Marco Pataracchia

A multiplex PCR assay for the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae.

A multiplex PCR assay for the identification of serotypes Ia to IX of Streptococcus agalactiae was developed. By using a single PCR reaction containing a mix of 19 primers the assay identified each serotype by the analysis of the unique two or three bands pattern on agarose gel.

Molecular Epidemiology and Distribution of Serotypes, Surface Proteins, and Antibiotic Resistance among Group B Streptococci in Italy

ABSTRACT Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm(B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes.

Therapeutic Failures of Antibiotics Used To Treat Macrolide-Susceptible Streptococcus pyogenes Infections May Be Due to Biofilm Formation

ABSTRACT Streptococcus pyogenes infections often fail to respond to antibiotic therapy, leading to persistent throat carriage and recurrent infections. Such failures cannot always be explained by the occurrence of antibiotic resistance determinants, and it has been suggested that S. pyogenes may enter epithelial cells to escape antibiotic treatment. We investigated 289 S. pyogenes strains isolated from different clinical sources to evaluate their ability to form biofilm as an alternative method to escape antibiotic treatment and host defenses. Up to 90% of S. pyogenes isolates, from both invasive and noninvasive infections, were able to form biofilm. Specific emm types, such as emm6, appeared to be more likely to produce biofilm, although variations within strains belonging to the same type might suggest biofilm formation to be a trait of individual strains rather than a general attribute of a serotype. Interestingly, erythromycin-susceptible isolates formed a significantly thicker biofilm than resistant isolates (P < 0.05). Among resistant strains, those carrying the erm class determinants formed a less organized biofilm than the mef(A)-positive strains. Also, prtF1 appeared to be negatively associated with the ability to form biofilm (P < 0.01). Preliminary data on a selection of strains indicated that biofilm-forming isolates entered epithelial cells with significantly lower efficiency than biofilm-negative strains. We suggest that prtF1-negative macrolide-susceptible or mef(A)-carrying isolates, which are poorly equipped to enter cells, may use biofilm to escape antimicrobial treatments and survive within the host. In this view, biofilm formation by S. pyogenes could be responsible for unexplained treatment failures and recurrences due to susceptible microorganisms.

emm Types, Virulence Factors, and Antibiotic Resistance of Invasive Streptococcus pyogenes Isolates from Italy: What Has Changed in 11 Years?

ABSTRACT To investigate the epidemiology and characteristics of invasive group A streptococcal (GAS) disease over 11 years in Italy, this study compared the emm types and the superantigen toxin genes speA and speC as well as the erythromycin, clindamycin, and tetracycline susceptibilities of 207 invasive GAS strains collected during two national enhanced surveillance periods (1994 to 1996 and 2003 to 2005) and the time between each set of surveillance periods. The present study demonstrated that emm1 strains were consistently responsible for about 20% of invasive GAS infections, while variations in the frequencies of the other types were noted, although the causes of most cases of invasive infections were restricted to emm1, emm3, emm4, emm6, emm12, and emm18. During the 1994 to 1996 surveillance period, an emm89 epidemic clone spread across the northern part of Italy. A restricted macrolide resistance phenotype-type distribution of the bacteriophage-encoded speA toxin as well as of macrolide resistance genes was noted over time. Indeed, the recent acquisition of macrolide resistance in previously susceptible emm types was observed.

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)].

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.

Detection of genes encoding internalization-associated proteins in Streptococcus pyogenes isolates from patients with invasive diseases and asymptomatic carriers

ABSTRACT A total of 161 Streptococcus pyogenes isolates from patients with invasive infections or from asymptomatic carriers were examined for genes (prtF1, prtF2, and fba) coding for fibronectin-binding proteins to evaluate their involvement in the pathogenesis of different streptococcal manifestations. We found no significant differences in the presence of these three genes between the two groups. Overall, the prtF2 gene was present in similar percentages among strains from both sources (61% versus 63%). Strains carrying the gene fba were slightly more common among those isolated from asymptomatic carriers (72.6% versus 65%). Also, the prtF1 gene was present in a higher, but not significant, percentage among strains from throat swabs than among isolates from invasive infections (75% versus 64.9%). However, this more detailed characterization of the genes encoding fibronectin-binding proteins allowed us to identify a strong association of genes of the erm class, coding for macrolide resistance, with prtF1 and prtF2 rather than with prtF1 alone. Since macrolide resistance was significantly associated with throat swab isolates, it may be hypothesized that proteins coded by prtF1 and prtF2 genes may be synergic in providing support for cell invasion and/or colonizing or persistence efficiency.

Genetic diversity and virulence properties of Streptococcus dysgalactiae subsp. equisimilis from different sources.

A recent increase in virulence of pathogenic Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been widely proposed. Such an increase may be partly explained by the acquisition of new virulence traits by horizontal gene transfer from related streptococci such as Streptococcus pyogenes (GAS) and Streptococcus agalactiae (GBS). A collection of 54 SDSE strains isolated in Italy in the years 2000-2010 from different sources (paediatric throat carriage, invasive and non-invasive diseases) was characterized by emm typing and pulsed-field gel electrophoresis (PFGE) analysis. The virulence repertoire was evaluated by PCR for the presence of GAS superantigen (spe) genes, the streptolysin S (sagA) gene, the group G fibronectin-binding protein (gfbA) gene and GAS-GBS alpha-like protein family (alp) genes; moreover, the ability to invade human epithelial cells was investigated. Resistance to tetracycline, erythromycin and clindamycin was assessed. The combined use of emm typing and PFGE proved to be a reliable strategy for the epidemiological analysis of SDSE isolates. The most frequent emm types were the same as those more frequently reported in other studies, thus indicating the diffusion of a limited number of a few successful emm types fit to disseminate in humans. The speG gene was detected in SDSE strains of different genetic backgrounds. Erythromycin resistance determined by the erm(T) gene, and the unusual, foggy MLSB phenotype, observed in one and seven strains, respectively, have never previously, to our knowledge, been reported in SDSE. Moreover, a new member of the alp family was identified. The identification of new antibiotic and virulence determinants, despite the small size of the sample analysed, shows the importance of constant attention to monitoring the extent of lateral gene transfer in this emerging pathogen.

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.

Identification and molecular discrimination of toxigenic and nontoxigenic diphtheria Corynebacterium strains by combined real-time polymerase chain reaction assays☆

With the recognition of several diphtheria outbreaks and the emergence of nontoxigenic corynebacteria strains, there has been renewed interest in the development of laboratory diagnostic methods. Previously reported polymerase chain reaction (PCR) assays can have low diagnostic sensitivity or give species misidentifications among clinical isolates. The aim of the present study was the development of combined real-time PCR assays, based on the tox and rpoB genes, for the detection and differentiation of toxigenic and nontoxigenic corynebacteria. By the PCR tox assay, it was possible to perform the direct identification of DT tox gene of Corynebacterium diphtheriae and Corynebacterium ulcerans, while the PCR rpoB assay differentiated C. diphtheriae from C. ulcerans, irrespective of their toxigenic status. In addition, we detected the DT toxin of Corynebacterium pseudotuberculosis for the first time. These assays revealed high sensitivity, specificity, and reproducibility, and the availability of plasmid controls will facilitate further research into the diagnostics of diphtheria corynebacteria.