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Purpose: Staphylococcus aureus isolates, collected from various clinical samples, were analysed to evaluate the contribution of the genetic background of both erythromycin-resistant (ERSA) and -susceptible (ESSA) S. aureus strains to biofilm formation., Methods: A total of 66 ESSA and 43 ERSA clinical isolates were studied for adhesiveness and biofilm formation under different atmospheres. All isolates were evaluated for phenotypic and genotypic macrolide resistance, and for clonal relatedness by pulsed-field gel electrophoresis (PFGE), and by spa typing on representative isolates., Results: A high genetic heterogeneity was encountered, although 10 major PFGE types accounted for 86 % with a few small spatially and temporally related clusters. Overall, biofilm formation under anoxia was significantly lower than under oxic and micro-aerophilic atmospheres. Biofilm formation by ESSA was significantly higher compared to ERSA under oxic and micro-aerophilic conditions. Adhesiveness to plastic was significantly higher among respiratory tract infection isolates under micro-aerophilic conditions, while surgical site infection isolates formed significantly higher biomass of biofilm under oxic and micro-aerophilic atmospheres compared to anoxia. Pulsotype 2 and 4 strains formed significantly higher biofilm biomass than pulsotype 1, with strains belonging to CC8 forming significantly more compared to those belonging to CC5, under both oxic and micro-aerophilic atmospheres., Conclusions: S. aureus biofilm formation appears to be more efficient in ESSA than ERSA, associated with specific S. aureus lineages, mainly CC8 and CC15, and affected by atmosphere. Further studies investigating the relationship between antibiotic resistance and biofilm formation could prove useful in the development of new strategies for the management of S. aureus infections.

The role of Streptococcus pneumoniae in cystic fibrosis (CF) is poorly understood. The pneumococcal population has changed over time after the introduction of the heptavalent conjugate vaccine (PCV7) and, more recently, the 13-valent conjugate vaccine (PCV13). Although serotypes and clones causing invasive pneumococcal disease or colonizing healthy children have been extensively analysed, little is known so far on the serotypes and clones of pneumococci in CF patients. The aim of this work was to investigate serotypes, antibiotic susceptibilities, genotypes and biofilm production of CF pneumococcal isolates. Overall, 44 S. pneumoniae strains collected from 32 paediatric CF patients from January 2010 to May 2012 in a large Italian CF Centre were tested for antimicrobial susceptibility testing by Etest, serotyped by the Quellung reaction and genotyped by a combination of different molecular typing methods, including pbp gene restriction profiling, pspA restriction profiling and sequencing, PFGE and multilocus sequence typing. Biofilm production by pneumococcal strains was also assessed. Penicillin non-susceptibility was 16 %. High resistance rates (>56 %) were observed for erythromycin, clindamycin and tetracycline. The most frequent serotype recovered was serotype 3 (31.8 %). The coverage of PCV7 and PCV13 was 6.8 and 47.7 %, respectively. More than 80 % of CF strains belonged to Pneumococcal Molecular Epidemiology Network (PMEN) reference clones, the most common being Netherlands(3)-ST180 (28.2 %), and Greece(21)-30/ST193 (15.4 %). All strains produced biofilm in vitro, although with large variability in biofilm formation efficiency. No correlation was found between biofilm levels and serotype, clone or antibiotic resistance. The high isolation rate of antibiotic-resistant serotype 3 pneumococci from CF patients suggests that PCV13 could increase protection from pneumococcal colonization and infection., (© 2014 The Authors.)

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.

Streptococcus pyogenes (group A streptococci; GAS) recovered from paediatric pharyngitis (101 isolates) and asymptomatic children (79 isolates) in the same geographical area and period, as well as isolates collected during an enhanced national surveillance programme for GAS invasive diseases (79 isolates), were screened for the incidence of the streptococcal pyrogenic exotoxin (spe) genes speA and speC, as well as the macrolide-resistance genes erm(B), erm(A) subclass erm(TR) and mef(A), and typed by emm sequencing. The speA gene was detected with comparable incidence among throat isolates (13.9 % of asymptomatic children and 16.8 % of pharyngitis isolates) and in 25 % of invasive cases; in contrast, speC incidence was, surprisingly, higher in paediatric populations (55.4 % in pharyngitis isolates and 65.8 % in asymptomatic children) than in invasive isolates (30 %; P < 0.0001). Macrolide resistance was detected in 26.6, 38.0 and 37.6 % of strains belonging to invasive, asymptomatic and pharyngitis populations, respectively. The different incidences of exotoxin and antibiotic-resistance genes among populations did not appear to have an intrinsic clinical significance, but may reflect the propensity of these traits to be associated with certain emm types independent of the source from which the strains were isolated. Further investigations with larger emm-type populations are warranted to confirm this.

A collection of Enterococcus faecalis strains from clinical isolates, healthy individuals and the environment was screened for the presence of virulence factor genes, such as those for collagen-binding protein (ace), endocarditis antigen (efaA), haemolysin activator (cylA), gelatinase (gelE), aggregation substances (asa1 and asa373), a surface protein (esp) and two novel putative surface antigens (EF0591 and EF3314). Apart from some genes that were present in all strains (ace, efaA and EF3314), the gelE gene was the most common factor, although its presence did not correlate with its expression. The genes that encode Esp and CylA were never detected in endocarditis isolates, whereas an association was noted between the esp gene and isolates from urinary tract infection (UTI) and bacteraemia. An aggregation substance gene was always present in commensal strains. As for gelatinase, the presence of the cylA and asa genes did not correlate completely with their phenotypic expression. Generally, isolates from endocarditis, biliary stents and the environment were equipped with fewer virulence factors than isolates from other sources. UTI strains possessed the highest number of factors.

One hundred and sixty-eight isolates of Escherichia coli obtained in Italy from 112 children with diarrhoea and 56 age-matched controls were examined by the HEp-2 cell adhesion assay. Sixteen strains showed localised adherence (LA), 29 showed diffuse adherence (DA) and eight strains showed aggregative adherence (AA). No adhesion pattern was significantly associated with disease. Strains that showed LA or AA were further characterised by serotyping, fluorescent actin staining (FAS) test and hybridisation with the EPEC adherence factor (EAF), E. coli attaching and effacing (eae) and enteroaggregative (EAgg) DNA probes. Strains that showed poor LA were FAS-negative, did not belong to EPEC serotypes and did not hybridise with EPEC probes. Conversely, the two strains that showed a good LA pattern belonged to serotype O128:H2, were FAS positive and hybridised with the eae probe. No isolate hybridised with the EAF probe. Only three of the eight strains with the AA pattern hybridised with the EAgg probe. Probe positivity correlated with the ability to produce clumps at the surface of the liquid culture and to agglutinate rat erythrocytes. In two of these EAgg probe-positive strains, electronmicroscopy revealed the presence of fibrillar bundles which seem to mediate bacterial aggregation.