Phylogenetic analysis and accessory genome diversity reveal insight into the evolutionary history of Streptococcus dysgalactiae.

Streptococcus dysgalactiae (SD) is capable of infecting both humans and animals and causing a wide range of invasive and non-invasive infections. With two subspecies, the taxonomic status of subspecies of SD remains controversial. Subspecies equisimilis (SDSE) is an important human pathogen, while subspecies dysgalactiae (SDSD) has been considered a strictly animal pathogen; however, occasional human infections by this subspecies have been reported in the last few years. Moreover, the differences between the adaptation of SDSD within humans and other animals are still unknown. In this work, we provide a phylogenomic analysis based on the single-copy core genome of 106 isolates from both the subspecies and different infected hosts (animal and human hosts). The accessory genome of this species was also analyzed for screening of genes that could be specifically involved with adaptation to different hosts. Additionally, we searched putatively adaptive traits among prophage regions to infer the importance of transduction in the adaptation of SD to different hosts. Core genome phylogenetic relationships segregate all human SDSE in a single cluster separated from animal SD isolates. The subgroup of bovine SDSD evolved from this later clade and harbors a specialized accessory genome characterized by the presence of specific virulence determinants (e.g., cspZ) and carbohydrate metabolic functions (e.g., fructose operon). Together, our results indicate a host-specific SD and the existence of an SDSD group that causes human-animal cluster infections may be due to opportunistic infections, and that the exact incidence of SDSD human infections may be underestimated due to failures in identification based on the hemolytic patterns. However, more detailed research into the isolation of human SD is needed to assess whether it is a carrier phenomenon or whether the species can be permanently integrated into the human microbiome, making it ready to cause opportunistic infections.

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.

Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability.

Capsular Type, Sequence Type and Microbial Resistance Factors Impact on DNase Activity of Streptococcus agalactiae Strains from Human and Bovine Origin.

Extracellular deoxyribonucleases (DNases) contribute to the spread of pathogenic bacteria through the evasion from host innate immunity. Our main objective was to evaluate the production of extracellular DNases by human and bovine Streptococcus agalactiae clinical strains and perform a correlation of genetic lineages and DNase activity with capsular type, genetic determinants, clinical origin (colonization and infection), and host (human or bovine). DNase activity was evaluated by qualitative and quantitative assays for a collection of 406 human (n = 285) and bovine (n = 121) strains. All (121/121) bovine were isolated from mastitis and revealed to be DNase (+), indicating a putative pathogenic role in this clinical scenario. From the human S. agalactiae strains, 86% (245/285) showed DNase activity, among which all strains belonging to capsular types, namely, Ia, Ib, III-2, and IV. All CC17 strains (n = 58) and 56/96 (58.3%) of the CC19 displayed DNase activity. DNase (-) strains belonged to the CC19 group. However, the subcharacterization of CC19 S. agalactiae strains through multiple-locus variable number tandem repeat analysis (MLVA), antibiotic resistance, mobile elements, and surface proteins did not provide any distinction among DNase producers and non-producers. The production of DNases by all human CC17 strains, about two-fifths of human CC19, and all bovine strains, suggest an important contribution of DNases to hypervirulence.

Clinical strains of Streptococcus agalactiae carry two different variants of pathogenicity island XII.

Streptococcus agalactiae or Group B streptococci (GBS) are a common cause of serious diseases of newborns and adults. GBS pathogenicity largely depends on genes located on the accessory genome including several pathogenicity islands (PAI). The present paper is focused on the structure and molecular epidemiological analysis of one of the GBS pathogenicity islands-the pathogenicity island PAI XII (Glaser et al. Mol Microbiol 45(6):1499-1513, 2002). This PAI was found to be composed of three different mobile genetic elements: a composite transposon (PAI-C), a genomic islet (PAI-B), and a pathogenicity island associated with gene sspB1 (PAI-A). PAI-A in GBS has a homolog--PAI-A1 with similar, but a different genetic constellation. PCR-based analysis of GBS collections from different countries revealed that a strains lineage with PAI-A is less common than PAI-A1 and was determined to be present only among the strains obtained from Russia. Our results suggest that PAI-A and PAI-A1 have the same progenitor, which evolved independently and appeared in the GBS genome as separate genetic events. Results of this study reflect specific geographical distribution of the GBS strains with the mobile genetic element under study.

Persistence of a dominant bovine lineage of group B Streptococcus reveals genomic signatures of host adaptation.

Group B Streptococcus (GBS) is a host-generalist species, most notably causing disease in humans and cattle. However, the differential adaptation of GBS to its two main hosts, and the risk of animal to human infection remain poorly understood. Despite improvements in control measures across Europe, GBS is still one of the main causative agents of bovine mastitis in Portugal. Here, by whole-genome analysis of 150 bovine GBS isolates we discovered that a single CC61 clone is spreading throughout Portuguese herds since at least the early 1990s, having virtually replaced the previous GBS population. Mutations within an iron/manganese transporter were independently acquired by all of the CC61 isolates, underlining a key adaptive strategy to persist in the bovine host. Lateral transfer of bacteriocin production and antibiotic resistance genes also underscored the contribution of the microbial ecology and genetic pool within the bovine udder environment to the success of this clone. Compared to strains of human origin, GBS evolves twice as fast in bovines and undergoes recurrent pseudogenizations of human-adapted traits. Our work provides new insights into the potentially irreversible adaptation of GBS to the bovine environment.

The Madeira Archipelago As a Significant Source of Marine-Derived Actinomycete Diversity with Anticancer and Antimicrobial Potential.

Marine-derived actinomycetes have demonstrated an ability to produce novel compounds with medically relevant biological activity. Studying the diversity and biogeographical patterns of marine actinomycetes offers an opportunity to identify genera that are under environmental pressures, which may drive adaptations that yield specific biosynthetic capabilities. The present study describes research efforts to explore regions of the Atlantic Ocean, specifically around the Madeira Archipelago, where knowledge of the indigenous actinomycete diversity is scarce. A total of 400 actinomycetes were isolated, sequenced, and screened for antimicrobial and anticancer activities. The three most abundant genera identified were Streptomyces, Actinomadura, and Micromonospora. Phylogenetic analyses of the marine OTUs isolated indicated that the Madeira Archipelago is a new source of actinomycetes adapted to life in the ocean. Phylogenetic differences between offshore (>100 m from shore) and nearshore (< 100 m from shore) populations illustrates the importance of sampling offshore in order to isolate new and diverse bacterial strains. Novel phylotypes from chemically rich marine actinomycete groups like MAR4 and the genus Salinispora were isolated. Anticancer and antimicrobial assays identified Streptomyces, Micromonospora, and Salinispora as the most biologically active genera. This study illustrates the importance of bioprospecting efforts at unexplored regions of the ocean to recover bacterial strains with the potential to produce novel and interesting chemistry.

Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a prosthetic joint infection and an infective endocarditis were described in humans. To unravel if bovine SDSD isolates are able to infect human cells, the adherence and internalization to human primary keratinocytes of two bovine SDSD strains isolated from milk collected from udder were analyzed. Bacterial adhesion assays and confocal microscopy indicate a high adherence and internalization of SDSD isolates to human cells, suggesting for the first time the ability of bovine isolates to infect human cells.

Natural Mutations in Streptococcus agalactiae Resulting in Abrogation of ß Antigen Production.

Streptococcus agalactiae genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of bac gene, coding for ß antigen at transcriptional level. Inactivation of bgrR abrogated bac gene expression and increased virulence properties of S. agalactiae. In this study, a total of 140 strains were screened for the presence of bac gene, and the TCS bgrR and bgrS genes. A total of 53 strains carried the bac, bgrR and bgrS genes. Most of them (48 strains) expressed ß antigen, while five strains did not express ß antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of ß antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of ß antigen indicating the crucial regulatory role of TCS BgrRS. The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of S. agalactiae infection compared to parental strain A49V. In conclusion we showed that disruption of ß antigen expression is associated with: i) insertion of ISSa4 upstream the bac gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the bac gene and a truncated form of ß antigen; iii) single deletion (G) in position 439 of the bgrR gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in bgrR and bgrS genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of S. agalactiae gives a novel clue for understanding of S. agalactiae adaptation to the human.

Pulsed-field gel electrophoresis (PFGE): application in population structure studies of bovine mastitis-causing streptococci.

Pulsed-field gel electrophoresis (PFGE) separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens and is a valuable typing scheme to detect and differentiate strains. Particularly, the contour-clamped homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of many bacterial pathogens. Here we describe a PFGE protocol that was applicable to the study of bovine streptococci, namely, Streptococcus agalactiae (group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (group C Streptococcus, GCS), and Streptococcus uberis-which are relevant pathogens causing mastitis, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production.

Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.

Streptococcus agalactiae (Group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (Group C Streptococcus, GCS) and Streptococcus uberis are relevant mastitis pathogens, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production. The aims of this study were the evaluation of antimicrobial drug resistance patterns, particularly important for streptococcal mastitis control and the identification of strain molecular features. Antimicrobial resistance was assessed by disk diffusion against amoxicillin-clavulanic acid, cefazolin, cefoperazone, pirlimycin-PRL, rifaximin, streptomycin, chloramphenicol, erythromycin-ERY, gentamicin, tetracycline-TET and vancomycin. Genotypic relationships were identified using pulsed-field gel electrophoresis (PFGE), macrolide and/or tetracycline resistance gene profiling, GBS capsular typing, GBS virulence gene profiling and GBS and S. uberis multi locus sequence typing (MLST). The majority of the isolates were susceptible to all drugs except to aminoglycoside, macrolide, lincosamide and tetracycline. Close to half of the TET resistant isolates have tetO and tetK and almost all ERY-PRL resistant isolates have ermB. A high degree of intra-species polymorphism was found for GCS. The GBS belonged to ST-2, -554, -61, -23 lineages and five new molecular serotypes and human GBS insertion sequences in the cpsE gene were found. Also, GBS of serotype V with scpB and lmb seem to be related with GBS isolates of human origin (same ST-2 and similar PFGE). Overall our results suggested that different therapeutic programs may have been implemented in the different farms and that in most cases clones were herd-specific.

Molecular epidemiology of group B streptococcal meningitis in children beyond the neonatal period from Angola.

Streptococcus agalactiae is a major pathogen of neonates and immunocompromised adults. Prior studies have demonstrated that, beyond the neonatal period, S. agalactiae rarely causes invasive infections in children. However, during 2004-2005, S. agalactiae was the causative agent of 60 meningitis episodes in children aged 3 months to 12 years from Angola. To identify and study the specific causative genetic lineages of S. agalactiae childhood meningitis, which lack characterization to date, we conducted an extensive molecular analysis of the recovered isolates (n = 21). This constitutes what we believe to be the first molecular study of the population structure of invasive S. agalactiae isolates from Africa. A low genetic diversity was observed among the isolates, where the majority belonged to clonal complex (CC) 17 presenting the capsular subtype III-2 (86 % of cases) and marked by the intron group II GBSi1, which has previously been observed to be associated with neonatal hosts. The predominance of single-locus variants of sequence type (ST) 17 suggested the local diversification of this hypervirulent clone, which displayed novel alleles of the fbsB and sip virulence genes. The absence of the scpB-lmb region in two S. agalactiae isolates with the Ia/ST23 genotype is more typical of cattle than human isolates. Globally, these data provide novel information about the enhanced invasiveness of the CC17 genetic lineage in older children and suggest the local diversification of this clone, which may be related to the future emergence of a novel epidemic clone in Angola.

Virulence gene pool detected in bovine group C Streptococcus dysgalactiae subsp. dysgalactiae isolates by use of a group A S. pyogenes virulence microarray.

A custom-designed microarray containing 220 virulence genes of Streptococcus pyogenes (group A Streptococcus [GAS]) was used to test group C Streptococcus dysgalactiae subsp. dysgalactiae (GCS) field strains causing bovine mastitis and group C or group G Streptococcus dysgalactiae subsp. equisimilis (GCS/GGS) isolates from human infections, with the latter being used for comparative purposes, for the presence of virulence genes. All bovine and all human isolates carried a fraction of the 220 genes (23% and 39%, respectively). The virulence genes encoding streptolysin S, glyceraldehyde-3-phosphate dehydrogenase, the plasminogen-binding M-like protein PAM, and the collagen-like protein SclB were detected in the majority of both bovine and human isolates (94 to 100%). Virulence factors, usually carried by human beta-hemolytic streptococcal pathogens, such as streptokinase, laminin-binding protein, and the C5a peptidase precursor, were detected in all human isolates but not in bovine isolates. Additionally, GAS bacteriophage-associated virulence genes encoding superantigens, DNase, and/or streptodornase were detected in bovine isolates (72%) but not in the human isolates. Determinants located in non-bacteriophage-related mobile elements, such as the gene encoding R28, were detected in all bovine and human isolates. Several virulence genes, including genes of bacteriophage origin, were shown to be expressed by reverse transcriptase PCR (RT-PCR). Phylogenetic analysis of superantigen gene sequences revealed a high level (>98%) of identity among genes of bovine GCS, of the horse pathogen Streptococcus equi subsp. equi, and of the human pathogen GAS. Our findings indicate that alpha-hemolytic bovine GCS, an important mastitis pathogen and considered to be a nonhuman pathogen, carries important virulence factors responsible for virulence and pathogenesis in humans.

Emergence of ciprofloxacin-nonsusceptible Streptococcus pyogenes isolates from healthy children and pediatric patients in Portugal.

We describe 66 ciprofloxacin-nonsusceptible Streptococcus pyogenes isolates recovered from colonized and infected children. The ParC S79A substitution was frequent and associated with the emm6/sequence type 382 (emm6/ST382) lineage. The ParC D83G substitution was detected in two isolates (emm5/ST99 and emm28/ST52 lineages). One isolate (emm89/ST101) had no quinolone resistance-determining region codon substitutions or other resistance mechanisms. Five of 66 isolates were levofloxacin resistant. Although fluoroquinolones are not used in children, they may be putative disseminators of fluoroquinolone-nonsusceptible strains in the community.

Human group A streptococci virulence genes in bovine group C streptococci.

Phage-encoded virulence genes of group A streptococci were detected in 10 (55.6%) of 18 isolates of group C streptococci that had caused bovine mastitis. Bovine isolates carried other genetic determinants, such as composite transposon Tn1207.3/F10394.4 (100%) and antimicrobial drug resistance genes erm(B)/erm(A) (22.2%), linB (16.6%), and tet(M)/tet(O) (66.7%), located on mobile elements.

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.

High rates of transmission of and colonization by Streptococcus pneumoniae and Haemophilus influenzae within a day care center revealed in a longitudinal study.

Day care centers (DCCs) are unique settings where young children are at increased risk for colonization by pneumococci and Haemophilus influenzae. Although point prevalence studies in DCCs are frequent, only a few longitudinal studies on the dynamics of colonization have been published. We conducted a 1-year longitudinal study with 11 sampling periods on nasopharyngeal carriage of pneumococci and H. influenzae among 47 children who attended a single DCC. All isolates were antibiotyped and genotyped by pulsed-field gel electrophoresis. Pneumococci were also serotyped. Of the 414 samples obtained, 61.4% contained pneumococci, and 87% contained H. influenzae. Only 8.3% of the samples were negative for both species. Twenty-one pneumococcal clones and 47 H. influenzae clones were identified. Introduction of clones occurred during all year. Ninety-eight percent and 96% of all pneumococcal and H. influenzae isolates, respectively, belonged to clones shared by more than one child. Children were sequentially colonized with up to six pneumococcal clones (mean, 3.6) and five serotypes and nine H. influenzae clones (mean, 7.1). Clones with increased capacity for transmission and/or prolonged colonization were identified in both species. These two fitness properties appeared to be independent. In conclusion, among DCC attendees, a high rate of acquisition and turnover of strains was observed, and all children were overwhelmingly colonized by clones shared with others. DCCs are units where permanent introduction of new clones occurs, and attendees, as a whole, provide a pool of hosts where the fittest clones find privileged opportunities to persist and expand.

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.