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C G Dowson

Publications and source records attributed to C G Dowson.

At least 19 recordsLinked to original sources
PubMed2005

Multilocus sequence typing of Staphylococcus aureus isolated from high-somatic-cell-count cows and the environment of an organic dairy farm in the United Kingdom.

An outbreak of Staphylococcus aureus intramammary infections on an organic dairy farm was monitored for 10 months. Environmental and milk samples were collected from a total of 26 cows and a group of 21 purchased heifers about to be introduced into the milking herd. There was variation in the rate of isolation of S. aureus (9.5 to 43.8%) from individual mammary quarters, although no S. aureus isolates were detected in the milk samples collected from the heifers. One hundred ninety-one S. aureus isolates were detected from cow milk samples (n = 182), milking machine clusters (n = 4), farm personnel (n = 4), and the environment (n = 1). Multilocus sequence typing (MLST) had a typeability of 100% when it was applied to the 191 isolates. Among the 191 isolates there was limited strain diversity, with seven sequence types (STs) dominated by two strains with closely related STs that differed at a single locus. Within individual mammary quarters there were naturally occurring dual infections, although this was identified in only 0.4% of milk samples. Different strains were associated with variable persistence within quarters. MLST is clearly a very suitable tool for the differentiation and analysis of S. aureus populations detected on dairy cattle farms.

Animals↗
PubMed2005

Multilocus sequence typing of intercontinental bovine Staphylococcus aureus isolates.

A total of 258 bovine-associated Staphylococcus aureus isolates from the United States, Chile, and the United Kingdom, plus the reference isolate S. aureus Newbould 305 (NCIMB 702892), were analyzed by multilocus sequence typing (MLST). A collection of previously characterized United Kingdom isolates were also included in the analysis. The results demonstrated that MLST is suitable for the differentiation of bovine S. aureus isolates from various sites (milk, teat skin, milking machine unit liners, hands, and bedding) and countries. The theory of the host specificity of S. aureus is supported by the detection of a previously undescribed clonal complex that comprised 87.4% of the isolates studied, with representatives from all geographic locations investigated. This suggests that a single clonal group has achieved a widespread distribution and is responsible for the majority of infections. Some sequence types (STs; ST25, ST115, ST124, and ST126) demonstrated site specificity, as they were significantly (P < 0.05) associated with milk or teat skin.

Animals↗
PubMed2001

Spontaneous sequence duplication within an open reading frame of the pneumococcal type 3 capsule locus causes high-frequency phase variation.

The molecular genetic basis of high-frequency serotype 3 capsule phase variation in Streptococcus pneumoniae (the pneumococcus) was investigated. Pneumococci were grown in sorbarod biofilms at 34 degrees C to mimic nasopharyngeal carriage. Different type 3 pneumococci commonly associated with invasive disease generated apparently random tandem duplications of 11-239 bp segments within the cap3A gene of the type 3 capsule locus. These duplications alone were found to be responsible for high-frequency capsule phase variation, in which (phase off) acapsular variants possessed duplications within cap3A, and (phase on) capsular revertants possessed wild-type cap3A genes, indicating the precise excision of the duplication. Additionally, the frequency of phase reversion (off to on) was found to exhibit a linear relationship between (log) frequency of reversion and (log) length of duplication. This apparently random duplication giving rise to phase variation is in stark contrast to the 'preprogrammed' contingency genes in many Gram-negative organisms that possess homopolymeric sequence repeats or motifs for site-specific recombination.

Bacterial Capsules↗
PubMed2001

Distribution and genetic diversity of suilysin in Streptococcus suis isolated from different diseases of pigs and characterization of the genetic basis of suilysin absence.

Streptococcus suis is an economically important pathogen of pigs responsible for a variety of diseases including meningitis, septicemia, arthritis, and pneumonia, although little is known about the mechanisms of pathogenesis or virulence factors associated with this organism. Here, we report on the distribution and genetic diversity of the putative virulence factor suilysin, a member of the thiol-activated toxin family of gram-positive bacteria. On the basis of PCR analysis of over 300 isolates of S. suis, the suilysin-encoding gene, sly, was detected in 69.4% of isolates. However, sly was present in a considerably higher proportion of isolates obtained from cases of meningitis, septicemia, and arthritis (>80%) and isolates obtained from asymptomatic tonsillar carriage (>90%) than lung isolates associated with pneumonia (44%). With the exception of serotypes 1, 14, and 1/14, there was no strong correlation between the presence of suilysin and serotype. Analysis of the genetic diversity of suilysin by restriction fragment length polymorphism and sequence analysis found that the suilysin gene, where present, is highly conserved with a maximum of 1.79% diversity at the nucleotide level seen between sly alleles. Assays of hemolytic activity and hybridization analysis provided no evidence for a second member of the thiol-activated toxin family in S. suis. Inverse PCR was used to characterize regions flanking sly, which in turn allowed the first characterization of the equivalent region in a strain lacking sly. Sequence comparison of these regions from sly-positive (P1/7) and sly-negative (DH5) strains indicated that two alternative arrangements are both flanked by genes with highest similarity to haloacid dehalogenase-like hydrolases (5' end) and putative N-acetylmannosamine-6-phosphate epimerases (3' end). However, sly appears to be completely absent from the alternative arrangement, and a gene of unknown function is located in the equivalent position. Finally, PCR analysis of multiple sly-positive and -negative strains indicated that these two alternative genetic arrangements are conserved among many S. suis isolates.

Amino Acid Sequence↗
PubMed2000

Expression of resistance to tetracyclines in strains of methicillin-resistant Staphylococcus aureus.

A diverse collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates resistant to tetracycline was screened by PCR for the presence of the resistance determinants tetK, tetL, tetM or tetO. Twenty-four of 66 isolates had tetM alone, 21 had tetK alone and 21 had both tetK and tetM (tetKM). All isolates were tetL- and tetO-negative. MICs of tetracycline, doxycycline and minocycline were evaluated for all isolates with or without preincubation in the presence of subinhibitory concentrations of tetracycline or minocycline. All isolates with one or more tetracycline resistance determinants were resistant to tetracycline 8 mg/L without induction of resistance. Some MRSA isolates of each of these three genotypes showed an unexpected lack of resistance to tetracyclines when the disc diffusion or agar dilution method was applied to uninduced cells. Resistance to tetracycline and doxycycline was greater (two- to four-fold) in tetK cells preincubated with tetracycline (tetK MRSA isolates were susceptible to minocycline </=0.25 mg/L under all conditions tested). For isolates with tetM alone, preincubation with tetracycline or minocycline gave up to a four-fold increase in the level of resistance to doxycycline and minocycline. Induction of doxycycline and minocycline resistance was clearly observed for tetKM isolates when cells were preincubated with minocycline. This study suggests that, despite the results of susceptibility testing, all tetracycline-resistant S. aureus isolates should be treated as resistant to doxycycline, and all tetM-positive isolates should be treated as resistant to all tetracyclines. A double disc diffusion method has been developed to identify inducible resistance to minocycline and to distinguish between tetK, tetM and tetKM isolates.

Genes, Bacterial↗
PubMed2000

Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae.

The aim of the present study was to examine the stability and evolution of tet(M)-mediated resistance to tetracyclines among members of different clonal lineages of Streptococcus pneumoniae. Thirty-two tetracycline-resistant isolates representing three national (Spanish serotype 14, Spanish serotype 15, and Polish serotype 23F) and one international (Spanish serotype 23F) multidrug-resistant epidemic clones were all found to be tet(M) positive and tet(O), tet(K), and tet(L) negative. These isolates all carried the integrase gene, int, which is associated with the Tn1545-Tn916 family of conjugative transposons. High-resolution restriction analysis of tet(M) products identified six alleles, tet(M)1 to tet(M)6: tet(M)1 to tet(M)3 and tet(M)5 in isolates of the Spanish serotype 14 clone, tet(M)4 in both the Spanish serotype 15 and 23F clones, and tet(M)6, the most divergent allele, in the Polish 23F clone. This indicates that tet(M) variation can occur at the inter- and intraclone levels in pneumococci. Two alleles of int were identified, with int1 being found in all isolates apart from members of the international Spanish 23F clone, which carried int2. Susceptibility to tetracycline, doxycycline, and minocycline was evaluated for all isolates with or without preincubation in the presence of subinhibitory concentrations of tetracyclines. Resistance to tetracyclines was found to be inducible in isolates of all clones; however, the strongest induction was observed in the Spanish serotype 15 and 23F clones carrying tet(M)4. Tetracycline was found to be the strongest inducer of resistance, and minocycline was found to be the weakest inducer of resistance.

Bacterial Proteins↗
PubMed2000

Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of "Atypical" pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes.

The oral streptococcal group (mitis phylogenetic group) currently consists of nine recognized species, although the group has been traditionally difficult to classify, with frequent changes in nomenclature over the years. The pneumococcus (Streptococcus pneumoniae), an important human pathogen, is traditionally distinguished from the most closely related oral streptococcal species Streptococcus mitis and Streptococcus oralis on the basis of three differentiating characteristics: optochin susceptibility, bile solubility, and agglutination with antipneumococcal polysaccharide capsule antibodies. However, there are many reports in the literature of pneumococci lacking one or more of these defining characteristics. Sometimes called "atypical" pneumococci, these isolates can be the source of considerable confusion in the clinical laboratory. Little is known to date about the genetic relationships of such organisms with classical S. pneumoniae isolates. Here we describe these relationships based on sequence analysis of housekeeping genes in comparison with previously characterized isolates of S. pneumoniae, S. mitis, and S. oralis. While most pneumococci were found to represent a closely related group these studies identified a subgroup of atypical pneumococcal isolates (bile insoluble and/or "acapsular") distinct from, though most closely related to, the "typical" pneumococcal isolates. However, a large proportion of isolates, found to be atypical on the basis of capsule reaction alone, did group with typical pneumococci, suggesting that they have either lost capsule production or represent as-yet-unrecognized capsular types. In contrast to typical S. pneumoniae, isolates phenotypically identified as S. mitis and S. oralis, which included isolates previously characterized in taxonomic studies, were genetically diverse. While most of the S. oralis isolates did fall into a well-separated group, S. mitis isolates did not cluster into a well-separated group. During the course of these studies we also identified a number of potentially important pathogenic isolates, which were frequently associated with respiratory disease, that phenotypically and genetically are most closely related to S. mitis but which harbor genes encoding the virulence determinants pneumolysin and autolysin classically associated with S. pneumoniae.

Bacterial Proteins↗
PubMed2000

Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation.

Interspecies genetic exchange is an important evolutionary mechanism in bacteria. It allows rapid acquisition of novel functions by transmission of adaptive genes between related species. However, the frequency of homologous recombination between bacterial species decreases sharply with the extent of DNA sequence divergence between the donor and the recipient. In Bacillus and Escherichia, this sexual isolation has been shown to be an exponential function of sequence divergence. Here we demonstrate that sexual isolation in transformation between Streptococcus pneumoniae recipient strains and donor DNA from related strains and species follows the described exponential relationship. We show that the Hex mismatch repair system poses a significant barrier to recombination over the entire range of sequence divergence (0.6 to 27%) investigated. Although mismatch repair becomes partially saturated, it is responsible for 34% of the observed sexual isolation. This is greater than the role of mismatch repair in Bacillus but less than that in Escherichia. The remaining non-Hex-mediated barrier to recombination can be provided by a variety of mechanisms. We discuss the possible additional mechanisms of sexual isolation, in view of earlier findings from Bacillus, Escherichia, and Streptococcus.

Base Pair Mismatch↗
PubMed1999

Molecular characterization of equine isolates of Streptococcus pneumoniae: natural disruption of genes encoding the virulence factors pneumolysin and autolysin.

Although often considered a strict human pathogen, Streptococcus pneumoniae has been reported to infect and cause pneumonia in horses, although the pathology appears restricted compared to that of human infections. Here we report on the molecular characterization of a group of S. pneumoniae isolates obtained from horses in England and Ireland. Despite being obtained from geographically distinct locations, the isolates were found to represent a tight clonal group, virtually identical to each other but genetically distinguishable from more than 120 divergent isolates of human S. pneumoniae. A comprehensive analysis of known pneumococcal virulence determinants was undertaken in an attempt to understand the pathogenicity of equine pneumococci. Surprisingly, equine isolates appear to lack activities associated with both the hemolytic cytotoxin pneumolysin, often considered a major virulence factor of pneumococci, and the major autolysin gene lytA, also considered an important virulence factor. In support of phenotypic data, molecular studies demonstrated a deletion of parts of the coding sequences of both lytA and ply genes in equine pneumococci. The implications of these findings for the evolution and pathogenicity of equine S. pneumoniae are discussed.

Animals↗
PubMed1999

The autolysin-encoding gene (lytA) of Streptococcus pneumoniae displays restricted allelic variation despite localized recombination events with genes of pneumococcal bacteriophage encoding cell wall lytic enzymes.

The lytA-encoded autolysin (N-acetylmuramoyl-L-alanine amidase) of Streptococcus pneumoniae is believed to play an important role in the pathogenesis of pneumococcal infection and has been identified as a putative vaccine target. Allelic diversity of lytA in an extensive collection of clinical isolates was assessed by restriction fragment length polymorphism and confirmatory sequencing studies. Genetic diversity within lytA is limited, especially compared to the high levels of diversity seen in other pneumococcal virulence factor genes, although small blocks generating mosaic structure were identified. Sequence comparisons with genes encoding cell wall lytic enzymes of pneumococcal bacteriophage suggest that localized recombination events have occurred between host lytA and these bacteriophage genes. These results confirm earlier suggestions that recombination between DNA encoding bacteriophage autolytic enzymes and chromosomally encoded lytA might be important in the evolution of lytA. The implications of these findings for understanding the evolution of lytA and the potential utility of LytA as a vaccine target are discussed.

Alleles↗
PubMed1999

Genetic diversity of the streptococcal competence (com) gene locus.

The com operon of naturally transformable streptococcal species contains three genes, comC, comD, and comE, involved in the regulation of competence. The comC gene encodes a competence-stimulating peptide (CSP) thought to induce competence in the bacterial population at a critical extracellular concentration. The comD and comE genes are believed to encode the transmembrane histidine kinase and response regulator proteins, respectively, of a two-component regulator, with the comD-encoded protein being a receptor for CSP. Here we report on the genetic variability of comC and comD within Streptococcus pneumoniae isolates. Comparative analysis of sequence variations of comC and comD shows that, despite evidence for horizontal gene transfer at this locus and the lack of transformability of many S. pneumoniae strains in the laboratory, there is a clear correlation between the presence of a particular comC allele and the cognate comD allele. These findings effectively rule out the possibility that the presence of noncognate comC and comD alleles may be responsible for the inability to induce competence in many isolates and indicate the importance of a functional com pathway in these isolates. In addition, we describe a number of novel CSPs from disease-associated strains of S. mitis and S. oralis. The CSPs from these isolates are much more closely related to those from S. pneumoniae than to most CSPs previously reported from S. mitis and S. oralis, suggesting that these particular organisms may be a potential source of DNA in recombination events generating the mosaic structures commonly reported in genes of S. pneumoniae that are under strong selective pressure.

Alleles↗
PubMed1998

Molecular evolution of rifampicin resistance in Streptococcus pneumoniae.

Rifampicin resistance has arisen in several different species of bacteria because of alterations to one or more regions in the target of the antibiotic, the beta-subunit of RNA polymerase encoded by rpoB. Nucleotide sequence analysis of a 270 bp fragment of rpoB from 16 clinical rifampicin-susceptible isolates of Streptococcus pneumoniae, 8 clinical rifampicin-resistant isolates, and 3 spontaneous rifampicin-resistant mutants, has revealed that, as with previously examined species, point mutations within the cluster I region of rpoB, at sites encoding Asp516 and HiS526, also confer resistance to rifampicin in this important human pathogen. Moreover, the residues within cluster I, that were altered within the rifampicin-resistant mutants of S. pneumoniae, were in the same position as those previously found to alter in resistant isolates of Escherichia coli and Mycobacterium tuberculosis. Sequence analysis of rpoB, both from these isolates of S. pneumoniae and from two strains of S. mitis, reveals that, among a number of clinical isolates, resistance to rifampicin in S. pneumoniae has arisen by point mutation. However, the nucleotide sequence of rpoB from one isolate examined suggests that interspecies gene transfer may also have played a role in the evolution of rifampicin-resistance in S. pneumoniae.

Amino Acid Sequence↗
PubMed1996

The tetracycline resistance gene tet(M) exhibits mosaic structure.

Tetracycline resistance genes of the M class, tet(M), are typically found on mobile genetic elements as the conjugative transposons of gram-positive bacteria. By comparing the sequences of eight different tet(M) genes (from Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Ureaplasma urealyticum, and Neisseria), a mosaic structure was detected which could be traced to two distinct alleles. The two alleles displayed a divergence of 8% and a different G/C content. The block structure of these genes provides evidence for the contribution of homologous recombination to the evolution and the heterogeneity of the tet(M) locus. Unlike described cases of chromosomally located mosaic loci, tet(M) is a relatively recently acquired determinant in the species examined and it would appear that mosaic structure within tet(M) has evolved after acquisition of the gene by the mobile genetic elements upon which it is located.

Bacterial Proteins↗
PubMed1995

Homeologous recombination and mismatch repair during transformation in Streptococcus pneumoniae: saturation of the Hex mismatch repair system.

The ability of the Hex generalized mismatch repair system to prevent recombination between partially divergent (also called homeologous) sequences during transformation in Streptococcus pneumoniae was investigated. By using as donor in transformation cloned fragments 1.7-17.5% divergent in DNA sequence from the recipient, it was observed that the Hex system prevents chromosomal integration of the least and the most divergent fragments but frequently fails to do so for other fragments. In the latter case, the Hex system becomes saturated (inhibited) due to an excess of mismatches: it is unable to repair a single mismatch located elsewhere on the chromosome. Further investigation with chromosomal donor DNA, carrying only one genetically marked divergent region, revealed that a single divergent fragment can lead to saturation of the Hex system. Increase in cellular concentration of either HexA, the MutS homologue that binds mismatches, or HexB, the MutL homologue for which the essential role in repair as yet remains obscure, was shown to restore repair ability in previously saturating conditions. Investigation of heterospecific transformation by chromosomal DNA from two related streptococcal species, Streptococcus oralis and Streptococcus mitis, also revealed complete saturation of the Hex system. Therefore the Hex system is not a barrier to interspecies recombination in S. pneumoniae. These results are discussed in light of those described for the Mut system of Escherichia coli.

Adenosine Triphosphatases↗
PubMed1995

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae.

Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 micrograms benzylpenicillin ml-1. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.

Bacterial Proteins↗
PubMed1995

Genetics and molecular biology of beta-lactam-resistant pneumococci.

Penicillin-resistant pneumococci have been reported with increasing frequency in recent years. Isolates with high-level resistance are now found in many countries, and in some countries they constitute a substantial proportion of all isolates. A worrying development is the recent emergence of pneumococci with high-level resistance to third-generation cephalosporins. Resistance to beta-lactam antibiotics in pneumococci is due entirely to the development of altered forms of the high-molecular-weight penicillin-binding proteins (PBPs) that have decreased affinity for the antibiotics. High-level resistance to third-generation cephalosporins has occurred by the development of altered forms of PBP1a and 2x, whereas high-level penicillin resistance additionally requires alterations of PBP2b. Altered PBPs are encoded by mosaic genes that have emerged by recombinational events between the pbp genes of pneumococci and their homologs in closely related streptococcal species. Horizontal gene transfer, presumably mediated by genetic transformation, has also resulted in the dissemination of altered pbp genes, and possibly capsular biosynthetic genes, between different pneumococcal lineages to produce new resistant clones.

Aminoacyltransferases↗