Jodi A. Lindsay

Genome Sequence of a Recently Emerged, Highly Transmissible, Multi-Antibiotic- and Antiseptic-Resistant Variant of Methicillin-Resistant Staphylococcus aureus, Sequence Type 239 (TW)

The 3.1-Mb genome of an outbreak methicillin-resistant Staphylococcus aureus (MRSA) strain (TW20) contains evidence of recently acquired DNA, including two large regions (635 kb and 127 kb). The strain is resistant to a wide range of antibiotics, antiseptics, and heavy metals due to resistance genes encoded on mobile genetic elements and also mutations in housekeeping genes.

Evolutionary Genomics of Staphylococcus aureus Reveals Insights into the Origin and Molecular Basis of Ruminant Host Adaptation

Phenotypic biotyping has traditionally been used to differentiate bacteria occupying distinct ecological niches such as host species. For example, the capacity of Staphylococcus aureus from sheep to coagulate ruminant plasma, reported over 60 years ago, led to the description of small ruminant and bovine S. aureus ecovars. The great majority of small ruminant isolates are represented by a single, widespread clonal complex (CC133) of S. aureus, but its evolutionary origin and the molecular basis for its host tropism remain unknown. Here, we provide evidence that the CC133 clone evolved as the result of a human to ruminant host jump followed by adaptive genome diversification. Comparative whole-genome sequencing revealed molecular evidence for host adaptation including gene decay and diversification of proteins involved in host–pathogen interactions. Importantly, several novel mobile genetic elements encoding virulence proteins with attenuated or enhanced activity in ruminants were widely distributed in CC133 isolates, suggesting a key role in its host-specific interactions. To investigate this further, we examined the activity of a novel staphylococcal pathogenicity island (SaPIov2) found in the great majority of CC133 isolates which encodes a variant of the chromosomally encoded von Willebrand-binding protein (vWbpSov2), previously demonstrated to have coagulase activity for human plasma. Remarkably, we discovered that SaPIov2 confers the ability to coagulate ruminant plasma suggesting an important role in ruminant disease pathogenesis and revealing the origin of a defining phenotype of the classical S. aureus biotyping scheme. Taken together, these data provide broad new insights into the origin and molecular basis of S. aureus ruminant host specificity.

Anti-MRSA Agent Discovery Using Diversity-Oriented Synthesis†

Antibacterial drugs have played an essential role in the global increase in quality of life and life expectancy. However, these gains are at serious risk owing to bacterial drug resistance by so-called “superbugs”, such as methicillin-resistant Staphylococcus aureus (MRSA). The discovery of new antibiotics with novel modes of action is vital to tackle the threat of multidrug-resistant bacteria. Traditionally, antibiotics have been discovered from natural sources; however, there are many disadvantages to using extracts (e.g. limited availability, bioactive constituent identification, and complex analogue synthesis). These problems have led to a complementary approach of synthesizing structurally diverse, natural-product-like small molecules directly and efficiently, an approach known as diversity-oriented synthesis (DOS). Whereas compound collections of a common scaffold decorated with diverse building blocks have been synthesized efficiently, there are limited examples of the synthesis of small molecules with a high degree of skeletal diversity (usually by a build–couple–pair strategy). Previously, we have used a diazoacetate starting unit to mimic nature8s divergent synthetic strategy with acetyl CoA (by a pluripotent functional-group strategy) to synthesize compounds with natural-product scaffolds (e.g. cocaine and warfarin). Herein, we report the use of a solid-supported phosphonate unit to synthesize 242 drug-like compounds based on 18 natural-product-like scaffolds in two to five steps and their use in discovering a new structural class of antibiotic with anti-MRSA activity. The solid-supported phosphonate 1 (Scheme 1) was identified as an attractive DOS starting unit for three key reasons. First, the reactive phosphonate functionality permits the stereoselective formation of a,b-unsaturated acyl imidazolidinones (2) that could be used to generate enantioselectively a wide range of scaffolds that can be diversified further. Second, the imidazolidinone linker not only enables twopoint binding of chiral catalysts but also permits divergent cleavage of the exocyclic acyl group (hydrolysis, reduction, esterification, and amide formation). Thirdly, immobilization of 1 on a silyl polystyrene support simplified reaction optimization and work-up procedures in the multistep parallel synthesis (total of over 1000 individual steps), thereby allowing the efficient production of milligram quantities of 242 compounds without the requirement for automation equipment. In the first step of the diversity-oriented synthesis, 1 was treated with aldehyde building blocks (aryl, heteroaryl, and alkyl; see the Supporting Information) to deliver twelve a,bunsaturated acyl imidazolidinones (2). The second steps of the solid-supported synthesis exploited three catalytic, enantioselective, divergent reaction pathways (Scheme 1): 1) [2+3] cycloaddition (reaction b, ee 60–65%, de 7899%), 2) dihydroxylation (reaction c, ee 88–91%), and 3) [4+2] cycloaddition (reaction d, ee 89–98%, de 74– 74%). Similar selectivities were observed when repeating the reactions in solution with a triisopropylsilyl-protected linker (as opposed to the diisopropylpolystyrene group; see the Supporting Information). The reactions were also conducted with achiral catalysts to give racemic products, which were used for the later steps of the synthesis. This procedure enabled the diversity-oriented synthesis to be streamlined to half the size, yet permitted the enantioselective synthesis of hits during the structure–activity relationship stages of this [*] Dr. G. L. Thomas, R. J. Spandl, F. G. Glansdorp, Dr. M. Ladlow, Dr. D. R. Spring Department of Chemistry, University of Cambridge Lensfield Road, Cambridge, CB2 1EW (UK) Fax: (+44) 1223-336362 E-mail: drspring@ch.cam.ac.uk Homepage: http://www-spring.ch.cam.ac.uk/

Major families of multiresistant plasmids from geographically and epidemiologically diverse staphylococci.

Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20–30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.

An outbreak of severe infections with community-acquired MRSA carrying the Panton-Valentine leukocidin following vaccination.

Background Infections with community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are emerging worldwide. We investigated an outbreak of severe CA-MRSA infections in children following out-patient vaccination. Methods and Findings We carried out a field investigation after adverse events following immunization (AEFI) were reported. We reviewed the clinical data from all cases. S. aureus recovered from skin infections and from nasal and throat swabs were analyzed by pulse-field gel electrophoresis, multi locus sequence typing, PCR and microarray. In May 2006, nine children presented with AEFI, ranging from fatal toxic shock syndrome, necrotizing soft tissue infection, purulent abscesses, to fever with rash. All had received a vaccination injection in different health centres in one District of Ho Chi Minh City. Eight children had been vaccinated by the same health care worker (HCW). Deficiencies in vaccine quality, storage practices, or preparation and delivery were not found. Infection control practices were insufficient. CA-MRSA was cultured in four children and from nasal and throat swabs from the HCW. Strains from children and HCW were indistinguishable. All carried the Panton-Valentine leukocidine (PVL), the staphylococcal enterotoxin B gene, the gene complex for staphylococcal-cassette-chromosome mec type V, and were sequence type 59. Strain HCM3A is epidemiologically unrelated to a strain of ST59 prevalent in the USA, although they belong to the same lineage. Conclusions We describe an outbreak of infections with CA-MRSA in children, transmitted by an asymptomatic colonized HCW during immunization injection. Consistent adherence to injection practice guidelines is needed to prevent CA-MRSA transmission in both in- and outpatient settings.

Lack of transmission of methicillin-resistant Staphylococcus aureus (MRSA) between apparently healthy dogs in a rescue kennel

Although it is widely accepted that methicillin-resistant Staphylococcus aureus (MRSA) can be transmitted between humans and animals in both directions, little is known about the dynamics of animal-to-animal transfer. This study aimed to investigate aspects of dog-to-dog MRSA transfer in a rescue facility in the South-East of England during an MRSA outbreak. One hundred and twenty-nine apparently healthy dogs, mostly housed in pairs, were swabbed at nasal, oral, axillary and perianal sites. Swabs were enriched in selective broth and staphylococci identified using standard biological methods. MRSA isolates were confirmed by demonstration of the thermonuclease gene (nuc) and mecA. After initial swabbing, a dog excluded from the study design but housed at the same facility was discovered to have a wound infection due to MRSA. MRSA carriage was identified in 10/129 dogs (7.8%) and all isolates were of the same lineage as the one isolated from the infected dog. All carrier dogs lived in shared kennels and their 16 kennel partners sampled negative on two occasions. Concurrently with successful antimicrobial treatment of the infected patient, MRSA carriage resolved spontaneously in all dogs within two weeks. In conclusion, MRSA did not transmit readily between apparently healthy dogs, MRSA carriage was not supported for long periods in a regularly cleaned environment and exposure alone may not lead to MRSA acquisition by dogs without the presence of additional risk factors.

The importance of location

Staphylococcus aureus is one of the most common causes of both nosocomial- and community-acquired infection, causing bacteraemia, surgical site and wound infections, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, food poisoning and many other diseases. Strains that are resistant to multiple antibiotics are prevalent in many hospitals, and treatment options are often limited. Over 30 toxins and virulence factors produced by S. aureus have been described and are thought to contribute to its ability to adapt to so many environments in vivo. Streptococcus pneumoniae is the major cause of bacterial pneumonia but also causes meningitis and otitis media in children. It too produces a number of extracellular toxins and adhesins. Both are low G+C Gram-positive bacteria, a group that is phylogenetically different to the Gram-negative bacteria whose pathogenic mechanisms have been elucidated in far more detail.Two papers describe the application of signature tagged mutagenesis (STM) to S. aureus (by Pathogenesis Corporation, CA, USA)1xStaphylococcus aureus genetic loci impacting growth and survival in multiple infection environments. Coulter, S.N. et al. Mol. Microbiol. 1998; 30: 393–404Crossref | PubMed | Scopus (196)See all References1 and S. pneumoniae (by Glaxo Wellcome, Verona, Italy)2xLarge-scale identification of virulence genes from Streptococcus pneumoniae. Polissi, A. et al. Infect. Immun. 1998; 66: 5620–5629PubMedSee all References2, pathogens whose genomic sequences are not available in the public domain. STM allows the large-scale identification of bacterial genes that are required for virulence in vivo. A pathogenic strain is randomly mutagenized, using transposons or homologous recombination, and is labelled with specific tags. A collection of mutants with different tags are simultaneously inoculated into an animal model and the resulting isolates are screened for weak or non-survivors. Although this method is biased against the isolation of mutants in extracellular toxins and essential genes, both studies have been strengthened by the use of more than one animal model.In S. aureus 237, and in S. pneumoniae 126, attenuated strains were identified. Although some of the genes encoded proteins that had been previously identified as virulence determinants, many of the remaining genes were involved in transportation or biosynthesis of amino acids, peptides and energy metabolism, emphasizing the need for bacteria to survive in vivo to cause infection. Mutations in regions that did not have significant homology to the genomic databases might represent novel virulence determinants.The most unexpected and fascinating result generated from both studies is that only a fraction of genes that were required for virulence in one animal model were also required in other models (10% of S. aureus in all three models, 70% of S. pneumoniae in both models). This shows that in different infection types, bacteria are exposed to different environmental conditions and rely on different factors for survival and disease generation.Therefore, we must conclude that virulence mechanisms of S. aureus and S. pneumoniae are highly dependent on the location and type of infection. In order to further our understanding of the pathogenic processes involved, we must regard each infection type as unique rather than making generalizations about disease caused by a particular bacterial species.

Reduced Sensitivity of Oxacillin-Screening Agar for Detection of MRSA ST398 from Colonized Pigs

Methicillin-resistant Staphylococcus aureus (MRSA) bacteria are opportunistic, zoonotic pathogens presenting major challenges for health care providers. The pig-adapted lineage, MRSA ST398, is now recognized worldwide ([8][1]), and pigs are increasingly considered an important source of community-

Typing pathogenic bugs on the net

Multilocus sequence typing (MLST) involves comparing the sequences of five to seven housekeeping genes in order to epidemiologically type bacterial and parasitic strains. Identification of point mutations and small, horizontal recombinations allows a series of allele numbers to be assigned to each strain. The real advantage of this typing method is that sequence information is highly reproducible, and easily deposited into growing banks of MLST sequence on the internet (for example, see http://mlst.zoo.ox.ac.uk). Furthermore, each laboratory will not need to obtain reference strains or conduct extensive validation before typing their own strains.MLST typing has highlighted the different mutation rates in various bacteria, and this makes the technique suitable for different purposes in each species. This is emphasized by two recent publications.Neisseria meningitidis has a rapid mutation rate that is clearly observed in housekeeping genes. The mutations are stable enough to be useful for tracking epidemiological outbreaks. Feavers and colleagues1xMultilocus sequence typing and antigen gene sequencing in the investigation of a meningococcal disease outbreak. Feavers, I.M. et al. J. Clin. Microbiol. 1999; 37: 3883–3887PubMedSee all References1 applied MLST to a recent outbreak of meningococcal meningitis in a British university, and were able to demonstrate that more than one clone was responsible, and that several other clones were circulating in the student population without causing disease. They have also suggested that it might be possible to MLST-type an isolate that could not actually be cultured, as PCR amplification might be possible.In contrast, the mutation rate in housekeeping genes of Yersinia is extremely low. Achtman and colleagues2xYersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Achtman, M. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14043–14048Crossref | PubMed | Scopus (598)See all References2 showed that all strains of Y. pestis tested were identical, and very similar to Y. pseudotuberculosis strains. Y. enterocolitica strains are also highly conserved but are quite distinct from the former two species. This supports other evidence that Y. pestis and Y. pseudotuberculosis are closely related and should be re-classified as two subspecies. Clinical and historical considerations have prevented this so far. As the mutation rate is so low, Achtman and colleagues2xYersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Achtman, M. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14043–14048Crossref | PubMed | Scopus (598)See all References2 were able to estimate the point at which Y. pestis is likely to have diverged from Y. pseudotuberculosis. Their estimate suggests a date of between 1500 and 20 000 years ago, some time prior to the first plague pandemic in the sixth century.Potentially, MLST could be applied to any two genomes in order to determine their relatedness, provided that there is enough background information available to make meaningful interpretations. This process is under way for a number of microbes, aided by the rapid deposition and dissemination of information on the internet.

Conserved virulence-control in Gram-positive bacteria?

The accessory gene regulator (agr) locus is the major regulator of virulence-factor gene expression in staphylococci, and controls the upregulation of multiple extracellular proteins including toxins and proteases. The agr locus is autoinduced by a quorum sensing mechanism: it encodes a peptide pheromone that is secreted by the bacterial cell, and, once extracellular levels accumulate to a threshold level (quorum), they are detected by the agr signal transduction system, activating the gene regulation response. Because peptides produced by different staphylococcal species and subspecies inhibit the signal transduction step, such inhibitory peptides are proposed adjuncts to antibacterial therapy, and can be administered in vivo to reduce disease.Now, Qin and colleagues1xEffects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence. Qin, X. et al. Infect. Immun. 2000; 68: 2579–2586Crossref | PubMed | Scopus (201)See all References1 have reported that a similar mechanism for controlling virulence factor expression is present in Enterococcus faecalis, an important cause of hospital acquired infection. The E. faecalis regulator (fsr) locus is highly homologous to agr, it is autoregulated, and controls the upregulation of the exoproteins gelatinase and serine protease. Importantly, fsr mutants are attenuated in virulence. Although there is still much to do, this discovery suggests that similar signal transduction systems to agr might be widespread in pathogenic Gram-positive bacteria. These organisms are particularly difficult to treat in hospitals, because of their resistance to multiple antibiotics, so adjunct therapies that impact on virulence might be particularly useful. Whether a broad spectrum approach to encompass all Gram-positive bacteria can be developed awaits further characterization of this locus and investigation of other pathogens.