TY - JOUR AU - Sanchez, C.J. AU - Shivshankar, P. AU - Stol, K. AU - Trakhtenbroit, S. AU - Sullam, P.M. AU - Sauer, K. AU - Hermans, P.W.M. AU - Orihuela, C.J. PY - 2010 UR - https://hdl.handle.net/2066/87784 AB - The Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10) on the surface of lung cells through amino acids 273-341 located in the Basic Region (BR) domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (r)BR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122-166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs) of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae) may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection. TI - The pneumococcal serine-rich repeat protein is an intra-species bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms. EP - pii: E1001044 SN - 1553-7366 IS - iss. 8 SP - pii: E1001044 JF - Plos Pathogens VL - vol. 6 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/87784/87784.pdf?sequence=1 ER - TY - JOUR AU - Burghout, P.J. AU - Cron, L.E. AU - Gradstedt, H. AU - Quintero, B. AU - Simonetti, E.R. AU - Bijlsma, J.J. AU - Bootsma, H.J. AU - Hermans, P.W.M. PY - 2010 UR - https://hdl.handle.net/2066/88184 AB - The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO(2)) it encounters during transmission, colonization, and infection. Since CO(2) is important for various cellular processes, factors that allow optimal CO(2) sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO(2)-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO(2) to bicarbonate (HCO(3)(-)), an essential step to prevent the cellular release of CO(2). The addition of unsaturated fatty acids (UFAs) reversed the CO(2)-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Deltapca), indicating that PCA-mediated CO(2) fixation is at least associated with HCO(3)(-)-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO(2) fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO(2)-independent and naturally circulating CO(2)-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO(2) fixation pathways for pneumococcal growth and survival. TI - Carbonic anhydrase is essential for Streptococcus pneumoniae growth in environmental ambient air. EP - 4062 SN - 0021-9193 IS - iss. 15 SP - 4054 JF - Journal of Bacteriology VL - vol. 192 N1 - 1 augustus 2010 DO - https://doi.org/10.1128/JB.00151-10 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/88184/88184.pdf?sequence=1 ER - TY - JOUR AU - Hira, V. AU - Sluijter, M. AU - Goessens, W.H.F. AU - Ott, A. AU - Groot, R. de AU - Hermans, P.W.M. AU - Kornelisse, R.F. PY - 2010 UR - https://hdl.handle.net/2066/89077 AB - Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in neonatal intensive care units (NICU) worldwide. Infecting strains of these commensal bacteria may originate from NICU personnel. Therefore, we studied the characteristics of CoNS isolates from NICU personnel and compared them to those of isolates from the general population and from sepsis patients. Furthermore, we studied the epidemiological effect on CoNS carriage of NICU personnel after a period of absence. In our study, we isolated CoNS from the thumbs of NICU personnel every 2 weeks during the summer of 2005 and sampled personnel returning from vacation and a control group from the general population. Furthermore, we collected sepsis isolates from this period. Isolates were tested for antibiotic resistance, mecA and icaA carriage, biofilm production, and genetic relatedness. We found that mecA and icaA carriage as well as penicillin, oxacillin, and gentamicin resistance were significantly more prevalent in CoNS strains from NICU personnel than in community isolates. Similar trends were observed when postvacation strains were compared to prevacation strains. Furthermore, genetic analysis showed that 90% of the blood isolates were closely related to strains found on the hands of NICU personnel. Our findings revealed that CoNS carried by NICU personnel differ from those in the general population. Hospital strains are replaced by community CoNS after a period of absence. NICU personnel are a likely cause for the cross-contamination of virulent CoNS that originate from the NICU to patients. TI - Coagulase-negative staphylococcal skin carriage among neonatal intensive care unit personnel: from population to infection. EP - 3881 SN - 0095-1137 IS - iss. 11 SP - 3876 JF - Journal of Clinical Microbiology VL - vol. 48 N1 - 1 november 2010 DO - https://doi.org/10.1128/JCM.00967-10 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/89077/89077.pdf?sequence=1 ER - TY - JOUR AU - Bart, M.J. AU - Gent, M. van AU - Heide, H.G. van der AU - Boekhorst, J. AU - Hermans, P.W.M. AU - Parkhill, J. AU - Mooi, F.R. PY - 2010 UR - https://hdl.handle.net/2066/89571 AB - BACKGROUND: Despite vaccination since the 1950s, pertussis has persisted and resurged. It remains a major cause of infant death worldwide and is the most prevalent vaccine-preventable disease in developed countries. The resurgence of pertussis has been associated with the expansion of Bordetella pertussis strains with a novel allele for the pertussis toxin (Ptx) promoter, ptxP3, which have replaced resident ptxP1 strains. Compared to ptxP1 strains, ptxP3 produce more Ptx resulting in increased virulence and immune suppression. To elucidate how B. pertussis has adapted to vaccination, we compared genome sequences of two ptxP3 strains with four strains isolated before and after the introduction vaccination. RESULTS: The distribution of SNPs in regions involved in transcription and translation suggested that changes in gene regulation play an important role in adaptation. No evidence was found for acquisition of novel genes. Modern strains differed significantly from prevaccination strains, both phylogenetically and with respect to particular alleles. The ptxP3 strains were found to have diverged recently from modern ptxP1 strains. Differences between ptxP3 and modern ptxP1 strains included SNPs in a number of pathogenicity-associated genes. Further, both gene inactivation and reactivation was observed in ptxP3 strains relative to modern ptxP1 strains. CONCLUSIONS: Our work suggests that B. pertussis adapted by successive accumulation of SNPs and by gene (in)activation. In particular changes in gene regulation may have played a role in adaptation. TI - Comparative genomics of prevaccination and modern Bordetella pertussis strains. EP - 627 SN - 1471-2164 SP - 627 JF - BMC Genomics VL - vol. 11 DO - https://doi.org/10.1186/1471-2164-11-627 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/89571/89571.pdf?sequence=1 ER - TY - JOUR AU - Simoes, A.S. AU - Sa-Leao, R. AU - Eleveld, M.J. AU - Tavares, D.A. AU - Carrico, J.A. AU - Bootsma, H.J. AU - Hermans, P.W.M. PY - 2010 UR - https://hdl.handle.net/2066/88330 AB - While performing surveillance studies in Oeiras, Portugal, designed to describe the impact of pneumococcal conjugate vaccine on colonization, we observed an increase from 0.7% in 2003 to 5% in 2006 in the prevalence of penicillin resistance (MIC of 2 to 6 mg/liter) among presumptively identified pneumococcal isolates. Although 15 of the 22 penicillin-resistant isolates obtained in 2006 were optochin resistant, they were bile soluble and thus considered to be bona fide pneumococci. This study aimed to clarify the nature of these isolates by using a combination of phenotypic and genotypic approaches that included routine strategies for pneumococcal identification, multilocus sequence analysis (MLSA), and comparative genomic hybridization (CGH). By MLSA, all isolates were classified as "streptococci of the mitis group" that, however, were distinct from typical Streptococcus pneumoniae or Streptococcus mitis. A single isolate was identified as Streptococcus pseudopneumoniae. CGH confirmed these findings and further indicated that a considerable part of the proposed pneumococcal core genome is conserved in these isolates, including several pneumococcal virulence genes (e.g., pavA, spxB, cbpE, and cbpD). These results suggest that among pneumococci and closely related streptococci, universal unique phenotypic and genetic properties that could aid species identification are virtually impossible to define. In pneumococcal colonization studies, when atypical strains are found, MLSA and CGH are informative tools that can be used to complement routine tests. In our study, after correct identification of the penicillin-resistant true pneumococci, we found that penicillin resistance levels among pneumococci remained stable from 2003 to 2006. TI - Highly penicillin-resistant multidrug-resistant pneumococcus-like strains colonizing children in Oeiras, Portugal: genomic characteristics and implications for surveillance. EP - 246 SN - 0095-1137 IS - iss. 1 SP - 238 JF - Journal of Clinical Microbiology VL - vol. 48 DO - https://doi.org/10.1128/JCM.01313-09 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/88330/88330.pdf?sequence=1 ER - TY - JOUR AU - Vries, S.P.W. de AU - Hijum, S.A.F.T. van AU - Schueler, W. AU - Riesbeck, K. AU - Hays, J.P. AU - Hermans, P.W.M. AU - Bootsma, H.J. PY - 2010 UR - https://hdl.handle.net/2066/88539 AB - Moraxella catarrhalis is an emerging human-restricted respiratory tract pathogen that is a common cause of childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. Here, we report the first completely assembled and annotated genome sequence of an isolate of M. catarrhalis, strain RH4, which originally was isolated from blood of an infected patient. The RH4 genome consists of 1,863,286 nucleotides that form 1,886 protein-encoding genes. Comparison of the RH4 genome to the ATCC 43617 contigs demonstrated that the gene content of both strains is highly conserved. In silico phylogenetic analyses based on both 16S rRNA and multilocus sequence typing revealed that RH4 belongs to the seroresistant lineage. We were able to identify almost the entire repertoire of known M. catarrhalis virulence factors and mapped the members of the biosynthetic pathways for lipooligosaccharide, peptidoglycan, and type IV pili. Reconstruction of the central metabolic pathways suggested that RH4 relies on fatty acid and acetate metabolism, as the genes encoding the enzymes required for the glyoxylate pathway, the tricarboxylic acid cycle, the gluconeogenic pathway, the nonoxidative branch of the pentose phosphate pathway, the beta-oxidation pathway of fatty acids, and acetate metabolism were present. Moreover, pathways important for survival under challenging in vivo conditions, such as the iron-acquisition pathways, nitrogen metabolism, and oxidative stress responses, were identified. Finally, we showed by microarray expression profiling that approximately 88% of the predicted coding sequences are transcribed under in vitro conditions. Overall, these results provide a foundation for future research into the mechanisms of M. catarrhalis pathogenesis and vaccine development. TI - Genome analysis of Moraxella catarrhalis strain RH4, a human respiratory tract pathogen. EP - 3583 SN - 0021-9193 IS - iss. 14 SP - 3574 JF - Journal of Bacteriology VL - vol. 192 N1 - 1 juli 2010 DO - https://doi.org/10.1128/JB.00121-10 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/88539/88539.pdf?sequence=1 ER - TY - JOUR AU - Bello Gonzalez, T. AU - Rivera Olivero, I.A. AU - Pocaterra, L. AU - Spadola, E. AU - Araque, M. AU - Hermans, P.W.M. AU - Waard, J.H. de PY - 2010 UR - https://hdl.handle.net/2066/87365 AB - In North America, the indigenous groups have been identified as a population with increased risk of pneumococcal colonization and pneumococcal invasive disease. However, little information is available from South American natives. In the present study we evaluated the nasopharyngeal carriage and serotype distribution of Streptococcus pneumoniae in mothers and children of the Panare people from Venezuela. In May 2008, in 8 distinct geographically isolated communities, 148 nasopharyngeal samples were obtained from 64 healthy mothers and 84 healthy Panare children under 5 years of age. S. pneumoniae was isolated and identified by standard techniques. Strains were typified by multiplex PCR and resistance patterns were determined by the disk diffusion method. A total of 65 strains were isolated; 11% of the mothers and 69% of the children carried S. pneumoniae. Serotypes 6B (48%), 33F (21,5%), 6A (6%), 19A (3,1%) and 23F (1,5%) were the most predominant. Of the 6 colonized mother-child pairs, 3 pairs (2 with 6B), were colonized with the same serotype. All strains were sensitive to penicillin and 13,7% were resistant to macrolides. The high colonization rates in the Panare people suggest that the children are at increased risk of pneumococcal invasive disease and could benefit from vaccination. Four conjugate vaccine serotypes (6B, 6A, 19A and 23F) representing 58 % of all strains were present in the population at the moment of sampling. Resistance to antibiotics is (still) not a problem. TI - [Pneumococcal carriage in mothers and children of the Panare Amerindians from the State of Bolivar, Venezuela]. EP - 34 SN - 0325-7541 IS - iss. 1 SP - 30 JF - Revista Argentina de Microbiologia VL - vol. 42 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/87365/87365.pdf?sequence=1 ER - TY - JOUR AU - Leegaard, T.M. AU - Bootsma, H.J. AU - Caugant, D.A. AU - Eleveld, M.J. AU - Mannsaker, T. AU - Froholm, L.O. AU - Gaustad, P. AU - Hoiby, E.A. AU - Hermans, P.W.M. PY - 2010 UR - https://hdl.handle.net/2066/89734 AB - Accurate differentiation between pneumococci and other viridans streptococci is essential given their differences in clinical significance. However, classical phenotypic tests are often inconclusive, and many examples of atypical reactions have been reported. In this study, we applied various phenotypic and genotypic methods to discriminate between a collection of 12 streptococci isolated from the upper respiratory tract of HIV-seropositive individuals in 1998 and 1999. Conventional phenotypic characterization initially classified these streptococci as Streptococcus pneumoniae, as they were all sensitive to optochin and were all bile soluble. However, they did not agglutinate with anti-pneumococcal capsular antibodies and were also far more resistant to antimicrobial agents than typeable pneumococci isolated in the same period. Genotypic characterization of these isolates and control isolates by both multilocus sequence analysis (MLSA) and comparative genomic hybridization (CGH) showed that only a single isolate was genetically considered to be a true S. pneumoniae isolate, and that the remaining 11 non-typable isolates were indeed distinct from true pneumococci. Of these, 10 most closely resembled a subgroup of Streptococcus mitis isolates genetically, while one strain was identified as a Streptococcus pseudopneumoniae isolate. CGH also showed that a considerable part of the proposed pneumococcal core genome, including many of the known pneumococcal virulence factors, was conserved in the non-typable isolates. Sequencing of part of the 16S rRNA gene and investigation for the presence of ply by PCR corroborated these results. In conclusion, our findings confirm the close relationship between streptococci of the Mitis group, and show that both MLSA and CGH enable pneumococci to be distinguished from other Mitis group streptococci. TI - Phenotypic and genomic characterization of pneumococcus-like streptococci isolated from HIV-seropositive patients. EP - 848 SN - 0026-2617 IS - iss. Pt 3 SP - 838 JF - Microbiology (New York) VL - vol. 156 N1 - 1 maart 2010 DO - https://doi.org/10.1099/mic.0.035345-0 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/89734/89734.pdf?sequence=1 ER -