Evidence for the evolutionary steps leading to mecA-mediated ß-lactam resistance in staphylococci

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA–an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all β-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the β-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to β-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of β-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics

Staphylococcus aureus in former Portuguese colonies from Africa and the Far East: missing data to help fill the world map

AbstractThe aim of the present study was to determine the prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage among patients and healthcare workers in Angola (ANG), São Tomé and Príncipe (STP), Cape Verde (CV) and East Timor (ET), and to characterize the antimicrobial susceptibility, virulence content and population structure of all S. aureus. Despite the importance of MRSA as a major human pathogen, data from these former Portuguese colonies in Africa and Asia are scarce. A total of 2065 nasal swabs recovered between 2010–14 were included in the study. Antimicrobial susceptibility testing and molecular characterization of S. aureus showed: (i) a very high MRSA prevalence in ANG (61.6%), moderate in STP (25.5%), low in CV (5.6%) and null in ET; (ii) a high prevalence of Panton–Valentine leukocidin in STP (36.8%), ET (29.2%) and CV (28.3%) contrasting with ANG (7.9%); (iii) ST5-SCCmecIVa, ST8-IV/V and ST5-VI were the major MRSA clones in ANG (65.2%), STP (44.8%) and CV (50%), respectively; (iv) a high resistance to trimethoprim-sulfamethoxazole in ANG (66.5%) and STP (50.9%), to rifampin in ANG (77.3%), and to tetracycline in STP (26.3%) and ET (20.8%); (v) three major methicillin-susceptible S. aureus clones (ST15, ST508, ST152) were present in all four countries. Age <18 years (OR 2.03, 95% CI 1.24–3.31), previous surgery (OR 2.45, 95% CI 1.24–4.83), no smoking (OR 4.04, 95% CI 1.05–15.50), and longer hospitalization (OR 2.53, 95% CI 1.49–4.28) were risk factors for MRSA carriage. This study provided the first comprehensive overview on MRSA in former Portuguese colonies in Africa and Asia, missing data in the world map

MAGNETIC FORCES IN ORTHODONTICS

BACKGROUND: Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity. RESULTS: A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements. CONCLUSIONS: NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies

Community-Associated Methicillin-Resistant Staphylococcus Aureus Lacking PVL, as a Cause of Severe Invasive Infection Treated with Linezolid

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging public health problem worldwide. Severe invasive infections have been described, mostly associated with the presence of Panton-Valentine leukocidin (PVL). In Portugal limited information exists regarding CA-MRSA infections. In this study we describe the case of a previously healthy 12-year-old female, sport athlete, who presented to the hospital with acetabulofemoral septic arthritis, myositis, fasciitis, acetabulum osteomyelitis, and pneumonia.The MRSA isolated from blood and synovial fluid was PVL negative and staphylococcal enterotoxin type P (SEP) and type L (SEL) positive, with a vancomycin MIC of 1.0mg/L and resistant to clindamycin and ciprofloxacin. The patient was submitted to multiple surgical drainages and started on vancomycin, rifampicin, and gentamycin. Due to persistence of fever and no microbiological clearance, linezolid was started with improvement. This is one of the few reported cases of severe invasive infection caused by CA-MRSA in Portugal,which was successfully treated with linezolid. In spite of the severity of infection, the MRSA isolate did not produce PVL

Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI

In order to obtain insights into the methicillin-resistant Staphylococcus aureus (MRSA) population structure in the Azores archipelago, 106 MRSA isolates were collected from patients attending an Azorean central hospital between January 2007 and February 2008. Antimicrobial resistance was determined for all isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), staphylococcal chromosome cassette mec (SCCmec) typing and the presence of Panton–Valentine leukocidin (PVL). The majority of the isolates (87%, n = 92) belonged to the EMRSA-15 clone (ST22, SCCmec-IVh), followed by the Pediatric clone (ST5-VI/IVc) (11%, n = 12). The Berlin clone (ST45-IVa) and a new clone (spa type t1839, ST1339 and SCCmec V variant) were represented by single isolates. All of the isolates carried SCCmec types IV, V or VI and a non-multiresistant antibiotic profile, resembling the currently emerging community MRSA. Moreover, PVL was described for the first time to be associated with the Pediatric clone carrying SCCmec type VI. We provided the first description of the population structure of MRSA in the Azores islands, which seems to be shaped by genetic events occurring locally, as well as by the regular population exchange between the islands, continental Portugal, the United Kingdom and the United States

The blp locus of Streptococcus pneumoniae plays a limited role in the selection of which strains can co-colonize the human nasopharynx.

Nasopharyngeal colonization is important for Streptococcus pneumoniae evolution, providing the opportunity for horizontal gene transfer when multiple strains co-occur. Although colonization with more than one strain of pneumococcus is common, the factors that influence the ability of strains to co-exist are not known. A highly variable blp (bacteriocin-like peptide) locus has been identified in all sequenced strains of S. pneumoniae This locus controls the regulation and secretion of bacteriocins, small peptides that target other bacteria. In this study, we analyzed a series of co-colonizing isolates to evaluate the impact of the blp locus on human colonization to determine whether competitive phenotypes of bacteriocin secretion restrict co-colonization.We identified a collection of 135 nasopharyngeal samples with two or more strains totaling 285 isolates. The blp locus of all strains was characterized genetically with regards to pheromone type, bacteriocin/immunity content and potential for locus functionality. Inhibitory phenotypes of bacteriocin secretion and locus activity were assessed through overlay assays. Isolates from single colonization (n=298) were characterized for comparison.Co-colonizing strains had a high diversity of blp cassettes; approximately one third displayed an inhibitory phenotype in vitro Despite in vitro evidence of competition, pneumococci co-colonized individuals independently of their blp pheromone type (p=0.577), bacteriocin/immunity content, blp locus activity (p=0.798) and inhibitory phenotype (p=0.716). In addition, no significant differences were observed when single and co-colonizing strains were compared.Despite clear evidence of blp-mediated competition in experimental models, our study suggests that the blp locus plays a limited role in restricting pneumococcal co-colonization in humans. IMPORTANCE: Nasopharyngeal colonization with Streptococcus pneumoniae (pneumococcus) is important for pneumococcal evolution as it represents the major site for horizontal gene transfer when multiple strains co-occur, a phenomenon known as co-colonization. Understanding how pneumococcal strains interact within the competitive environment of the nasopharynx is of chief importance in the context of pneumococcal ecology. In this study we used an unbiased collection of naturally co-occurring pneumococcal strains and showed that a biological process frequently used by bacteria for competition - bacteriocin production - is not decisive in the co-existence of pneumococci in the host, contrary to what has been shown in experimental models

Disease isolates of Streptococcus pseudopneumoniae and non-typeable S. pneumoniae presumptively identified as atypical S. pneumoniae in Spain

We aimed to obtain insights on the nature of a collection of isolates presumptively identified as atypical Streptococcus pneumoniae recovered from invasive and non-invasive infections in Spain. One-hundred and thirty-two isolates were characterized by: optochin susceptibility in ambient and CO2-enriched atmosphere; bile solubility; PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802, aliB-like ORF2, and a specific 16S rRNA region; multilocus sequence analysis; and antimicrobial susceptibility. By multilocus sequence analysis, 61 isolates were S. pseudopneumoniae, 34 were pneumococci, 13 were S. mitis, and 24 remained unclassified as non-pneumococci. Among S. pseudopneumoniae isolates, 51 (83.6%) were collected from respiratory tract samples; eight isolates were obtained from sterile sources. High frequency of non-susceptibility to penicillin (60.7%) and erythromycin (42.6%) was found. Only 50.8% of the S. pseudopneumoniae isolates displayed the typical optochin phenotype originally described for this species. None harbored the cpsA gene or the pneumococcal typical lytA restriction fragment length polymorphism. The Spn9802 and the specific 16S rRNA regions were detected among the majority of the S. pseudopneumoniae isolates (n = 59 and n = 49, respectively). The ply and pspA genes were rarely found. A high genetic diversity was found and 59 profiles were identified. Among the S. pneumoniae, 23 were capsulated and 11 were non-typeable. Three non-typeable isolates, associated to international non-capsulated lineages, were recovered from invasive disease sources. In conclusion, half of the atypical pneumococcal clinical isolates were, in fact, S. pseudopneumoniae and one-fourth were other streptococci. We identified S. pseudopneumoniae and non-typeable pneumococci as cause of disease in Spain including invasive disease