Identification and molecular characterization of a S. agalactiae strain lacking the capsular locus.

During a national surveillance program on Group B streptococci (GBS) maternal carriage and neonatal infections, a GBS strain isolated from a pregnant woman's vagino-rectal swab was non typable by either serological or molecular methods. Further molecular characterization demonstrated that the strain lacked the entire capsular locus, possibly by a recombination event that excised a 14,1 Kbase pairs genomic fragment extending from the regulatory protein cpsX gene to the neuA gene. The natural loss of the capsular locus by GBS isolated from a human has never been described so far. Such an event, while possibly a dead-end from the evolutionary point of view, leaves a still able-to-colonize organism unrecognizable by the vaccines currently under development.

Invasive neonatal GBS infections from an area-based surveillance study in Italy.

During an area-based study, 75 group B streptococcus (GBS) strains isolated both from early-onset disease (EOD, 37 strains) and from late-onset disease (LOD, 38 strains) were analysed for serotype, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing profiles, protein markers and antibiotic resistance. Serotype III, possessing the rib gene, was the most frequent (54 strains, 72%) and responsible for 89.5% and 54% of LOD and EOD, respectively. Forty-six serotype III strains belonged to the same PFGE type and clonal complex 17, already described as an over-represented clone in neonatal invasive GBS infections. Other serotypes were Ia (9.3%), II (6.7%), Ib (5.3%), V (5.3%) and IV (1.3%). Seventeen PFGE groups were identified comprising strains with related sequence types; conversely, strains displaying the same sequence type could belong to different PFGE groups. When both neonate and maternal strains from vaginorectal swabs and/or milk were available (eight cases), they were indistinguishable. Resistance to erythromycin (12%) was associated with a constitutive resistance to clindamycin in five cases (four carrying the erm(B) gene and one both the erm(B) and mef(E) genes) and with an inducible clindamycin resistance in two cases (one possessing the erm(A) gene, the other the erm(T) gene). Two isolates displayed the M phenotype (mef(E) gene). All strains but five were resistant to tetracycline, mostly mediated by the tet(M) gene (97.1%). The study underlined the importance of an active surveillance system for the elucidation of a GBS population structure causing neonatal infections and allowed the detection of rare antibiotic resistance determinants [erm(T)].

Characterization of macrolide efflux pump mef subclasses detected in clinical isolates of Streptococcus pyogenes isolated between 1999 and 2005.

The macrolide efflux mechanism of resistance, mef, was characterized in community-acquired respiratory tract infections with Streptococcus pyogenes. Fifty-four (4.6%) M phenotype isolates were screen tested as negative for mef(A). Of these 54 isolates, 5 (0.4%), 27 (2.3%), and 1 (0.1%) were considered to be mef(I) positive, a novel mosaic variant of mef, or a novel subclass of mef, respectively. This study shows (i) the definitive presence of mef(E) in S. pyogenes and its global distribution, (ii) the presence of a mosaic variant of mef composed of mef(A) and mef(E), (iii) the previously undescribed presence of mef(I) in S. pyogenes, and (iv) the presence of a novel subclass of mef in S. pyogenes.

The Alpha-like surface proteins: an example of an expanding family of adhesins.

The Alpha-like protein (Alp) family, repeat-containing surface proteins once thought to be important adhesion factors confined to pathogenic streptococci and enterococci, is broader than previously known. Analysis of the annotated microbial genomes has identified new potential members of the Alp family not only in other Gram- positive opportunistic pathogens but also in commensal microflora of the human gut and the skin. This finding has highlighted the importance of genome sequencing projects for unraveling in greater detail lateral gene transfer events involving virulence factors between pathogens and commensals. These should receive constant attention not only as part of infectious disease prevention programs, but also in the food and biotechnology industries.

Underestimated collateral effects of antibiotic therapy in prosthesis-associated bacterial infections.

Antibiotic treatment of infections associated with the use of indwelling medical devices in ageing and/or severely ill patients represents a significant healthcare problem due to the difficulty of treating such infections and to the various collateral effects that may be observed following the often aggressive therapy. We summarize some effects of antibiotics on the expression of virulence factors of the microorganisms which cause such infections. These effects, particularly those resulting in a stimulation of bacterial virulence, might be usefully included among the other well-known collateral effects of antibiotic therapy.

Group B streptococcus: early- and late-onset infections.

Group B streptococcus has emerged as a prominent neonatal pathogen in developed countries since the late 1960s. The incidence of disease remained fairly constant until the 1990 s, when prevention efforts increased. American consensus guidelines were endorsed in the mid 1990 s; since then a decrease in disease incidence has been reported in the United States. This review summarizes the main issues regarding the prevention of neonatal infection and presents aspects of group B streptococcal disease with the first population data recently obtained in a northern region of Italy.

Virulence factors in enterococcal infections of orthopedic devices.

Enterococci are opportunistic pathogens which today represent one of the leading causes of nosocomial infections. We have examined a collection of 52 Enterococcus faecalis isolated from orthopedic infections to determine if they were characterized by a specific pattern of virulence factors. The isolates were evaluated for biofilm formation, presence of genes coding the enterococcal surface protein (esp) and gelatinase (gelE), as well as for gelatinase production. While the rate of esp-positive isolates was comparable to that found among strains from other clinical sources, we found a significantly higher rate of strong biofilm formers and gelatinase producers. Particularly high was the rate of gelE-carrying strains expressing the gene. Data suggest that these two factors in particular may play an important role in enterococcal infections associated with biomaterials.

Pathogenesis of implant infections by enterococci.

Enterococci are commensals of human and animal intestinal tract that have emerged in the last decades as a major cause of nosocomial infections of bloodstream, urinary tract and in infected surgical sites. Enterococcus faecalis is responsible for ca. 80% of all enterococcal infections while Enterococcus faecium accounts for most of the others; among the most relevant risk factors for development of enterococcal infections is the presence of implanted devices. The pathogenesis of such infections is poorly understood, but several virulence factors have been proposed. Among them, the ability to form biofilm has recently been shown to be one of the most prominent features of this microorganism, allowing colonization of inert and biological surfaces, while protecting against antimicrobial substances, and mediating adhesion and invasion of host cells and survival within professional phagocytes. Biofilm formation has been shown to be particularly important in the development of prosthetic valve enterococcal endocarditis and stent occlusion. Enterococci are also able to express other surface factors that may support colonization of both inert and biological surfaces, and that may be involved in the invasion of, and survival within, the host cell.

Clonality among Enterococcus faecium clinical isolates.

In the course of a survey to determine the epidemiology of enterococcal infections in Italy, a sudden increment, in a 1-year time, was noted in the number of glycopeptide resistant Enterococcus faecium isolated from different wards of the University Hospital in Rome, Italy. The isolates were characterized for clonal relatedness by comparing SmaI gel electropherotypes, presence of vancomycin-resistance genes, and expression of virulence factors. PFGE identified in a single pulsed type all the glycopeptide-resistant isolates but one. Resistance to high levels of aminoglycosides was expressed by these same isolates, which also included a majority of non biofilm-forming strains. Two esp gene-carrying strains were also identified in different PFGE types. Data indicates that a specific clone acquired, in the clinical setting, the genetic determinant for glycopeptide resistance, thus improving environmental adaptation and favoring its persistence and spread.

SWIFT (sequence-wide investigation with Fourier transform): a software tool for identifying proteins of a given class from the unannotated genome sequence.

BACKGROUND: The ever increasing number of sequenced genomes calls for new analysis techniques, which can benefit from the methodologies developed in the field of signal processing. METHODS: The present paper addresses the question of searching a pattern of amino acids (not necessarily completely specified) by means of the cross-correlation of complex sequences, obtained after suitable coding of the original amino acid sequence. Subsequently, the proposed algorithm provides a flexible strategy in setting the border between the accepted and rejected ORFs, by means of the k-means clustering of the candidate ORFs. The search for the class of proteins specified by the pattern is carried out from the most basic level, i.e. the DNA sequence, without sifting through an ensemble of previously determined ORFs. Thus, an exhaustive examination of all the occurrences of the pattern in the genome is performed. RESULTS: The application of the method to the search of surface proteins in Gram-positive bacteria witnesses its efficacy, in terms of both sensitivity and specificity. The comparison with the usual (and somewhat arbitrary) choice of setting a fixed value for the threshold length of the putative ORF confirms the validity of the proposed approach.

Analysis of virulence factors in cases of enterococcal endocarditis.

Eleven isolates of Enterococcus faecalis causing endocarditis were screened for possible virulence factors with PCR and phenotypic assays. The gene coding for the enterococcal surface protein (esp) was detected in one isolate only, and haemolysin was produced by two isolates. Aggregation substance, biofilm formation and gelatinase were present in seven, nine and eight isolates, respectively. Predisposing factors, particularly hospitalisation and multiple antibiotic therapy, appeared to be more relevant to the development of enterococcal endocarditis following bloodstream infections than the pattern of virulence factors.

Characterisation of group A streptococcal (GAS) isolates from children with tic disorders.

BACKGROUND & OBJECTIVES: An association between the onset or recrudescence of some neuropsychiatric disorders in children such as tic disorders and group A streptococcal (GAS) infections has been suggested. No information is available on the characterization of GAS strains associated with such disorders. The present study was undertaken to characterize the GAS strains isolated from children with tic disorders and to determine and correlate the antistreptolysin O (ASO) titre with the presence of GAS. METHODS: During 1996-2001, 368 children with tic disorders were investigated for possible exposition to streptococcal antigens. All children, at the time of the first visit and during the follow up visits were apparently healthy and showed no clinical evidence of streptococcal infections or post streptococcal sequelae. Blood and throat swab samples were collected and serological and bacteriological tests done. The isolates obtained were investigated for T pattern, M protein and emm type, as well as for the production of protease. RESULTS: Of the 800 throat swabs studied 100, corresponding to 67 patients, were positive for GAS; 49 children were found positive for GAS only once during the study, 18 had more than one sample positive for different serotypes, 8 were positive twice or more for the same type. ASO titres of these children were, in general, elevated. Five types, namely type M12, 3, 13, 11, 1, accounted for 39 per cent of the isolates, M12 being the most common, but a large number of different types were also found. A large number of isolates (62%) showed an elevated prodution of protease in the casein plate assay. INTERPRETATION & CONCLUSION: Despite the high level of ASO titres found, the results were not in favour of a particular virulence or invasivity of the isolates. Only a few colonies per sample were found indicating that factors different from the microbial virulence play a role in this type of disease.

Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci.

Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, source-specific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy.

Enterococcus spp. produces slime and survives in rat peritoneal macrophages.

Enterococcal clinical isolates were investigated for the ability to form biofilm on inert surfaces, as a measure of slime production, in an attempt to find new possible virulence factors for these microorganisms. This property was commonly found among Enterococcus faecalis. Also E. faecium isolates were able to form biofilm, although to a lesser extent; for this species, however, biofilm formation seemed more frequently associated with isolates from infection rather than with environmental strains or isolates from healthy individuals. Biofilm formation was strongly affected by the presence of an additional carbohydrate source in the medium, or by iron deprivation, indicating a role of slime for survival in stressful conditions. Slime-producing E. faecalis were able to survive inside peritoneal macrophages for extended periods compared to slime-negative strains or to slime-positive bacteria grown in conditions depressing slime production. In particular, slime-producing and slime-negative cells showed a decrease of 1 and 2 log units, respectively, at 1 h after infection; slime-negative cells were then rapidly killed, with clearance of bacterial cells at 24 h. Slime-producing bacteria persisted up to 48 h, which was the last time point examined, as after that time viability of both infected and non-infected macrophages started to decline. Scanning electron microscopy observations showed the presence of abundant amorphous extracellular material, of possible polysaccharide nature, embedding bacterial cells to form a multilayered biofilm. Even in conditions not supporting biofilm formation, bacterial cells appeared capsulated, suggesting that capsule and slime might represent different structures. Genes belonging to the epa locus or to a putative icaA homolog did not seem to be involved in synthesis and export of slime.

Mosaicism in the alpha-like protein genes of group B streptococci.

Members of a family of repeat-containing surface proteins of group B streptococci (GBS) defined by the alpha C and Rib proteins exhibit size variability and cross-reactivity and have been studied as potential vaccine components. We report evidence of horizontal DNA transfer with subsequent recombination as a mechanism generating diversity within this antigen family. Alp2 and Alp3 are additional members of the alpha C protein family identified in strains of the emerging GBS serotypes V and VIII. Each contains an overall genetic organization highly similar to that of the alpha C and Rib proteins, including a tandem repeat region and conserved N- and C-terminal regions. Among different strains, protein size varies according to the number of tandem repeats within the corresponding gene. Unlike the alpha C and Rib proteins, however, the newly described alpha-like proteins contain other regions, including one similar to the IgA-binding region of the GBS beta C protein, a nontandem repeat region, and an isolated repeat highly homologous to the alpha C repeat. Sequence analysis of the regions flanking the alpha C protein gene on a 13.7-kb insert reveals several ORFs that are likely to be involved in basic metabolic pathways. Analysis of corresponding flanking regions in other GBS strains, including the parent strains of the newly described alpha-like proteins, shows striking conservation among all strains studied. These findings indicate that the alpha-like proteins are encoded by mosaic variants at a single genomic locus and suggest that recombination after horizontal DNA transfer is a means of generating diversity within this protein family.

The archaea monophyly issue: A phylogeny of translational elongation factor G(2) sequences inferred from an optimized selection of alignment positions.

A global alignment of EF-G(2) sequences was corrected by reference to protein structure. The selection of characters eligible for construction of phylogenetic trees was optimized by searching for regions arising from the artifactual matching of sequence segments unique to different phylogenetic domains. The spurious matchings were identified by comparing all sections of the global alignment with a comprehensive inventory of significant binary alignments obtained by BLAST probing of the DNA and protein databases with representative EF-G(2) sequences. In three discrete alignment blocks (one in domain II and two in domain IV), the alignment of the bacterial sequences with those of Archaea-Eucarya was not retrieved by database probing with EF-G(2) sequences, and no EF-G homologue of the EF-2 sequence segments was detected by using partial EF-G(2) sequences as probes in BLAST/FASTA searches. The two domain IV regions (one of which comprises the ADP-ribosylatable site of EF-2) are almost certainly due to the artifactual alignment of insertion segments that are unique to Bacteria and to Archaea-Eucarya. Phylogenetic trees have been constructed from the global alignment after deselecting positions encompassing the unretrieved, spuriously aligned regions, as well as positions arising from misalignment of the G' and G" subdomain insertion segments flanking the "fifth" consensus motif of the G domain (AE varsson, 1995). The results show inconsistencies between trees inferred by alternative methods and alternative (DNA and protein) data sets with regard to Archaea being a monophyletic or paraphyletic grouping. Both maximum-likelihood and maximum-parsimony methods do not allow discrimination (by log-likelihood difference and difference in number of inferred substitutions) between the conflicting (monophyletic vs. paraphyletic Archaea) topologies. No specific EF-2 insertions (or terminal accretions) supporting a crenarchaeal-eucaryal clade are detectable in the new EF-G(2) sequence alignment.

Discontinuous occurrence of the hsp70 (dnaK) gene among Archaea and sequence features of HSP70 suggest a novel outlook on phylogenies inferred from this protein.

Occurrence of the hsp70 (dnaK) gene was investigated in various members of the domain Archaea comprising both euryarchaeotes and crenarchaeotes and in the hyperthermophilic bacteria Aquifex pyrophilus and Thermotoga maritima representing the deepest offshoots in phylogenetic trees of bacterial 16S rRNA sequences. The gene was not detected in 8 of 10 archaea examined but was found in A. pyrophilus and T. maritima, from which it was cloned and sequenced. Comparative analyses of the HSP70 amino acid sequences encoded in these genes, and others in the databases, showed that (i) in accordance with the vicinities seen in rRNA-based trees, the proteins from A. pyrophilus and T. maritima form a thermophilic cluster with that from the green nonsulfur bacterium Thermomicrobium roseum and are unrelated to their counterparts from gram-positive bacteria, proteobacteria/mitochondria, chlamydiae/spirochetes, deinococci, and cyanobacteria/chloroplasts; (ii) the T. maritima HSP70 clusters with the homologues from the archaea Methanobacterium thermoautotrophicum and Thermoplasma acidophilum, in contrast to the postulated unique kinship between archaea and gram-positive bacteria; and (iii) there are exceptions to the reported association between an insert in HSP70 and gram negativity, or vice versa, absence of insert and gram positivity. Notably, the HSP70 from T. maritima lacks the insert, although T. maritima is phylogenetically unrelated to the gram-positive bacteria. These results, along with the absence of hsp70 (dnaK) in various archaea and its presence in others, suggest that (i) different taxa retained either one or the other of two hsp70 (dnaK) versions (with or without insert), regardless of phylogenetic position; and (ii) archaea are aboriginally devoid of hsp70 (dnaK), and those that have it must have received it from phylogenetically diverse bacteria via lateral gene transfer events that did not involve replacement of an endogenous hsp70 (dnaK) gene.