Sluijter, M.

Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium (Article)

2011-12-01T00:00:00Z

Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the Holliday junction migration motor protein RuvB. In both M. pneumoniae and M. genitalium, genes have been identified that have the capacity to encode RuvB homologs (MPN536 and MG359, respectively). Here, the characteristics of the MPN536- and MG359-encoded proteins (the RuvB proteins from M. pneumoniae strain FH [RuvBFH] and M. genitalium [RuvBMge], respectively) are described. Both RuvBFHand RuvBMgewere found to have ATPase activity and to bind DNA. In addition, both proteins displayed divalent cation- and ATP-dependent DNA helicase activity on partially double-stranded DNA substrates. The helicase activity of RuvBMge, however, was significantly lower than that of RuvBFH. Interestingly, we found RuvBFHto be expressed exclusively by subtype 2 strains of M. pneumoniae. In strains belonging to the other major subtype (subtype 1), a version of the protein is expressed (the RuvB protein from M. pneumoniae strain M129 [RuvBM129]) that differs from RuvBFHin a single amino acid residue (at position 140). In contrast to RuvBFH, RuvBM129displayed only marginal levels of DNAunwinding activity. These results demonstrate that M. pneumoniae strains (as well as closely related Mycoplasma spp.) can differ significantly in the function of components of their DNA recombination and repair machinery.

Identification of amino acid residues critical for catalysis of holliday junction resolution by Mycoplasma genitalium RecU (Article)

2011-08-01T00:00:00Z

The RecU protein from Mycoplasma genitalium, RecUMge, is a 19.4-kDa Holliday junction (HJ) resolvase that binds in a nonspecific fashion to HJ substrates and, in the presence of Mn2+, cleaves these substrates at a specific sequence (5'-G/TC2C/TTA/GG-3'). To identify amino acid residues that are crucial for HJ binding and/or cleavage, we generated a series of 16 deletion mutants (9 N- and 7 C-terminal deletion mutants) and 31 point mutants of RecUMge. The point mutations were introduced at amino acid positions that are highly conserved among bacterial RecU-like sequences. All mutants were purified and tested for the ability to bind to, and cleave, HJ substrates. We found the five N-terminal and three C-terminal amino acid residues of RecUMgeto be dispensable for its catalytic activities. Among the 31 point mutants, 7 mutants were found to be inactive in both HJ binding and cleavage. Interestingly, in 12 other mutants, these two activities were uncoupled; while these proteins displayed HJ-binding characteristics similar to those of wild-type RecUMge, they were unable to cleave HJ substrates. Thus, 12 amino acid residues were identified (E11, K31, D57, Y58, Y66, D68, E70, K72, T74, K76, Q88, and L92) that may play either a direct or indirect role in the catalysis of HJ resolution.

Variation in a surface-exposed region of the Mycoplasma pneumoniae P40 protein as a consequence of homologous DNA recombination between RepMP5 elements (Article)

2011-02-01T00:00:00Z

Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory tract infections. The first step in infection is adherence of the bacteria to the respiratory epithelium. This step is mediated by a specialized organelle, which contains several proteins (cytadhesins) that have an important function in adherence. Two of these cytadhesins, P40 and P90, represent the proteolytic products from a single 130 kDa protein precursor, which is encoded by the MPN142 gene. Interestingly, MPN142 contains a repetitive DNA element, termed RepMP5, of which homologues are found at seven other loci within the M. pneumoniae genome. It has been hypothesized that these RepMP5 elements, which are similar but not identical in sequence, recombine with their counterpart within MPN142 and thereby provide a source of sequence variation for this gene. As this variation may give rise to amino acid changes within P40 and P90, the recombination between RepMP5 elements may constitute the basis of antigenic variation and, possibly, immune evasion by M. pneumoniae. To investigate the sequence variation of MPN142 in relation to inter-RepMP5 recombination, we determined the sequences of all RepMP5 elements in a collection of 25 strains. The results indicate that: (i) inter-RepMP5 recombination events have occurred in seven of the strains, and (ii) putative RepMP5 recombination events involving MPN142 have induced amino acid changes in a surface-exposed part of the P40 protein in two of the strains. We conclude that recombination between RepMP5 elements is a common phenomenon that may lead to sequence variation of MPN142-encoded proteins.

Coagulase-negative staphylococcal skin carriage among neonatal intensive care unit personnel: From population to infection (Article)

2010-11-01T00:00:00Z

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.

Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing (Article)

2010-09-01T00:00:00Z

The first choice antibiotics for treatment of Mycoplasma pneumoniae infections are macrolides. Several recent studies, however, have indicated that the prevalence of macrolide (ML)-resistance, which is determined by mutations in the bacterial 23S rRNA, is increasing among M. pneumoniae isolates. Consequently, it is imperative that ML-resistance in M. pneumoniae is rapidly detected to allow appropriate and timely treatment of patients. We therefore set out to determine the utility of pyrosequencing as a convenient technique to assess ML-resistance. In addition, we studied whether pyrosequencing could be useful for molecular typing of M. pneumoniae isolates. To this end, a total of four separate pyrosequencing assays were developed. These assays were designed such as to determine a short genomic sequence from four different sites, i.e. two locations within the 23S rRNA gene, one within the MPN141 (or P1) gene and one within the MPN528a gene. While the 23S rRNA regions were employed to determine ML-resistance, the latter two were used for molecular typing. The pyrosequencing assays were performed on a collection of 108 M. pneumoniae isolates. The ML-resistant isolates within the collection (n = 4) were readily identified by pyrosequencing. Moreover, each strain was correctly typed as either a subtype 1 or subtype 2 strain by both the MPN141 and MPN528a pyrosequencing test. Interestingly, two recent isolates from our collection, which were identified as subtype 2 strains by the pyrosequencing assays, were found to carry novel variants of the MPN141 gene, having rearrangements in each of the two repetitive elements (RepMP4 and RepMP2/3) within the gene. In conclusion, pyrosequencing is a convenient technique for ML-resistance determination as well as molecular typing of M. pneumoniae isolates.

The Mycoplasma genitalium MG352-encoded protein is a Holliday junction resolvase that has a non-functional orthologue in Mycoplasma pneumoniae (Article)

2010-09-01T00:00:00Z

Recombination between repeated DNA elements in the genomes of Mycoplasma species appears to lie at the basis of antigenic variation of several essential surface proteins. It is therefore imperative that the DNA recombinatorial pathways in mycoplasmas be unravelled. Here, we describe the proteins encoded by the Mycoplasma genitalium MG352 and Mycoplasma pneumoniae MPN528a genes (RecUMgeand RecUMpnrespectively), which share sequence similarity with RecU Holliday junction (HJ) resolvases. RecUMgewas found to: (i) bind HJ substrates and large double-stranded DNA molecules and (ii) cleave HJ substrates at the sequence 5′-G/TC↓C/TTA/GG- 3′ in the presence of Mn2+. Interestingly, RecUMpn(from M. pneumoniae subtype 2 strains) did not possess obvious DNA binding or cleavage activities, which was found to be caused by the presence of a glutamic acid residue at position 67 of the protein, which is not conserved in RecUMge. Additionally, RecUMpnappears not to be expressed by subtype 1 M. pneumoniae strains, as these possess a TAA translation termination codon at position 181-183 of MPN528a. We conclude that RecUMgeis a HJ resolvase that may play a central role in recombination in M. genitalium.

The Mycoplasma pneumoniae MPN490 and Mycoplasma genitalium MG339 genes encode RecA homologs that promote homologous DNA strand exchange (Article)

2009-11-01T00:00:00Z

The P1, P40, and P90 proteins of Mycoplasma pneumoniae and the MgPa and P110 proteins of Mycoplasma genitalium are immunogenic adhesion proteins that display sequence variation. Consequently, these proteins are thought to play eminent roles in immune evasive strategies. For each of the five proteins, a similar underlying molecular mechanism for sequence variation was hypothesized, i.e., modification of the DNA sequences of their respective genes. This modification is thought to result from homologous recombination of parts of these genes with repeat elements (RepMp and MgPar elements in M. pneumoniae and M. genitalium, respectively) that are dispersed throughout the bacterial genome. Proteins that are potentially involved in homologous DNA recombination have been suggested to be implicated in recombination between these repeat elements and thereby in antigenic variation. To investigate this notion, we set out to study the function of the RecA homologs that are encoded by the M. pneumoniae MPN490 and M. genitalium MG339 genes. Both proteins, which are 79% identical on the amino acid level, were found to promote recombination between homologous DNA substrates in an ATP-dependent fashion. The recombinational activities of both proteins were Mg2+and pH dependent and were strongly supported by the presence of single-stranded DNA binding protein, either from M. pneumoniae or from Escherichia coli. We conclude that the MPN490- and MG339-encoded proteins are RecA homologs that have the capacity to recombine homologous DNA substrates. Thus, they may play a central role in recombination between repetitive elements in both M. pneumoniae and M. genitalium. Copyright

The Mycoplasma pneumoniae MPN229 gene encodes a protein that selectively binds single-stranded DNA and stimulates Recombinase A-mediated DNA strand exchange (Article)

2008-10-31T00:00:00Z

Background. Mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. In spite of this genetic stability, homologous DNA recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, P1, of M. pneumoniae. In order to identify the proteins that may be involved in homologous DNA recombination in M. pneumoniae, we set out to characterize the MPN229 open reading frame (ORF), which bears sequence similarity to the gene encoding the single-stranded DNA-binding (SSB) protein of other micro-organisms. Results. The MPN229 ORF has the capacity to encode a 166-amino acid protein with a calculated molecular mass of 18.4 kDa. The amino acid sequence of this protein (Mpn SSB) is most closely related to that of the protein predicted to be encoded by the MG091 gene from Mycoplasma genitalium (61% identity). The MPN229 ORF was cloned, and different versions of Mpn SSB were expressed in E. coli and purified to > 95% homogeneity. The purified protein was found to exist primarily as a homo-tetramer in solution, and to strongly and selectively bind single-stranded DNA (ssDNA) in a divalent cation- and DNA substrate sequence-independent manner. Mpn SSB was found to bind with a higher affinity to ssDNA substrates larger than 20 nucleotides than to smaller substrates. In addition, the protein strongly stimulated E. coli Recombinase A (RecA)-promoted DNA strand exchange, which indicated that Mpn SSB may play an important role in DNA recombination processes in M. pneumoniae. Conclusion. The M. pneumoniae MPN229 gene encodes a protein, Mpn SSB, which selectively and efficiently binds ssDNA, and stimulates E. coli RecA-promoted homologous DNA recombination. Consequently, the Mpn SSB protein may play a crucial role in DNA recombinatorial pathways in M. pneumoniae. The results from this study will pave the way for unraveling these pathways and assess their role in antigenic variation of M. pneumoniae.

Pneumococcal carriage among indigenous Warao children in Venezuela: Serotypes, susceptibility patterns, and molecular epidemiology (Article)

2007-12-01T00:00:00Z

Little attention has been paid to pneumococcal carriage and disease in Amerindians from Latin America. The Warao people, an indigenous population from Venezuela, live in the delta of the Orinoco River in geographically isolated communities with difficult access to medical care. To obtain insight into pneumococcal carriage and the theoretical coverage of pneumococcal vaccines in this population, we investigated pneumococcal colonization, serotype, and genotype distribution among Warao children in 9 distinct, geographically isolated communities in the Delta Amacuro area in the northeast of Venezuela. From April 2004 through January 2005, a total of 161 Streptococcus pneumoniae isolates were recovered from single nasopharyngeal swab samples obtained from 356 children aged 0-72 months. The overall pneumococcal carriage rate was 49%, ranging from 13% to 76%, depending on the community investigated and the age of the children (50% among children aged <2 years and 25% among children aged >2 years). The most frequent serotypes were 23F (19.5% of isolates), 6A (19.5%), 15B (10.4%), 6B (9.1%), and 19F (7.2%). The theoretical coverage of the 7-valent pneumococcal conjugate vaccine, including the cross-reactive nonvaccine serotype 6A, was 65%. A total of 26% of the isolates were resistant to first-line antibiotics, with 70% of these strains being covered by the 7-valent pneumococcal conjugate vaccine. Restriction fragment end labelling analysis revealed 65 different genotypes, with 125 (80%) of the isolates belonging to 27 different genetic clusters, suggesting a high degree of horizontal spread of pneumococcal strains in and between the villages. The high colonization rates and high (registered) acute respiratory tract infection morbidity and mortality in this part of Venezuela suggest that Warao children are at increased risk for pneumococcal disease and, therefore, benefit from vaccination.

Clinical and molecular epidemiologic characteristics of coagulase-negative staphylococcal bloodstream infections in intensive care neonates (Article)

2007-07-01T00:00:00Z

OBJECTIVES: This study aimed to determine clinical characteristics of coagulase-negative staphylococcal (CoNS) sepsis in neonates, to assess the molecular epidemiology and biofilm forming properties of isolated strains, and to assess antibiotic susceptibility of clonal compared with incidentally occurring strains. METHODS: We performed a retrospective study on late-onset CoNS sepsis in infants in the neonatal intensive care unit of a Dutch university hospital in 2003. CoNS isolates were genotyped by restriction fragment end labeling and pulsed-field gel electrophoresis. Resistance profiles, biofilm production, and the presence of mecA and icaA were determined. RESULTS: Twenty-six percent of all 339 infants developed late-onset sepsis, 66% of these with CoNS sepsis. Eighty-six percent of all CoNS sepsis occurred in very low birth weight infants. Sixty-six CoNS strains were isolated. In multivariate analysis, small for gestational age and prolonged hospitalization were associated with CoNS sepsis. Among 3 restriction fragment end labeling clusters, we found 1 large cluster comprising 32% of the isolates. Biofilm producing Staphylococcus epidermidis were more frequently icaA positive than nonbiofilm formers (74% vs. 12%; P < 0.001). In other species, this association was not found. Nearly all isolates were resistant to antibiotics. MecA was present in 87% of the isolates. Multiresistance occurred in 77% of all strains and in 73% of clustered strains. There was significantly less multiresistance among the largest cluster. CONCLUSIONS: Small for gestational age and prolonged hospitalization were associated with CoNS sepsis. The icaA gene is a predictor for biofilm formation in S. epidermidis, but not in other species. Multiresistance is not associated with clonality.

Molecular epidemiology of pneumococcal colonization in response to pneumococcal conjugate vaccination in children with recurrent acute otitis media. (Article)

2005-01-01T00:00:00Z

A randomized double-blind trial with a 7-valent pneumococcal conjugate vaccine was conducted in The Netherlands among 383 children, aged 1 to 7 years, with a history of recurrent acute otitis media. No effect of vaccination on the pneumococcal colonization rate was found. However, a shift in serotype distribution was clearly observed (R. Veenhoven et al., Lancet 361:2189-2195, 2003). We investigated the molecular epidemiology of 921 pneumococcal isolates retrieved from both the pneumococcal vaccine (PV) and control vaccine (CV) groups during the vaccination study. Within individuals a high turnover rate of pneumococcal restriction fragment end labeling genotypes, which was unaffected by vaccination, was observed. Comparison of the genetic structures before and after completion of the vaccination scheme revealed that, despite a shift in serotypes, there was clustering of 70% of the pneumococcal populations. The remaining isolates (30%) were equally observed in the PV and CV groups. In addition, the degree of genetic clustering was unaffected by vaccination. However, within the population genetic structure, nonvaccine serotype clusters with the serotypes 11, 15, and 23B became predominant over vaccine-type clusters after vaccination. Finally, overall pneumococcal resistance was low (14%), and, albeit not significant, a reduction in pneumococcal resistance as a result of pneumococcal vaccination was observed. Molecular surveillance of colonization in Dutch children shows no effect of pneumococcal conjugate vaccination on the degree of genetic clustering and the genetic structure of the pneumococcal population. However, within the genetic pneumococcal population structure, a clear shift toward nonvaccine serotype clusters was observed.

Host-pathogen interaction during pneumococcal infection in patients with chronic obstructive pulmonary disease (Article)

2004-01-01T00:00:00Z

Acute exacerbation is a frequent complication of chronic obstructive pulmonary disease (COPD). Recent studies suggested a role for bacteria such as Streptococcus pneumoniae in the development of acute exacerbation. For this study, we investigated the following in COPD patients: (i) the epidemiology of pneumococcal colonization and infection, (ii) the effect of pneumococcal colonization on the development of exacerbation, and (iii) the immunological response against S. pneumoniae. We cultured sputa of 269 COPD patients during a stable state and during exacerbation of COPD and characterized 115 pneumococcal isolates by use of serotyping. Moreover, we studied serum immunoglobulin G (IgG) antibody titers, antibody avidities, and functional antibody titers against the seven conjugate vaccine serotypes in these patients. Colonization with only pneumococci (monocultures) increased the risk of exacerbation, with a hazard ratio of 2.93 (95% confidence interval, 1.41 to 6.07). The most prevalent pneumococcal serotypes found were serotypes 19F, 3, 14, 9L/N/V, 23A/B, and 11. We calculated the theoretical coverage for the 7- and 11-valent pneumococcal vaccines to be 60 and 73%, respectively. All patients had detectable IgG levels against the seven conjugate vaccine serotypes. These antibody titers were significantly lower than those in vaccinated healthy adults. Finally, on average, a 2.5-fold rise in serotype-specific and functional antibodies in S. pneumoniae-positive sputum cultures was observed during exacerbation. Our data indicate that pneumococcal colonization in COPD patients is frequently caused by vaccine serotype strains. Moreover, pneumococcal colonization is a risk factor for exacerbation of COPD. Finally, our findings demonstrate that COPD patients are able to mount a significant immune response to pneumococcal infection. COPD patients may therefore benefit from pneumococcal vaccination.

Molecular epidemiology of penicillin-susceptible non-beta-lactam-resistant Streptococcus pneumoniae isolates from Greek children (Article)

2003-01-01T00:00:00Z

A total of 128 Streptococcus pneumoniae isolates that were susceptible to penicillin but resistant to non-beta-lactam agents were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and antibiotic resistance genotyping. The serotypes 6A/B (49%), 14 (14%), 19A/F (11%), 11A (9%), 23A/F (4%), 15B/C (2%), and 21 (2%) were most prevalent in this collection. Of the isolates, 65% were erythromycin resistant, while the remaining isolates were tetracycline and/or trimethoprim-sulfamethoxazole resistant. Fifty-nine distinct RFEL types were identified. Twenty different RFEL clusters, harboring 2 to 19 strains each, accounted for 76% of all strains. Confirmatory multilocus sequence typing analysis of the genetic clusters showed the presence of three international clones (Tennessee(23F)-4, England(14)-9, and Greece(6B)-22) representing 30% of the isolates. The erm(B) gene was present in 70% of the erythromycin-resistant isolates, whereas 18 and 8% contained the mef(A) and mef(E) genes, respectively. The pneumococci representing erm(B), erm(A), and mef genes belonged to distinct genetic clusters. In total, 45% of all isolates were tetracycline resistant. Ninety-six percent of these isolates contained the tet(M) gene. In conclusion, penicillin-susceptible pneumococci resistant to non-beta-lactams are a genetically heterogeneous group displaying a variety of genotypes, resistance markers, and serotypes. This suggests that multiple genetic events lead to non-beta-lactam-resistant pneumococci in Greece. Importantly, most of these genotypes are capable of disseminating within the community.

Molecular epidemiology of pneumococcal carriage among children with upper respiratory tract infections in Hanoi, Vietnam (Article)

2002-01-01T00:00:00Z

To investigate the molecular epidemiology of pneumococcal nasopharyngeal carriage in Hanoi, Vietnam, we studied 84 pneumococcal strains retrieved from children with upper respiratory tract infections. Serotypes 23F (32%), 19F (21%), 6B (13%), and 14 (10%) were found most often. A significant number of strains were antibiotic resistant. Fifty-two percent of the strains were (intermediate) resistant to penicillin, 87% were (intermediate) resistant to co-trimoxazole, 76% were resistant to tetracycline, 73% were resistant to erythromycin, and 39% were (intermediate) resistant to cefotaxime. Seventy-five percent were resistant to three or more classes of antibiotics. A high degree of genetic heterogeneity among the penicillin resistance genes was observed. In addition, the tetracycline resistance gene tet(M) and the erythromycin resistance gene erm(B) were predominantly observed among the isolates. Molecular analysis of the 84 isolates by restriction fragment end labeling (RFEL) revealed 35 distinct genotypes. Twelve of these genotypes represented a total of eight genetic clusters with 61 isolates (73%). The two largest clusters contained 24 and 12 isolates, and the isolates in those clusters were identical to the two internationally spreading multidrug-resistant clones Spain 23F-1 and Taiwan 19F-14, respectively. The remaining RFEL types were Vietnam specific, as they did not match the types in our reference collection of 193 distinct RFEL types from 16 countries. Furthermore, 57 of the 61 horizontally spreading isolates (93%) in the eight genetic clusters were covered by the seven-valent conjugate vaccine, whereas this vaccine covered only 43% of the isolates with unique genotypes. According to the serotype distribution of the nasopharyngeal pneumococcal isolates, this study suggests a high potential benefit of the seven-valent pneumococcal conjugate vaccine for children in Hanoi.

The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses (Article)

2000-01-01T00:00:00Z

Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl cis/trans isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of ppmA. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal ppmA-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines.

Genetic relatedness within serotypes of penicillin-susceptible Streptococcus pneumoniae isolates (Article)

2000-01-01T00:00:00Z

The molecular epidemiological characteristics of all Streptococcus pneumoniae strains isolated in a nationwide manner from patients with meningitis in The Netherlands in 1994 were investigated. Restriction fragment end labeling analysis demonstrated 52% genetic clustering among these penicillin-susceptible strains, a value substantially lower than the percentage of clustering among Dutch penicillin-nonsusceptible strains. Different serotypes were found within 8 of the 28 genetic clusters, suggesting that horizontal transfer of capsular genes is common among penicillin-susceptible strains. The degree of genetic clustering was much higher among serotype 3, 7F, 9V, and 14 isolates than among isolates of other serotypes, i.e., 6A, 6B, 18C, 19F, and 23F. We further studied the molecular epidemiological characteristics of pneumococci of serotype 3, which is considered the most virulent serotype and which is commonly associated with invasive disease in adults. Fifty epidemiologically unrelated penicillin-susceptible serotype 3 invasive isolates originating from the United States (n = 27), Thailand (n = 9), The Netherlands (n = 8), and Denmark (n = 6) were analyzed. The vast majority of the serotype 3 isolates (74%) belonged to two genetically distinct clades that were observed in the United States, Denmark, and The Netherlands. These data indicate that two serotype 3 clones have been independently disseminated in an international manner. Seven serotype 3 isolates were less than 85% genetically related to the other serotype 3 isolates. Our observations suggest that the latter isolates originated from horizontal transfer of the capsular type 3 gene locus to other pneumococcal genotypes. In conclusion, epidemiologically unrelated serotype 3 isolates were genetically more related than those of other serotypes. This observation suggests that serotype 3 has evolved only recently or has remained unchanged over long periods.

Multidrug-resistant Streptococcus pneumoniae in Poland: identification of emerging clones (Article)

1999-01-01T00:00:00Z

Penicillin resistance among Streptococcus pneumoniae isolates has rapidly emerged in Poland during the last decade and has reached prevalence levels of up to 14.4% in 1997. In order to investigate the nature of this increase, a molecular epidemiological analysis of non-penicillin-susceptible multidrug-resistant pneumococci isolated in 1995 and 1996 was conducted. Thirty-seven patients who suffered mainly from upper respiratory tract infections and pneumococcal pneumonia were enrolled in this study. The medical centers to which the patients were admitted were located in 16 Polish towns across the country. Eight distinct BOX PCR types were observed, representing 14 subtypes. Restriction fragment end labeling (RFEL) analysis divided the pneumococcal strains into 16 distinct types. By combining the BOX PCR and RFEL data, four genetically distinct clusters of strains were identified. Two clusters represented the genetic clones 23F and 9V, which have recently emerged all over the world. The two other genetic clusters, which represented serotypes 23F and 6B, clearly predominated in the analyzed collection of Polish non-penicillin-susceptible pneumococcal strains. Since the latter clusters did not match any of the 133 RFEL types of non-penicillin-susceptible pneumococci collected in 15 other countries, their Polish clonal origin is most likely.

Molecular characterization of pneumococcal nasopharynx isolates collected from children during their first 2 years of life (Article)

1998-01-01T00:00:00Z

Pneumococcal colonization was studied in 19 children monitored from birth through the age of 2 years. For this purpose, pneumococcal isolates were characterized by capsular typing, restriction fragment end labeling (RFEL), and penicillin-binding protein (PBP) genotyping. Fifty-eight isolates were collected and were found to belong to 10 capsular types, 31 RFEL types, and 7 PBP genotypes. Thirty-nine percent of the isolates had reduced susceptibility to penicillin. All seven highly resistant strains (MICs, > 1 microgram/ml) were identical to the pandemic clone 23F. Children were culture positive between one and eight times at 13 scheduled visits. Although the infants were frequently recolonized with different strains, colonization with one particular strain often persisted for several months. Isolation of a previously detected capsular type was common, and the chromosomal homogeneity tended to be high when it occurred. Horizontal transfer of capsular genes between strains of different RFEL types was demonstrated in one child. The ecological advantage of transfer of capsular genes is unclear unless survival of the organism on a mucosal surface may be linked to immunoprotective pressure against particular capsular types.

Novel BOX repeat PCR assay for high-resolution typing of Streptococcus pneumoniae strains (Article)

1996-01-01T00:00:00Z

Typing data obtained by specifically targeting a single, high-stringency PCR at the pneumococcal BOX repeat element for 28 strains of Streptococcus pneumoniae completely corroborated the resolutions attained by five genotypic procedures as described by Hermans et al. (P.W.M. Hermans, M. Sluijter, T. Hoogenboezem, H. Heersma, A. van Belkum, and R. de Groot, J. Clin. Microbiol. 33:1606-1612, 1995). All pairs of strains, except one, derived from both the cerebrospinal fluid and blood of the same individual were shown to be identical. Moreover, other, epidemiologically unrelated isolates were demonstrated to be unique. Considering the combined data from the five typing techniques applied previously as the "gold standard," the single BOX PCR test demonstrated excellent resolving powers while maintaining epidemiological linkage.

Genetic characterization of trimethoprim resistance in Haemophilus influenzae (Article)

1996-01-01T00:00:00Z

We previously demonstrated that trimethoprim (Tmp) resistance in Haemophilus influenzae is mediated by chromosomally encoded dihydrofolate reductase (DHFR) with a modified primary structure and distinct kinetic properties. To gain insight into the relationship of the DHFR structure and the level of Tmp resistance that it confers on the host bacterium, we cloned and characterized the folH genes of one Tmp-susceptible and two Tmp-resistant H. influenzae strains. Differences were observed between Tmp-susceptible and Tmp-resistant isolates both in the promoter region and in the coding sequences. The effect of differences between H. influenzae folH genes on Tmp susceptibility was investigated in Escherichia coli. Various folH gene hybrids were constructed, and their influence on Tmp susceptibility was determined. Resistance in E. coli mediated by folH from H. influenzae strain R1047 was associated with alterations in the promoter and the central part of folH. In contrast, the E. coli Tmp resistance phenotype associated with the folH gene of H. influenzae R1042 was characterized by alterations in one or more of three amino acid residues at the C-terminal part of the protein. These data indicate that Tmp resistance is not only related to alterations in the promoter region of the folH gene and the Tmp binding domains at the N-terminal and central part of DHFR. Alterations in the C-terminal part may also cause Tmp resistance, probably as a result of a change in secondary structure and the subsequent loss of Tmp binding affinity.

Comparative study of five different DNA fingerprint techniques for molecular typing of Streptococcus pneumoniae strains (Article)

1995-01-01T00:00:00Z

The aim of this study was to identify the strengths and weaknesses of five DNA fingerprint methods for epidemiological typing of Streptococcus pneumoniae. We investigated the usefulness of (i) ribotyping, (ii) BOX fingerprinting with the BOX repetitive sequence of S. pneumoniae as a DNA probe, (iii) PCR fingerprinting with a primer homologous to the enterobacterial repetitive intergenic consensus sequence, (iv) pulsed-field gel electrophoresis of large DNA fragments, and (v) restriction fragment end labeling to detect restriction fragment length polymorphism of small DNA fragments. Twenty-eight S. pneumoniae strains isolated from the blood and/or cerebrospinal fluid of 21 patients were analyzed. Genetic clustering among the 28 strains was independent of the DNA fingerprint technique used. However, the discriminatory power and the similarity values differed significantly among the individual techniques. BOX fingerprinting, pulsed-field gel electrophoresis, and restriction fragment end labeling provided the highest degree of discriminatory power. Furthermore, the ease with which computerized fingerprint analysis could be conducted also varied significantly among the techniques. Ribotyping, BOX fingerprinting, and restriction fragment end labeling were very suitable techniques for accurate computerized data analysis. Because of their high discriminatory potential and ease of accurate analysis, we conclude that BOX fingerprinting and restriction fragment end labeling are the most suitable techniques to type pneumococcal strains.