the flanking genes, is regulated by environmental factors in vitro. Flow cytometry used to measure green fluorescent protein expression from the virB promoter confirmed the data from slot blots. Fluorescence-activated cell sorter analysis and fluorescence microscopy showed that the virB promoter is induced in macrophages within 3 h after infection. Induction only occurred once the bacteria were inside the cells, and phagosome acidification was shown to be the major signal inducing intracellular expression. Because phagosome acidification is essential for the intracellular multiplication of Brucella, we suggest that it is the signal that triggers the secretion of unknown effector molecules. These effector molecules play a role in the remodeling of the phagosome to create the unique intracellular compartment in which Brucella replicates.