New Insights on Streptococcus dysgalactiae subsp. dysgalactiae Isolates.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) has been considered a strict animal pathogen. Nevertheless, the recent reports of human infections suggest a niche expansion for this subspecies, which may be a consequence of the virulence gene acquisition that increases its pathogenicity. Previous studies reported the presence of virulence genes of Streptococcus pyogenes phages among bovine SDSD (collected in 2002-2003); however, the identity of these mobile genetic elements remains to be clarified. Thus, this study aimed to characterize the SDSD isolates collected in 2011-2013 and compare them with SDSD isolates collected in 2002-2003 and pyogenic streptococcus genomes available at the National Center for Biotechnology Information (NCBI) database, including human SDSD and S. dysgalactiae subsp. equisimilis (SDSE) strains to track temporal shifts on bovine SDSD genotypes. The very close genetic relationships between humans SDSD and SDSE were evident from the analysis of housekeeping genes, while bovine SDSD isolates seem more divergent. The results showed that all bovine SDSD harbor Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas IIA system. The widespread presence of this system among bovine SDSD isolates, high conservation of repeat sequences, and the polymorphism observed in spacer can be considered indicators of the system activity. Overall, comparative analysis shows that bovine SDSD isolates carry speK, speC, speL, speM, spd1, and sdn virulence genes of S. pyogenes prophages. Our data suggest that these genes are maintained over time and seem to be exclusively a property of bovine SDSD strains. Although the bovine SDSD genomes characterized in the present study were not sequenced, the data set, including the high homology of superantigens (SAgs) genes between bovine SDSD and S. pyogenes strains, may indicate that events of horizontal genetic transfer occurred before habitat separation. All bovine SDSD isolates were negative for genes of operon encoding streptolysin S, except for sagA gene, while the presence of this operon was detected in all SDSE and human SDSD strains. The data set of this study suggests that the separation between the subspecies "dysgalactiae" and "equisimilis" should be reconsidered. However, a study including the most comprehensive collection of strains from different environments would be required for definitive conclusions regarding the two taxa.

Phage endolysins with broad antimicrobial activity against Enterococcus faecalis clinical strains.

Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.

Resistance to bacitracin in Streptococcus pyogenes from oropharyngeal colonization and noninvasive infections in Portugal was caused by two clones of distinct virulence genotypes.

During 2000-2007 in Lisbon, we identified 45 bacitracin-resistant Streptococcus pyogenes isolates among 1629 isolates: 24 from oropharyngeal healthy carriers (out of 1026), 21 from patients with noninvasive infections (out of 559) and zero from invasive infections (out of 44). Forty-four of those isolates, mainly of colonization, are low-level bacitracin-resistant members of the cMLS(B)-macrolide-resistant and tetracycline-susceptible emm28/ST52 clone previously detected in Europe, but only among clinical samples. One high-level bacitracin-resistant isolate, associated with a tonsillitis/pharyngitis episode, is cMLS(B)-macrolide-resistant and tetracycline-resistant member of the emm74/ST120 lineage, which was not previously known to include bacitracin-resistant isolates. The bcrABDR operon encoding an ATP-binding cassette transporter in Enterococcus faecalis was not detected among these bacitracin-resistant S. pyogenes strains. Virulence profiling indicated that genes coding for exotoxins and superantigens seem to be clone specific. This study provides an increased knowledge about specific bacitracin-resistant S. pyogenes strains, which may be useful in future investigations aiming to understand the mechanism(s) leading to bacitracin resistance and the cause(s) for differences in colonization and/or dissemination potential.

Properties of novel international drug-resistant pneumococcal clones identified in day-care centers of Lisbon, Portugal.

In this study, 61 drug-resistant Streptococcus pneumoniae strains were characterized by multilocus sequence typing (MLST). These strains were representatives of 26 major clones (defined using pulsed-field gel electrophoresis) accounting for 93% of the 1,285 drug-resistant Streptococcus pneumoniae isolates recovered from the nasopharynges of healthy children attending day-care centers in Lisbon during 2001 to 2003. Using MLST, 13 of the 26 clones were found to be identical or closely related to 11 Pneumococcal Molecular Epidemiology Network (PMEN) clones, 4 clones were found to be unique as there were no identical or highly related allelic profiles deposited in the MLST database, and the remaining 9 clones had sequence types that matched or differed at a single or double locus from allelic profiles available in the MLST database. These nine clones were of serotypes 33F, 10A, 19A, 19F, 6A, 20, 24F, and 3, one was nontypeable, and, by MLST, they were found to be identical or highly related to isolates from disease origin that were dispersed internationally. Since the majority of these clones had serotypes that are not included in the 7-valent conjugate pneumococcal vaccine, monitoring of these clones is important for surveying their possible spread in the future. We propose the inclusion of these novel international clones in the PMEN.

Effect of the seven-valent conjugate pneumococcal vaccine on carriage and drug resistance of Streptococcus pneumoniae in healthy children attending day-care centers in Lisbon.

AIMS: Prospective study to evaluate the impact of the 7-valent pneumococcal conjugate vaccine (Prevenar) on the nasopharyngeal (NP) carriage of drug-resistant Streptococcus pneumoniae (DRPn), by healthy children attending day-care centers (ages 6 months-6 years). METHODS: Vaccinees (238 children) who received vaccine and controls (457 children) were followed for carriage of total S. pneumoniae and DRPn and for the serotypes and genetic backgrounds of DRPn during 6 consecutive sampling periods between May 2001 and February 2003. RESULTS: We detected no significant differences between vaccinees and the control group in the total carriage rate of Pn (average, 68%) or in the frequency of carriage of DRPn (average, 38%), including the frequency of penicillin-nonsusceptible strains (average, 24%). In contrast, there was a decline in the carriage of DRPn with vaccine serotypes which was compensated by the appearance and gradual increase in the frequency of DRPn expressing unusual serotypes (6A, 10A, 15A and 15C, 19A, 23A, 33F) which were not present in the vaccine as well as an increase in nontypable strains. The majority of the DRPn with unusual serotypes showed different pulsed field gel electrophoresis patterns indicating replacement of the original resistant flora by other clonal types of drug-resistant bacteria. Antibiotic consumption and the frequency of respiratory tract infections were similar among the vaccinees and controls. CONCLUSIONS: Pneumococcal vaccination did not change the frequency of carriage of drug-resistant strains being the initially dominant vaccine serotypes replaced by others expressing nonvaccine serotypes. Reduction in the carriage of DRPn may require a combination of the conjugate vaccine and a decrease in antibiotic pressure.