Highly virulent M1 Streptococcus pyogenes isolates resistant to clindamycin.

CONTEXT: Emm1-type group A Streptococcus (GAS), or Streptococcus pyogenes, is mostly responsible for invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The recommended treatment of severe invasive GAS infections is a combination of clindamycin and penicillin. Until 2012, almost all emm1 isolates were susceptible to clindamycin. OBJECTIVES: We aimed to identify the phenotypic and genotypic characteristics of emm1 GAS clone resistant to clindamycin. METHODS: GAS strains were characterized by emm sequence typing, detection of genes encoding pyrogenic exotoxins or superantigens. Cluster analysis was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antibiotic susceptibility was assessed using disk diffusion and resistance genes were detected by PCR. RESULTS: A total of 1321 GAS invasive isolates were analyzed between January 2011 and December 2012. The overall number of invasive isolates resistant to clindamycin was 52 (3.9%); seven of them were emm1 isolates. All isolates had the same genomic markers: macrolide resistance due to the presence of the erm(B) gene, emm subtype 1.0, the same toxin or superantigen profile, PFGE pattern and sequence type. CONCLUSION: This is the first description of highly virulent GAS emm1 isolates resistant to clindamycin in France. This article strengthens the need for monitoring the epidemiology of invasive GAS strains as they could lead to changes in treatment guidelines.

Group B streptococcus neonatal invasive infections, France 2007-2012.

Streptococcus agalactiae (group B streptococcus (GBS)) is the leading cause of invasive infections among newborns in industrialized countries, with two described syndromes: early-onset disease (EOD) and late-onset disease (LOD). Since the introduction in many countries of intrapartum antibioprophylaxis (IAP), the incidence of EOD has dramatically decreased, whereas that of LOD remains unchanged. We describe the clinical and bacteriological characteristics of 438 GBS neonatal invasive infections notified to the French National Reference Centre for Streptococci in France from 2007 to 2012. Clinical data were retrieved from hospitalization reports or questionnaires. Capsular type, assignment to the hypervirulent clonal complex (CC)17 and antibiotic susceptibility profiles were determined. One hundred and seventy-four (39.7%) and 264 (60.3%) isolates were responsible for EOD, including death in utero, and LOD, respectively. EOD was associated with bacteraemia (n = 103, 61%) and LOD with meningitis (n = 145, 55%). EOD was mainly due to capsular polysaccharide (CPS) III isolates (n = 99, 57%) and CPS Ia isolates (n = 40, 23%), and CPS III isolates were responsible for 80% (n = 211) of LOD cases. CC17 accounted for 80% (n = 121) of CPS III isolates responsible for meningitis (n = 151; total cases of meningitis, 188). Bad outcome risk factors were low gestational age and low birthweight. LOD represents almost 60% of cases of neonatal GBS disease in France and other countries in which IAP has been implemented. This observation reinforces the need to develop new prevention strategies targeting CC17, which is predominant in GBS neonatal infections.

[Epidemiology of Streptococcus pyogenes invasive diseases in France (2007-2011)]. / Épidémiologie des infections invasives à Streptococcus pyogenes (France 2007-2011).

Group A Streptococcus (GAS) is a human pathogen responsible for a wide range of clinical manifestations. An increase of GAS invasive infections has been described since the mid 1980s. To study the French epidemiology of invasive infections (i) we characterized all GAS invasive strains received at the French National Reference Center for streptococci (CNR-Strep) between 2007 and 2011; (ii) we analyzed the epidemiological data on the corresponding strains. For each strain, emm genotype, superantigen genes and antibiotics susceptibility were determined. Among the 2 603 non redundant invasive GAS strains, 65.1 % (n=1 695) were isolated from blood culture. A streptococcal toxic shock syndrome (STSS) was described in 16.4 % (n=428) of cases, mostly associated with necrotizing fasciitis (NF), pleuropulmonary or osteoarticular infections (p ≤0.001). The case fatality rate was 10.6 %. A total of 102 different emm genotypes were identified. Three emm genotypes predominated, reaching nearly 60 % of the strains: emm 1 (26.7 %), emm 28 (16.4 %), and emm 89 (12.8 %). The proportion of each emm genotype varied according to the year and the age of patients. Among those < 15 years old, the three main genotypes were emm 1 (36.8 %), emm 12 (12.9 %) and emm 4 (9.5 %). The distribution of superantigen genes (SpeA, SpeC and Ssa) was restricted to several emm genotypes. Between 2007 and 2011, the rate of macrolides resistant GAS strains decreased from 7.8 to 5.5 %. emm 1 strains are still the most common especially in most severe clinical manifestations including STSS and NF.

Identification of CodY targets in Bacillus anthracis by genome-wide in vitro binding analysis.

In Gram-positive bacteria, CodY is an important regulator of genes whose expression changes under conditions of nutrient limitation. Bacillus anthracis CodY represses or activates directly or indirectly approximately 500 genes. Affinity purification of CodY-DNA complexes was used to identify the direct targets of CodY. Of the 389 DNA binding sites that were copurified with CodY, 132 sites were in or near the regulatory regions governing the expression of 197 CodY-controlled genes, indicating that CodY controls many other genes indirectly. CodY-binding specificity was verified using electrophoretic mobility shift and DNase I footprinting assays for three CodY targets. Analysis of the bound sequences led to the identification of a B. anthracis CodY-binding consensus motif that was found in 366 of the 389 affinity-purified DNA regions. Regulation of the expression of the two genes directly controlled by CodY, sap and eag, encoding the two surface layer (S-layer) proteins, was analyzed further by monitoring the expression of transcriptional lacZ reporter fusions in parental and codY mutant strains. CodY proved to be a direct repressor of both sap and eag expression. Since the expression of the S-layer genes is under the control of both CodY and PagR (a regulator that responds to bicarbonate), their expression levels respond to both metabolic and environmental cues.

Bacillus anthracis cell envelope components.

Bacillus anthracis is a Gram-positive bacterium harboring a complex parietal architecture. The cytoplasmic membrane is surrounded by a thick peptidoglycan of the A1 gamma type. Only one associated polymer, a polysaccharide composed of galactose, N-acetylglucosamine, and N-acetylmannosamine, is covalently linked to the peptidoglycan. Outside the cell wall is an S-layer. Two proteins can each compose the S-layer. They are noncovalently anchored to the cell wall polysaccharide by their SLH N-terminal domain. The poly-gamma-D-glutamate capsule, which covers the S-layer, has an antiphagocytic role and its synthesis is dependent on environmental factors mimicking the mammalian host, such as bicarbonate and a temperature of 37 degrees C.

Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure.

Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus, in some B. thuringiensis strains and in B. anthracis, an S-layer has been described. We investigated how the S-layer is distributed in B. cereus, and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non-B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis. Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis, except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance.

Anthrax.

Bacillus anthracis was shown to be the etiological agent of anthrax by R. Koch and L. Pasteur at the end of the nineteenth century. The concepts on which medical microbiology are based arose from their work on this bacterium. The link between plasmids and major virulence factors of B. anthracis was not discovered until the 1980s. The three toxin components are organized in two A-B type toxins, and the bacilli are covered by an antiphagocytic polyglutamic capsule. Structure-function analysis of the toxins indicated that the common B-domain binds to a ubiquitous cell receptor and forms a heptamer after proteolytic activation. One enzyme moiety is an adenylate cyclase and the other is a Zn(2+) metalloprotease, which is able to cleave MAPKKs. The capsule covers an S-layer sequentially composed of two distinct proteins. Knowledge of the toxins facilitates the design of safer veterinary vaccines. Spore-structure analysis could contribute to the improvement of human nonliving vaccines. The phylogeny of B. anthracis within the Bacillus cereus group is also reviewed.

The incompatibility between the PlcR- and AtxA-controlled regulons may have selected a nonsense mutation in Bacillus anthracis.

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are members of the Bacillus cereus group. These bacteria express virulence in diverse ways in mammals and insects. The pathogenic properties of B. cereus and B. thuringiensis in mammals results largely from the secretion of non-specific toxins, including haemolysins, the production of which depends upon a pleiotropic activator PlcR. In B. anthracis, PlcR is inactive because of a nonsense mutation in the plcR gene. This suggests that the phenotypic differences between B. anthracis on the one hand and B. thuringiensis and B. cereus on the other could result at least partly from loss of the PlcR regulon. We expressed a functional PlcR in B. anthracis. This resulted in the transcriptional activation of genes weakly expressed in the absence of PlcR. The transcriptional activation correlated with the induction of enzymatic activities and toxins including haemolysins. The toxicity of a B. anthracis PlcR+ strain was assayed in the mouse subcutaneous and nasal models of infection. It was no greater than that of the parental strain, suggesting that the PlcR regulon has no influence on B. anthracis virulence. The PlcR regulon had dramatic effects on the sporulation of a B. anthracis strain containing the virulence plasmid pXO1. This resulted from incompatible interactions with the major AtxA-controlled virulence regulon. We propose that the PlcR-controlled regulon in B. anthracis has been counterselected on account of its disadvantageous effects.

Characterization of a plasmid region involved in Bacillus anthracis toxin production and pathogenesis.

The germination of spores within the host is the initial step of anthrax infection. We have shown, using immunofluorescence staining, confocal scanning laser microscopy and image cytometry analysis, that the alveolar macrophage is the primary site of B. anthracis germination in a murine inhalation infection model. B. anthracis germinated inside macrophages, in vesicles derived from the phagosomal compartment. We have demonstrated that the toxin genes and their trans-activator, AtxA, are expressed within the macrophages after germination. It was also shown that the pXO1 plasmid strongly enhanced capsule formation and that this influence is mediated by AtxA. This indicates the existence of a regulon where AtxA is the regulatory protein acting on genes located on different plasmids. We identified a tricistronic germination operon gerX located between the pag and atxA genes on the 40-kb toxin-encoding fragment of pXO1 . Analysis of a gerX null mutant indicated that gerX-encoded proteins are involved in the virulence of B. anthracis.

Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation.

Several bacterial proteins are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non-covalent interaction between the SLH domain and peptidoglycan-associated polymers. Here we report the characterization of a two-gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH-containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan-associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH-containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.

Characterization of the operon encoding the alternative sigma(B) factor from Bacillus anthracis and its role in virulence.

The operon encoding the general stress transcription factor sigma(B) and two proteins of its regulatory network, RsbV and RsbW, was cloned from the gram-positive bacterium Bacillus anthracis by PCR amplification of chromosomal DNA with degenerate primers, by inverse PCR, and by direct cloning. The gene cluster was very similar to the Bacillus subtilis sigB operon both in the primary sequences of the gene products and in the order of its three genes. However, the deduced products of sequences upstream and downstream from this operon showed no similarity to other proteins encoded by the B. subtilis sigB operon. Therefore, the B. anthracis sigB operon contains three genes rather than eight as in B. subtilis. The B. anthracis operon is preceded by a sigma(B)-like promoter sequence, the expression of which depends on an intact sigma(B) transcription factor in B. subtilis. It is followed by another open reading frame that is also preceded by a promoter sequence similarly dependent on B. subtilis sigma(B). We found that in B. anthracis, both these promoters were induced during the stationary phase and induction required an intact sigB gene. The sigB operon was induced by heat shock. Mutants from which sigB was deleted were constructed in a toxinogenic and a plasmidless strain. These mutants differed from the parental strains in terms of morphology. The toxinogenic sigB mutant strain was also less virulent than the parental strain in the mouse model. B. anthracis sigma(B) may therefore be a minor virulence factor.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

Bacillus anthracis surface: capsule and S-layer.

Two abundant surface proteins, EA1 and Sap, are components of the Bacillus anthracis surface layer (S-layer). Their corresponding genes have been cloned, shown to be clustered on the chromosome and sequenced. EA1 and Sap each possess three 'S-layer homology' motifs. Single and double disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of both the non-capsulated and capsulated bacilli. When present, the capsule is exterior to, and completely covers, the S-layer proteins, which form an array beneath it. Nevertheless, the presence of these proteins is not required for normal capsulation of the bacilli. Thus both structures are compatible, and yet neither is required for the correct formation of the other. Bacillus anthracis has, therefore, a very complex cell wall organization for a gram-positive bacterium.

The S-layer homology domain as a means for anchoring heterologous proteins on the cell surface of Bacillus anthracis.

Bacillus anthracis synthesizes two S-layer proteins, each containing three S-layer homology (SLH) motifs towards their amino-terminus. In vitro experiments suggested that the three motifs of each protein were organized as a structural domain sufficient to bind purified cell walls. Chimeric genes encoding the SLH domains fused to the levansucrase of Bacillus subtilis were constructed and integrated on the chromosome. Cell fractionation and electron microscopy studies showed that both heterologous polypeptides were targeted to the cell surface. In addition, surface-exposed levansucrase retained its enzymatic and antigenic properties. Preliminary results concerning applications of this work are presented.

Cell surface-exposed tetanus toxin fragment C produced by recombinant Bacillus anthracis protects against tetanus toxin.

Bacillus anthracis, the causal agent of anthrax, synthesizes two surface layer (S-layer) proteins, EA1 and Sap, which account for 5 to 10% of total protein and are expressed in vivo. A recombinant B. anthracis strain was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and tetanus toxin fragment C (ToxC). This construct was expressed under the control of the promoter of the S-layer component gene. The hybrid protein was stably expressed on the cell surface of the bacterium. Mice were immunized with bacilli of the corresponding strain, and the hybrid protein elicited a humoral response to ToxC. This immune response was sufficient to protect mice against tetanus toxin challenge. Thus, the strategy developed in this study may make it possible to generate multivalent live veterinary vaccines, using the S-layer protein genes as a cell surface display system.

Co-existence of clpB and clpC in the Bacillaceae.

The gene encoding ClpC in Bacillus anthracis was amplified from the chromosome by polymerase chain reaction using degenerate oligonucleotide primers. These primers also amplified a second DNA fragment identified as a clpB homolog. Both genes were suggested to be functional. Contrary to Bacillus subtilis which possesses clpC but not clpB, many Bacillus species were found to harbor both clpC and clpB. We also found that Clostridium strains could possess clpB, clpC, or both. All the Gram-negative strains tested had clpB only.

Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S-layer proteins and the Bacillus subtilis levansucrase.

Many surface proteins of Gram-positive bacteria contain motifs, about 50 amino acids long, called S-layer homology (SLH) motifs. Bacillus anthracis, the causal agent of anthrax, synthesizes two S-layer proteins, each with three SLH motifs towards the amino-terminus. We used biochemical and genetic approaches to investigate the involvement of these motifs in cell surface anchoring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three-motif polypeptides were produced in Escherichia coli that were able to bind the B. anthracis cell walls in vitro, demonstrating that the three SLH motifs were organized into a cell surface anchoring domain. We also determined the function of these SLH domains by constructing chimeric genes encoding the SLH domains fused to the normally secreted levansucrase of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three-motif domain was sufficient for the efficient anchoring of levansucrase onto the cell surface. Proteins consisting of truncated SLH domains fused to levansucrase were unstable and associated poorly with the cell surface. Surface-exposed levansucrase retained its enzymatic and antigenic properties.

The capsule and S-layer: two independent and yet compatible macromolecular structures in Bacillus anthracis.

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulent bacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, are components of the B. anthracis surface layer (S-layer). The B. anthracis paracrystalline S-layer has previously only been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesized under conditions where the poly-gamma-D-glutamic acid capsule is present. Structural and immunological analyses show that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of single and double S-layer protein mutants shows that the presence of these proteins is not required for normal capsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in the presence of the capsule. Thus, both structures are compatible, and yet neither is required for the correct formation of the other.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen.

Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S-layer. An array remained when sap, a gene described as encoding an S-layer component, was deleted. The remaining S-layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be continuous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S-layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pl). Similar to Sap and other Gram-positive surface proteins, EA1 has three 'S-layer-homology' motifs immediately downstream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of the wild-type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S-layer, and is the major cell-associated antigen.