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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.

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.

Poly(N-vinylpyrrolidone) (PVP) nanofiber mats were electrospun from a solution containing silver nitrate and 254 nm UV light was used to, simultaneously, photo-crosslink the polymer and promote silver nanoparticles (AgNP) formation. PVP cross-linking gives rise to a peak at 290 nm in the UV–Vis spectrum while the surface plasmon resonance of AgNP appeared at 420 nm. PVP-AgNP mats showed inhibitory effects on bacteria and fungi. This work shows that a PVP-AgNP wound dressing that ensures antimicrobial protection can be produced following a simple route.

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.

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. This article is protected by copyright. All rights reserved.

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. This work was supported by Fundação para a Ciência e Tecnologia, Portugal [grant numbers PTDC/SAU-MII/105114/ 2008, PTDC/CVT-EPI/6685/2014, SFRH/BD/48231/2008, and SFRH/BD/118350/2016]. info:eu-repo/semantics/publishedVersion

International audience; 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.

FCT/MEC (PTDC/QUIQUI/119116/2010; IF/00700/2014; UID/QUI/50006/2013; UID/Multi/04378/2013) ERDF (POCI-01-0145-FEDER - 007265; POCI-01-0145-FEDER-007728) EU 7th Framework Programme (FP7) (PCOFUND-GA-2009-246542; 269138-NanoGuard) CONACYT 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. publishersversion published

Fundacao para a Ciencia e a Tecnologia, Grant/Award Number: ERDF under the PT2020 Partnership Agreement (POCI-, IF/00265/2015, PTDC/CVT-EPI/6685/2014, SFRH/BD/118350/2016, UID/MAR/04292/2013 and UID/Multi/04378/2013; Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO; FCT/MEC, Grant/Award Number: UID/Multi/04378/2013 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. publishersversion published

The seven-valent pneumococcal conjugate vaccine (PCV7) has been available in Portugal since June 2001, but is not included in the National Vaccination Plan. Its impact on colonization is unknown. A point-prevalence study to evaluate PCV7 usage was carried out in 2006 among day-care centre attendees from the Lisbon area. Pneumococcal carriage rates, serotypes, and antibiotypes were determined and compared with results from a similar study conducted in 2001 before vaccine approval. In 2001 and 2006, 717 and 571 children, respectively, were enrolled. In 2006, 45.9% of the participants were appropriately vaccinated and 11.5% were incompletely vaccinated. Carriage of pneumococci remained stable (64.9% in 2001; 68.7% in 2006). Vaccine types (VT) decreased from 53.1% of all pneumococci to 11.2% (p 0.05) due to significant increases in intermediate resistance to penicillin (from 5.5% to 17.8%), erythromycin (from 9.2% to 21.8%), clindamycin (from 6.4% to 19.3%) and tetracycline (from 8.3% to 15.8%) among NVT. Whereas in 2001 resistance among NVT was mostly associated with serotype 19A and NT strains, in 2006 resistance was also found among serotypes 6C, 15A, 24F and 33F. In conclusion, dramatic shifts in serotypes of colonizing pneumococci were observed among vaccinated and unvaccinated children. Rates of antibiotic resistance remained unchanged due to a balance between reduction in VT and an increase in antimicrobial-resistant NVT.

The molecular epidemiology and population structure of 30 bovine subclinical mastitis field isolates of Streptococcus uberis, collected from 6 Portuguese herds (among 12 farms screened) during 2002 and 2003, were examined by using pulsed-field gel electrophoresis (PFGE) for clustering of the isolates and multilocus sequence typing (MLST) to assess the relationship between PFGE patterns and to identify genetic lineages. The 30 isolates were clustered into 18 PFGE types, using a similarity cutoff of 80%, and 3 PFGE types accounted for almost half of the isolates (46.6%). These major types were herd specific, suggesting either cow-to-cow transmission or infection with isolates from the same environmental reservoirs. The remaining unrelated PFGE types of isolates were from different herds strongly suggesting environmental sources of Strep. uberis infection. All 30 isolates were analyzed by MLST and clustered into 14 sequence types (ST). These ST were found to be novel, either with 10 new alleles of 6 housekeeping genes or with different combinations of previously assigned alleles. Five of these ST were clustered into 3 clonal complexes (lineages), ST-143, ST-86, and ST-5, known to include bovine isolates from several geographic locations (Australia, New Zealand, United Kingdom, Sweden, and Denmark) and 9 singletons. To our knowledge, this is the first report that documents molecular typing studies of bovine isolates of Strep. uberis from Portugal, which were shown to represent novel genomic backgrounds of this pathogen.

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.

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 β 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 β antigen, while five strains did not express β antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of β antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of β 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 β 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 β 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.

In this study, we analyzed the antimicrobial resistance properties and T antigenic types of 511 isolates collected in Lisbon district, Portugal, from throat swabs of healthy subjects (n=341), during 2000-2002 and from diverse infection sites (n=170) of outpatients and inpatients, during 1999-2002. Erythromycin resistance was higher in tonsillitis/pharyngitis (27.4%) and skin infection isolates (21.1%), than in carriage and invasive isolates (or=10%). Differences in erythromycin resistance among children and adults were noticed only for carriage isolates (9.3% in children and 21.1% in adults). Most erythromycin-resistant isolates from carriage (82.4%) and tonsillitis/pharyngitis (71.9%) showed the M phenotype. All M phenotype isolates (n=53) carried mef(A), whereas all MLS(B) phenotype isolates (n=19) carried erm(B) and not erm(A). Resistance to tetracycline [mediated by tet(M) in most isolates] wasor=6% in tonsillitis/pharyngitis and carriage isolates, 36.8% in skin infection isolates, and 44.1% in invasive isolates. The M phenotype increased since 2000, linked to a decrease of tetracycline resistance, and was predominantly associated with T1 in 2000-2001 and T12 in 2002 among carriage isolates, and with T8/25/Imp19 through 2000-2002 among tonsillitis/pharyngitis isolates. The majority (53%) of the tetracycline-resistant invasive isolates were nontypable. All isolates were susceptible to penicillin and chloramphenicol. This study showed that tetracycline and macrolide resistance frequency and phenotypes differ among GAS from various origins and changed over time. Moreover, T typing suggested that most drug-resistant isolates causing oropharyngeal carriage are distinct from the majority of isolates causing noninvasive and invasive infection.

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.

Denmark and Iceland are countries where the frequency of methicillin-resistant Staphylococcus aureus is very low due to strict infection control and restrictive antibiotic use policies. In contrast, methicillin-resistant S. epidermidis (MRSE) continues to be isolated as a nosocomial pathogen. The molecular typing by pulsed-field gel electrophoresis (PFGE) of 136 MRSE isolates from five hospitals in Denmark and 94 MRSE isolates from one hospital in Iceland collected in 1997 and 1998 defined 40 different patterns. Closely related PFGE types were found in isolates recovered in Iceland, Denmark, Mexico, Uruguay, Greece, and Cape Verde, evidencing for the first time the geographic clonal dissemination of MRSE strains. The large majority (87.4%) of the MRSE isolates studied were multiresistant.

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.

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.

The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis , 157 of S. haemolyticus , 79 of S. aureus , 21 of S. hominis , 14 of S. saprophyticus , 8 of S. warneri , 6 of S. simulans , 2 of S. lugdunensis , and 1 each of S. caprae , S. carnosus , and S. cohnii . All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus , S. cohnii , S. gallinarum , S. xylosus , S. lentus , S. equorum , and S. chromogenes , the pair S. schleiferi and S. vitulus , and the pair S. piscifermentans and S. carnosus . Four species, S. aureus , S. caprae , S. haemolyticus , and S. lugdunensis , showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.

Four hundred ninety-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from 1996 to 1998 from 22 hospitals in five countries of Latin America—Argentina, Brazil, Chile, Uruguay and Mexico—were examined for antimicrobial susceptibility and clonal type in order to define the endemic clones in those hospitals. The hybridization of Cla I restriction digests with the mecA - and Tn 554 -specific DNA probes combined with pulsed-field gel electrophoresis of chromosomal Sma I digests ( Cla I- mecA :: Cla I-Tn 554 ::PFGE clonal types) documented not only the predominance and persistence of the Brazilian clone (XI::B::B) in Brazil (97%) and Argentina (86%) but also its massive dissemination to Uruguay (100%). Moreover, a close relative of the Brazilian clone (XI::κ::B) was highly represented in Chile (53%) together with a novel clone (47%) (II::E′::F) resistant to pencillin, oxacillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, and gentamicin. A unique clonal type (I::NH::M) was detected in Mexico among pediatric isolates and was resistant to penicillin, oxacillin, and gentamicin only. This study clearly documented the very large capacity for geographic expansion and the persistence of the Brazilian clone, contributing not only to the increasing uniformity of the MRSA in South America but worldwide as well.

Over half (259/503) of drug-resistant (DR) pneumococci colonizing healthy children attending day care centers in Lisbon were identified by molecular typing methods as representatives of several internationally spread clones. These included the 2 penicillin-resistant pandemic Spanish/USA and French/Spanish clones (21% of all DR pneumococci) and 5 new lineages with unusual antibiotypes (accounting for an additional 30% of all DR pneumococci). The most characteristic feature of the latter group was the high frequency of resistance to macrolides and tetracycline and very low or no resistance to penicillin. These observations provide support for the notion that the nasopharyngeal flora of children in day care centers may be a global reservoir of worldwide prevalent strains of DR pneumococci.

A national surveillance conducted in Colombia between 1994 and 1996 identified serotype 5 Streptococcus pneumoniae as the second most frequent cause of invasive disease in children younger than 5 years of age. All 43 serotype 5 isolates collected during this period were shown to be susceptible to penicillin, erythromycin, cefotaxime, and vancomycin, but most (38 of 43, or 88%) were highly resistant to chloramphenicol. In order to clarify a possible genetic relatedness among these isolates, additional microbiological and molecular characterizations were performed. Most (40 of 43, or 93%) of the isolates were found to be resistant to tetracycline. Pulsed-field gel electrophoresis (PFGE) patterns of chromosomal DNAs revealed that all the 43 isolates were closely related and that 38 of the 43 isolates were representatives of a “Colombian clone” of S. pneumoniae isolates which were recovered throughout the 3-year surveillance period from patients in 13 hospitals located in five Colombian cities. Isolates belonging to this Colombian clone were resistant to chloramphenicol and tetracycline, hybridized with the cat and tetM DNA probes in the same 340-kb SmaI fragment, and had identical PFGE patterns after both SmaI and ApaI digestions. Streptococcus pneumoniae is the leading bacterial cause of childhood pneumonia in the developing world (10). It is estimated that more than 1 million children die each year from pneumococcal pneumonia; approximately one-half of them are less than 1 year old (15). The high incidence of pneumococcal infections and the increasing emergence of drug-resistant isolates are the major reasons for the establishment of surveillance programs. Since 1994, an epidemiological surveillance study has been conducted in six Latin American countries in order to identify S. pneumoniae causing invasive disease in children less than 5 years old (10, 21). The study is part of an initiative (Regional

Objective To determine the nature (clonal type and antibiotic resistance pattern) of methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from the largest teaching hospital in Portugal and to detect temporal trends in clonal types during three consecutive surveillance periods in 1992-93, 1994-95 and 1996. Methods MRSA strains were characterized by chromosomal Sma I macrorestriction patterns using pulsed-field gel electrophoresis (PFGE) and by DNA fingerprints—applied to Cla I digests—capable of probing two specific areas of the staphylococcal chromosome: (1) the vicinity of the mecA gene, and (2) the attachment site(s) and copy number of transposon Tn 554 . The combination of these methods can generate ‘clonal types' useful for epidemiological tracking of MRSA strains. Results During the 1992-93 collection period, 65% of MRSA strains carried the mecA polymorph Tn 554 pattern E and PFGE pattern A (I::E::A)—a clonal type that was used to define the ‘Iberian MRSA', which is widely spread throughout southern Europe. The representation of this clone decreased to 42% in 1994-95 and to 20% in 1996. At the same time, a second multiresistant MRSA strain carrying mecA polymorph XI, Tn 554 type B and PFGE pattern B (XI::B::B)—a clonal type characteristic of the so-called ‘Brazilian MRSA'—increased from 5% in 1992-93 to 36% in 1994-95 and 29% in 1996. Conclusions Throughout the four years of surveillance, the Iberian and Brazilian MRSA types and their single subtype variants together have been responsible for the overwhelming majority (close to 90%) of all MRSA infections in the largest teaching hospital of Portugal. The mechanism of epidemicity of these two multiresistant international MRSA clones remains to be elucidated.

Mycobacterium avium subsp. hominissuis (MAH) is an important opportunistic pathogen, infecting humans and animals, notably pigs. Several methods have been used to characterize MAH strains. RFLP and PFGE typing techniques have been used as standard methods but are technically demanding. In contrast, the analysis of VNTR loci is a simpler, affordable and highly reliable PCR-based technique, allowing a numerical and reproductive digitalization of typing data. In this study, the analysis of Mycobacterium avium tandem repeats (MATRs) loci was adapted to evaluate the genetic diversity of epidemiological unrelated MAH clinical strains of human (n=28) and porcine (n=69) origins, collected from diverse geographical regions across mainland Portugal. These MAH isolates were found to be genetically diverse and genotypes are randomly distributed across the country. Some of the human strains shared identical VNTR profiles with porcine isolates. Our study shows that the VNTR genotyping using selected MATR loci is a useful analysis technique for assessing the genetic diversity of MAH isolates from Portugal. This typing method could be successfully applied in other countries toward the implementation of a worldwide open-access database of MATR-VNTR profiles of MAH isolates, allowing a better assessment of the global epidemiology traits of this important pathogenic species.

One hundred and eighty-three methicillin-resistant Staphylococcus aureus (MRSA) isolates from eight different Portuguese hospitals were genetically typed by random amplification of polymorphic DNA (RAPD) employing different oligonucleotide primers. Fourteen different RAPD genotypes were identified. A subset of the same strains was also characterized by pulsed-field gel electrophoresis (PFGE) and/or hybridization using mecA and Tn554 probes. In the majority of cases, the different genotyping methods have identified the same MRSA clones. However, PFGE combined with the DNA probes was clearly the method providing higher resolution. Most strains that have already been identified by PFGE and DNA probes as members of the widely spread Iberian clone of MRSA generated a common RAPD genotype. The most prevalent Iberian clone was not detected in a collection of MRSA from Poland that was also examined by RAPD. On the other hand, MRSA strains second most frequent in prevalence in the Portuguese and Polish collection appear to be identical by RAPD, indicating extensive geographic spread of this particular clone. No correlation was apparent between epidemic behavior and the number of protein A gene repeats in this particular collection of MRSA strains.

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.

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.

The asymptomatic oropharyngeal colonization rate by Streptococcus pyogenes was 10.7% in children (901 among 8,405 children 0–16 years old) and 3.3% in adults (37 among 1,126 households of children) in the Lisbon area during 2000–2006. Macrolide-resistant S. pyogenes from children (n = 149) was variable with time: 9.8–10.7% in 2000–2002, 28.1% in 2003, 19.6–2.7% in 2004–2005 and 14.6% in 2006. Eight lineages (97.3% of isolates) were identified based on at least 80% similarity of PFGE patterns, T types, emm types and multilocus sequence types (ST). The elevated frequency of macrolide resistance was associated with M phenotype lineages I (emm12/ST36) and V (emm4, emm75/ST39 and a novel emmstMrp6 type) and with one cMLSB lineage IV (emm28/ST52) known to be associated with upper respiratory tract and invasive infections. Significant associations (p 20%) of speC, prtF1 or ssa was probably caused either by clonal dissemination (speC), or to horizontal gene transfer events (prtF1 and ssa). This report contributes to a better understanding of the molecular epidemiology and evolution of macrolide-resistant S. pyogenes causing symptom-free oropharyngeal colonization. These colonizing strains carry macrolide resistance and virulence genes capable of being transferred to other bacterial species sharing the same niche.

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.

We describe 66 ciprofloxacin-nonsusceptible Streptococcus pyogenes isolates recovered from colonized and infected children. The ParC S79A substitution was frequent and associated with the emm 6/sequence type 382 ( emm 6/ST382) lineage. The ParC D83G substitution was detected in two isolates ( emm 5/ST99 and emm 28/ST52 lineages). One isolate ( emm 89/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.

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.

This study focussed on the clonal structure and temporal distribution of E. faecalis and E. faecium with high-level resistance to gentamicin (HLGR) and glycopeptides (GR) collected from clinical samples during 2004 to 2006 at a Portuguese Hospital. The findings were an E. faecalis-dominant and epidemic clone (PFGE-AO), the maintenance of a major epidemic E. faecium clone (PFGE-c) and a high prevalence of putative virulence genes--asa1 (aggregation substances), gelE (gelatinase), cylA (cytolysin), esp (enterococcal surface protein), and hyl (hyaluronidase)--most of them significantly associated with the major clones of both species. The E. faecalis GR isolates ST6 and the E. faecium GR isolates ST17, ST18 and ST280 belong to the clonal complexes E. faecalis-CC2 and E. faecium-CC17, which are well adapted to the nosocomial setting and are disseminated worldwide. This study highlights the need for continuous and active surveillance in this Portuguese hospital in order to follow the evolution of these epidemic and persistent clones.

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.

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.

Background EURIS (European Resistance Intervention Study) was launched as a multinational study in September of 2000 to identify the multitude of complex risk factors that contribute to the high carriage rate of drug resistant Streptococcus pneumoniae strains in children attending Day Care Centers in several European countries. Access to the very large number of data required the development of a web-based infrastructure – EURISWEB – that includes a relational online database, coupled with a query system for data retrieval, and allows integrative storage of demographic, clinical and molecular biology data generated in EURIS. Methods All components of the system were developed using open source programming tools: data storage management was supported by PostgreSQL, and the hypertext preprocessor to generate the web pages was implemented using PHP. The query system is based on a software agent running in the background specifically developed for EURIS. Results The website currently contains data related to 13,500 nasopharyngeal samples and over one million measures taken from 5,250 individual children, as well as over one thousand pre-made and user-made queries aggregated into several reports, approximately. It is presently in use by participating researchers from three countries (Iceland, Portugal and Sweden). Conclusion An operational model centered on a PHP engine builds the interface between the user and the database automatically, allowing an easy maintenance of the system. The query system is also sufficiently adaptable to allow the integration of several advanced data analysis procedures far more demanding than simple queries, eventually including artificial intelligence predictive models.

A pilot study was conducted to determine the genetic diversity of multiple colonies of pneumococci recovered from 37 nasopharyngeal (NP) samples of children. A total of 239 pneumococcal isolates (typically, six to eight colonies per sample) were typed by pulsed-field gel electrophoresis (PFGE). In most NP samples (89%) the multiple colonies shared common PFGE types and serotypes. However, four samples were heterogeneous (samples A through D): each contained two strains with different PFGE types, antibiotypes, and serotypes. Samples A and B each contained one strain of a vaccine capsular type and another expressing a non-vaccine type (according to the currently licensed seven-valent conjugate vaccine). In samples B and C the penicillin MIC for one strain was elevated and the other strain was susceptible. In each of the heterogeneous samples, one of the strains was a representative of an internationally disseminated clone. Samples A, C, and D contained strains which carried prophages that were inducible by mitomycin C and that could be visualized by electron microscopy. The comC gene allele (which encodes the competence-stimulating peptide) was the same in both strains found in each of samples A, B, and D. Carriage of multiple pneumococci with distinct properties should favor genetic exchange and provide a dynamic population structure for pneumococci in their ecological reservoir. Quantitative resolution of majority and minority components of the pneumococcal NP flora will be of importance for evaluation of the impact of intervention strategies such as vaccination or introduction of new antimicrobial agents.

Recent reports suggest that methicillin-resistant Staphylococcus aureus (MRSA) may be emerging as a community pathogen. In Portuguese hospitals, the incidence of MRSA among disease causing isolates is extremely high (48-50%). To determine the prevalence of MRSA in the Portuguese community, nasal samples were obtained from 823 draftees, 484 nonmedical university students, and 107 high-school students. In addition, throat samples were obtained from the 823 draftees and S. aureus isolates were also recovered from 283 (13%) nasopharyngeal samples obtained from 2,111 children attending day-care centers. The rate of nasal colonization of S. aureus was 34%, 25%, and 46% for draftees, nonmedical university students, and high-school students, respectively. The rate of pharyngeal colonization of the draftees was 33%. Of the 1,001 S. aureus isolates obtained, seven were MRSA and eight were borderline oxacillin-resistant S. aureus (BORSA). By molecular typing techniques, five of the seven MRSA were identified as belonging to one of three highly epidemic clones, the Brazilian, Iberian, and Pediatric clones of MRSA, which were identified as endemic in Portuguese hospitals. The eight BORSA were all members of clones previously identified in international samples. In spite of the extremely high prevalence of MRSA in Portuguese hospitals, the carriage rate of MRSA in healthy and young individuals remains low.

Characterization by antibiotype of the 1,096 Streptococcus pneumoniae recovered from 2,111 nasopharyngeal samples of children attending 16 day care centers (DCCs) in Lisbon, Portugal, and molecular typing of 413 drug-resistant pneumococci (DRPn) and 89 fully drug-susceptible pneumococci (DSPn) has allowed several conclusions. (i) There was an increase in the frequency of DRPn colonizing children in DCCs from 40% in 1996 to 45% in 1997 to 50% in 1998. (ii) Drug resistance spread by cross-transmission of DRPn clones. A few (8 out of 57) DRPn clones were repeatedly isolated from a large number of children in several DCCs and during each period of surveillance, suggesting the epidemic nature of these clones, which included lineages representing internationally spread S. pneumoniae clones. (iii) Dissemination of resistance determinants among pneumococci colonizing the nasopharynx occurred. Association of identical pulsed-field gel electrophoresis patterns with diverse antibiotypes among pneumococci colonizing children suggests that the high prevalence of DRPn involves not only cross-transmission of resistant strains but also dispersal of resistance genes through recombinational mechanisms. (iv) DCCs are autonomous epidemiological units. Among the 413 DRPn, 57 different lineages were detected; these lineages were dispersed among the 16 DCCs to produce unique microbiological profiles for each of the DCCs. Higher genetic diversity and less sharing of clonal types were observed among the DSPn.

Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

In an effort to establish the rate of carriage of antibiotic resistant respiratory pathogens in children attending urban day care centers (DCC) in Portugal, seven DCC in Lisbon were selected for determining the rate of nasopharyngeal colonization of children between the ages of 6 months to 6 years by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Of the 586 children studied between January and March 1996, 47% carried S. pneumoniae, 72% H. influenzae, and 54% M. catarrhalis. Twenty-four percent of the pneumococci had reduced susceptibility to penicillin, and most of these belonged to serogroups 19, 23, 14, and 6. An additional 19% were fully susceptible to penicillin but showed decreased susceptibility to other antimicrobials. These isolates expressed serogroups 6, 11, 14, 18, 19, and 34. The majority (96%) of M. catarrhalis and 20% of H. influenzae were penicillin resistant due to the production of beta-lactamases. Recent antimicrobial use was associated with carriage of penicillin non-susceptible pneumococci and beta-lactamase producing H. influenzae (p0.05). Individual DCC differed substantially from one another in their rates of carriage of antibiotic resistant H. influenzae and S. pneumoniae. Characterization of antibiotic resistant S. pneumoniae isolates by molecular fingerprinting techniques showed that each DCC had a unique microbiological profile, suggesting little, if any, exchange of the resistant microbial flora among them. An exception to this was the presence of isolates belonging to two internationally spread epidemic clones: the multiresistant Spanish/USA clone expressing serotype 23F, and the penicillin and sulfamethoxazole-trimethoprim resistant French/Spanish clone (serotype 14) which were detected in four and three DCC, respectively.

Close to half of the 878 methicillin-resistant Staphylococcus aureus (MRSA) strains recovered between 1992 and 1997 from the pediatric hospital in Lisbon were bacteria in which antibiotic resistance was limited to β-lactam antibiotics. The other half were multidrug resistant. The coexistence of MRSA with such unequal antibiotic resistance profiles prompted us to use molecular typing techniques for the characterization of the MRSA strains. Fifty-three strains chosen randomly were typed by a combination of genotypic methods. Over 90% of the MRSA strains belonged to two clones: the most frequent one, designated the “pediatric clone,” was reminiscent of historically “early” MRSA: most isolates of this clone were only resistant to β-lactam antimicrobials and remained susceptible to macrolides, quinolones, clindamycin, spectinomycin, and tetracycline. They showed heterogeneous and low-level resistance to methicillin (MIC, 1.5 to 6 μg/ml), carried the Cla I- mecA polymorph II, were free of the transposon Tn 554 , and showed macrorestriction pattern D (clonal type II::NH::D). The second major clone was the internationally spread and multiresistant “Iberian” MRSA with homogeneous and high-level resistance to methicillin (MIC, >200 μg/ml) and clonal type I::E::A. Surprisingly, the multidrug-resistant and highly epidemic Iberian MRSA did not replace the much less resistant pediatric clone during the 6 years of surveillance. The pediatric clone was also identified among contemporary MRSA isolates from Poland, Argentina, The United States, and Colombia, and the overwhelming majority of these were also associated with pediatric settings. We propose that the pediatric MRSA strain represents a formerly widely spread archaic clone which survived in some epidemiological settings with relatively limited antimicrobial pressure.