Biomedical subjects
Publications and source records attributed to Mark C Enright.
We evaluated three multilocus sequence typing (MLST) schemes for Staphylococcus epidermidis and selected the seven most discriminatory loci for the formation of a new, more powerful MLST scheme. This improved scheme gave 31 sequence types (STs) and 5 clonal complexes (CCs), whereas the other schemes delineate 16 to 24 STs and 1 to 3 CCs.
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The control of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) infection is of concern worldwide. Given the evidence that several pathogenic species replicate within amoebae and emerge more virulent and more resistant and the abundance of amoebae in healthcare settings, we investigated interactions of Acanthamoeba polyphaga with epidemic MRSA isolates. MRSA proliferated in the presence of amoebae, attributable partly to intracellular replication. Following 24 h of co-culture, confocal microscopy revealed that c. 50% amoebae had viable MRSA within phago-lysosomes and 2% of amoebae were heavily infected with viable cocci throughout the cytoplasm. Infection control strategies should recognize the contribution of protozoa.
Methicillin-resistant Staphylococcus aureus (MRSA) clones harboring the toxic shock syndrome toxin 1 (tst) gene have been detected in France and in Switzerland since 2002. During a passive survey conducted between 2002 and 2003, we collected 103 tst-positive S. aureus isolates from 42 towns in France, of which 27 were resistant to methicillin. The tst-positive MRSA belonged to two clones: a major clone comprising 25 isolates of sequence type (ST) 5 and agr group 2 and a minor clone comprising two isolates of ST30 and agr3. The tst-positive MRSA clones were associated with both hospital-acquired (12 cases) and community-acquired (8 cases) infections. The MRSA clones were mainly isolated from children (overall median age, 3 years). They caused a variety of clinical syndromes, including toxic shock syndrome and suppurative infections. Both clones were found to harbor a type IV staphylococcal chromosomal cassette mec (SCCmec) and to have similar antibiotic resistance profiles (usually resistant to oxacillin, kanamycin, and tobramycin and with intermediate resistance to fusidic acid). The origin of these clones is unclear. The tst-positive agr2 MRSA clone has the same sequence type (ST5) of two pandemic nosocomial MRSA clones, namely, the Pediatric clone and the New York/Japan clone. These findings suggest that all these clones are phylogenetically related. The pulsotype of the tst-positive MRSA clones differed from that of methicillin-sensitive S. aureus (MSSA) clones by a single band involving the SCCmec element. These findings suggest that the tst-positive MRSA clones may have emerged from their respective MSSA counterparts.
OBJECTIVES: To determine the efficacy of a novel antimicrobial compound, AQ+, against a genetically heterogeneous collection comprising 213 Staphylococcus aureus isolates from global sources. AQ+ is an aqueous preparation containing 0.5% 8-hydroxyquinoline. METHODS: MICs were found for all the isolates tested using the BSAC microdilution method. Time-kill studies were performed according to NCCLS guidelines. Transmission electron microscopy (TEM) was used to view the ultrastructural effects of AQ+. RESULTS: AQ+ was shown to strongly inhibit the growth of all isolates with a median MIC of 0.25% at a pH optimum of 9.2. Lowering the pH to 7.5 gave an approximately 4-fold reduction in efficacy and at pH 5.5 there was an approximately 8-fold reduction in efficacy. Methicillin-resistant S. aureus (MRSA) as well as vancomycin-intermediate S. aureus were shown to be as equally susceptible to AQ+ as methicillin-susceptible S. aureus. Time-kill curves for AQ+ were similar to those for gentamicin. TEM showed that AQ+ actively disrupts the cell wall of S. aureus leading to cell lysis. CONCLUSIONS: These results suggest that AQ+ has strong antimicrobial activity and may be useful in preparations to reduce nasal and skin carriage of MRSA.
Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered in Irish hospitals between 1971 and 2002 were characterized using multilocus sequence typing (MLST) (n = 130) and SCCmec typing (n = 172). Where atypical SCCmec typing results were obtained, PCR amplification of entire SCCmec elements, analysis of amplimer mobility, and nucleotide sequencing were undertaken. MLST revealed that 129/130 isolates had the same genotypes as internationally spread MRSA clones, including ST239, ST247, ST250, ST5, ST22, ST36, and ST8. A novel genotype, ST496, was identified in one isolate. Half of the isolates (86/172) had SCCmec type I, IA, II, III, or IV. The remaining 86 isolates harbored novel SCCmec variants in three distinct genetic backgrounds: (i) 74/86 had genotype ST8 and either one of five novel SCCmec II (IIA, IIB, IIC, IID, and IIE) or one of two novel SCCmec IV (IVE and IVF) variants; (ii) 3/86 had genotype ST239 and a novel SCCmec III variant; (iii) 9/86 had a novel SCCmec I variant associated with ST250. SCCmec IVE and IVF were similar to SCCmec IVc and IVb, respectively, but differed in the region downstream of mecA. The five SCCmec II variants were similar to SCCmec IVb in the region upstream of the ccr complex but otherwise were similar to SCCmec II, except for the following regions: SCCmec IIA and IID had a novel mec complex, A.4 (Delta mecI-IS1182-Delta mecI-mecR1-mecA-IS431mec); SCCmec IIC and IIE had a novel mec complex, A.3 (IS1182-Delta mecI-mecR1-mecA-IS431mec); SCCmec IID and IIE lacked pUB110; SCCmec IIC and IIE lacked a region of DNA between Tn554 and the mec complex; and SCCmec IIB lacked Tn554. This study has demonstrated a hitherto-undescribed degree of diversity within SCCmec.
The accessory gene regulator (agr) locus influences the expression of many virulence genes in the human pathogen Staphylococcus aureus. Four allelic groups of agr, which generally inhibit the regulatory activity of each other, have been identified within the species. Interference in virulence gene expression caused by different agr groups has been suggested to be a mechanism for isolating bacterial populations and a fundamental basis for subdividing the species. To test the hypothesis that the species is phylogenetically structured according to agr groups, we mapped agr groups onto a clone phylogeny inferred from partial sequences of 14 genes from 27 genetically diverse strains. Shimodaira-Hasegawa and parametric bootstrap tests rejected the hypotheses that the species is subdivided into three or five monophyletic agr groups but failed to reject the hypothesis that the species is subdivided into two groups that each consist of multiple clonal complexes and multiple agr groups. Additional evidence for agr recombination is found from clustered polymorphisms in complete agr sequences. However, agr recombination has not occurred frequently or randomly through time, because the topology and branch lengths of the clone phylogeny are reflected within each agr group. To account for these observations, we propose a new evolutionary model that involves a genetically polymorphic ancestral population of S. aureus that horizontally transferred agr groups between two subspecies groups near the time that these subspecies groups diverged.
The staphylococcal methicillin resistance determinant, mecA, resides on a mobile genetic element, staphylococcus chromosomal cassette mec (SCCmec). The distribution of SCCmec in nature is limited to relatively few clonal complexes of related methicillin-resistant Staphylococcus aureus (MRSA). We have previously reported that some genetic backgrounds are restrictive of mecA and penicillin-binding protein 2a expression, which could account for the restricted clonal distribution of SCCmec in nature. In this study, we investigate the potential role of the host chromosome in the transformability and expression of mecA in 103 naturally occurring methicillin-susceptible S. aureus clinical isolates. The isolates, which had been genotyped previously by multilocus sequence typing, were classified into one of two mutually exclusive categories based on whether the isolates belonged to "major" MRSA lineages or to "other" lineages that are never or occasionally MRSA. We introduced mecA expressed on the low-copy-number plasmid pYK20 into each MSSA strain and assayed the phenotype of resistance to nafcillin by population analysis to assess the relationship between the stability of mecA expression and genetic background. Strains from the major MRSA lineages were more transformable with pYK20 and better able to maintain the plasmid and express resistance in comparison to strains from other lineages. These data support the hypothesis that the presence of mecA within relatively few clonal complexes is partly due to genetic factors that are permissive of mecA and its gene product.
Staphylococcus aureus is an important nosocomial and community-acquired pathogen. Its genetic plasticity has facilitated the evolution of many virulent and drug-resistant strains, presenting a major and constantly changing clinical challenge. We sequenced the approximately 2.8-Mbp genomes of two disease-causing S. aureus strains isolated from distinct clinical settings: a recent hospital-acquired representative of the epidemic methicillin-resistant S. aureus EMRSA-16 clone (MRSA252), a clinically important and globally prevalent lineage; and a representative of an invasive community-acquired methicillin-susceptible S. aureus clone (MSSA476). A comparative-genomics approach was used to explore the mechanisms of evolution of clinically important S. aureus genomes and to identify regions affecting virulence and drug resistance. The genome sequences of MRSA252 and MSSA476 have a well conserved core region but differ markedly in their accessory genetic elements. MRSA252 is the most genetically diverse S. aureus strain sequenced to date: approximately 6% of the genome is novel compared with other published genomes, and it contains several unique genetic elements. MSSA476 is methicillin-susceptible, but it contains a novel Staphylococcal chromosomal cassette (SCC) mec-like element (designated SCC(476)), which is integrated at the same site on the chromosome as SCCmec elements in MRSA strains but encodes a putative fusidic acid resistance protein. The crucial role that accessory elements play in the rapid evolution of S. aureus is clearly illustrated by comparing the MSSA476 genome with that of an extremely closely related MRSA community-acquired strain; the differential distribution of large mobile elements carrying virulence and drug-resistance determinants may be responsible for the clinically important phenotypic differences in these strains.
The existence of bacterial clones was evident in early phenotypic studies that recognised high levels of similarity in geographically and temporally separated isolates. Multilocus sequence typing (MLST) has become the most common method for genetically characterizing clones of several bacterial pathogens, allowing the tracking of hypervirulent/antibiotic-resistant lineages. MLST has also been used to examine the way that bacterial populations, and in particular, bacterial clones evolve. Visualisation of MLST datasets has required the development of novel tools, such as 'eBURST', a key program in constructing evolutionary models that detail how methicillin resistant Staphylococcus aureus (MRSA) and other clones emerge and spread.
Multilocus sequence typing (MLST) of 48 methicillin-sensitive Staphylococcus aureus isolates from intravenous drug user abscesses/soft-tissue infections revealed 12 sequence types (STs) belonging to eight genetically distinct lineages. Only two novel STs were recovered (one isolate of each), indicating that isolates in this study were similar to those from previous studies of disease and carriage. However, ST59, the most common genotype recovered (from six individuals), may be adept at causing subcutaneous lesions in this patient population, as it is rare in carriage and disease. PCR detection of 22 toxin genes revealed a high prevalence of the gene for staphylococcal enterotoxin B compared with previous studies, indicating that this toxin may promote infections in this patient group.
Conjugative transfer and replacement of hundreds of kilobases of a bacterial chromosome can occur in vitro, but replacements in nature are either an order of magnitude smaller or involve the movement of mobile genetic elements. We discovered that two lineages of Staphylococcus aureus, including a pandemic methicillin-resistant lineage, were founded by single chromosomal replacements of at least approximately 244 and approximately 557 kb representing approximately 10 and approximately 20% of the chromosome, respectively, without the obvious involvement of mobile genetic elements. The replacements are unprecedented in natural populations of bacteria because of their large size and unique structure and may have a dramatic impact on bacterial evolution.
Methicillin-resistant Staphylococcus aureus (MRSA) with reduced vancomycin susceptibility vancomycin-intermediate S. aureus (VISA) has been reported from many countries. Whether resistance is evolving regularly in different genetic backgrounds or in a single clone with a genetic predisposition, as early results suggest, is unclear. We have studied 101 MRSA with reduced vancomycin susceptibility from nine countries by multilocus sequence typing (MLST), characterization of SCCmec (staphylococcal chromosomal cassette mec), and agr (accessory gene regulator). We found nine genotypes by MLST, with isolates within all five major hospital MRSA lineages. Most isolates (88/101) belonged to two of the earliest MRSA clones that have global prevalence. Our results show that reduced susceptibility to vancomycin has emerged in many successful epidemic lineages with no clear clonal disposition. Increasing antimicrobial resistance in genetically distinct pandemic clones may lead to MRSA infections that will become increasingly difficult to treat.
Recent advances in molecular epidemiological methods have elucidated the evolutionary pathways by which modern methicillin-resistant Staphylococcus aureus have evolved. Only five major lineages of hospital methicillin-resistant Staphylococcus aureus exist, but these are globally distributed and evolving resistance to even the newest antibiotics.
SCCmec is a mobile genetic element that carries the gene (mecA) mediating methicillin resistance in staphylococci. For Staphylococcus aureus, four SCCmec types have been described, one (type IV) of which has been associated with newly identified community-acquired methicillin-resistant S. aureus. However, the distribution of SCCmec types among S. epidermidis is not known. SCCmec typing of a collection of 44 methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered between 1973 and 1983 from the blood of patients with prosthetic valve endocarditis (PVE) was performed by PCR amplification of key genetic elements (mecA, mecI, IS1272, and ccrAB). Of the 44 isolates, 1 (2%) harbored SCCmec type I, 15 (34%) harbored type II, 12 (28%) harbored type III, and 16 (36%) harbored type IV. The complete nucleotide sequence of SCCmec type IV was determined for 16 isolates and found to be identical in size (24 kb) and 98% homologous to DNA sequences published for S. aureus. Type IV SCCmec was also common (5 of 10 isolates) among a geographically dispersed collection of 10 recent (1998 to 2001) S. epidermidis bloodstream isolates. Multilocus sequence typing (MLST) (using the same seven genes presently employed for S. aureus MLST) of these MRSE isolates and of 10 additional recent geographically dispersed methicillin-susceptible isolates demonstrated that all 16 PVE isolates and 2 of 5 recent isolates harboring type IV SCCmec were in three related clonal groups. All three MSSE PVE isolates recovered from patients between 1976 and 1979 were in the same clonal groups as type IV SCCmec MRSE isolates. These data support the hypothesis of intra- and interspecies transfer of type IV SCCmec and suggest that there are clonal associations in S. epidermidis that correlate with SCCmec type.
Five major lineages of methicillin-resistant Staphylococcus aureus (MRSA) have evolved since the introduction of methicillin for the treatment of infections caused by penicillin-resistant S. aureus in 1959. The clones of these lineages are responsible for the vast majority of hospital-acquired MRSA disease globally. We have constructed high-resolution evolutionary models for each lineage using a parsimony approach with 15 partial gene sequences from 147 geographically diverse isolates. On the basis of these models, we infer that MRSA has emerged at least 20 times upon acquisition of the methicillin resistance determinant, which is carried on a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec). The acquisition of SCCmec by sensitive clones was four times more common than the replacement of one SCCmec with another. Notably, SCCmec type IV was found in twice as many clones as any other SCCmec type, and it is this SCCmec type which is commonly found in clones from patients with community-acquired MRSA disease. Our findings suggest that most clones of MRSA arise by the acquisition of SCCmec type IV by methicillin-sensitive isolates.
Staphylococcus aureus is an important human pathogen and represents a growing public health burden owing to the emergence and spread of antibiotic-resistant clones, particularly within the hospital environment. Despite this, basic questions about the evolution and population biology of the species, particularly with regard to the extent and impact of homologous recombination, remain unanswered. We address these issues through an analysis of sequence data obtained from the characterization by multilocus sequence typing (MLST) of 334 isolates of S. aureus, recovered from a well-defined population, over a limited time span. We find no significant differences in the distribution of multilocus genotypes between strains isolated from carriers and those from patients with invasive disease; there is, therefore, no evidence from MLST data, which index variation within the stable "core" genome, for the existence of hypervirulent clones of this pathogen. Examination of the sequence changes at MLST loci during clonal diversification shows that point mutations give rise to new alleles at least 15-fold more frequently than does recombination. This contrasts with the naturally transformable species Neisseria meningitidis and Streptococcus pneumoniae, in which alleles change between 5- and 10-fold more frequently by recombination than by mutation. However, phylogenetic analysis suggests that homologous recombination does contribute toward the evolution of this species over the long term. Finally, we note a striking excess of nonsynonymous substitutions in comparisons between isolates belonging to the same clonal complex compared to isolates belonging to different clonal complexes, suggesting that the removal of deleterious mutations by purifying selection may be relatively slow.
Serotype changes among natural isolates of Streptococcus pneumoniae are well documented and occur by recombinational exchanges at the capsular biosynthetic locus. However, the frequency with which this phenomenon occurs within the nasopharynx of children is not clear and is likely to be highest in the nasopharynx of children, who have high rates of pneumococcal carriage. A birth cohort of 100 infants was studied, and pneumococci were recovered from nasopharyngeal samples taken at monthly intervals during the first 6 months of life and then at 2-monthly intervals until the age of 2 years. Among the 1,353 nasopharyngeal samples were 523 that contained presumptive pneumococci, and three colonies from each were serotyped. A total of 333 isolates, including all isolates of differing serotypes from the same child, were characterized by multilocus sequence typing. Sixty-eight children carried multiple serotypes during the first 2 years of life. Two children carried a typeable and a nonserotypeable pneumococcus of identical genotype, and five children carried genetically indistinguishable isolates of serotypes 15B and 15C. These isolates were considered, respectively, to be due to loss of capsule expression and the known ability of serotype 15B and 15C pneumococci to interconvert by loss or gain of an acetyl group on the capsular polysaccharide. In all other cases, isolates from the same children that differed in serotype also differed in genotype, indicating the acquisition of a different pneumococcal strain rather than a change in capsular type. There was therefore no evidence in this study for any change of serotype due to recombinational replacements at the capsular locus among the pneumococci carried within the nasopharynges of the children.