BackgroundUtilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci.ResultsSix barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species.ConclusionsThe use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.

BackgroundUtilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci.ResultsSix barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species.ConclusionsThe use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.

BackgroundHorizontal transfer of mobile genetic elements (MGEs) that carry virulence and antimicrobial resistance genes mediates the evolution of methicillin-resistant Staphylococcus aureus, and the emergence of new MRSA clones. Most MRSA lineages show an association with specific MGEs and the evolution of MGE composition following clonal expansion has not been widely studied.ResultsWe investigated the genomes of 1193 S. aureus bloodstream isolates, 1169 of which were MRSA, collected in the UK and the Republic of Ireland between 2001 and 2010. The majority of isolates belonged to clonal complex (CC)22 (n = 923), which contained diverse MGEs including elements that were found in other MRSA lineages. Several MGEs showed variable distribution across the CC22 phylogeny, including two antimicrobial resistance plasmids (pWBG751-like and SAP078A-like, carrying erythromycin and heavy metal resistance genes, respectively), a pathogenicity island carrying the enterotoxin C gene and two phage types Sa1int and Sa6int. Multiple gains and losses of these five MGEs were identified in the CC22 phylogeny using ancestral state reconstruction. Analysis of the temporal distribution of the five MGEs between 2001 and 2010 revealed an unexpected reduction in prevalence of the two plasmids and the pathogenicity island, and an increase in the two phage types. This occurred across the lineage and was not correlated with changes in the relative prevalence of CC22, or of any sub-lineages within in.ConclusionsAncestral state reconstruction coupled with temporal trend analysis demonstrated that epidemic MRSA CC22 has an evolving MGE composition, and indicates that this important MRSA lineage has continued to adapt to changing selective pressure since its emergence.

BackgroundHorizontal transfer of mobile genetic elements (MGEs) that carry virulence and antimicrobial resistance genes mediates the evolution of methicillin-resistant Staphylococcus aureus, and the emergence of new MRSA clones. Most MRSA lineages show an association with specific MGEs and the evolution of MGE composition following clonal expansion has not been widely studied.ResultsWe investigated the genomes of 1193 S. aureus bloodstream isolates, 1169 of which were MRSA, collected in the UK and the Republic of Ireland between 2001 and 2010. The majority of isolates belonged to clonal complex (CC)22 (n = 923), which contained diverse MGEs including elements that were found in other MRSA lineages. Several MGEs showed variable distribution across the CC22 phylogeny, including two antimicrobial resistance plasmids (pWBG751-like and SAP078A-like, carrying erythromycin and heavy metal resistance genes, respectively), a pathogenicity island carrying the enterotoxin C gene and two phage types Sa1int and Sa6int. Multiple gains and losses of these five MGEs were identified in the CC22 phylogeny using ancestral state reconstruction. Analysis of the temporal distribution of the five MGEs between 2001 and 2010 revealed an unexpected reduction in prevalence of the two plasmids and the pathogenicity island, and an increase in the two phage types. This occurred across the lineage and was not correlated with changes in the relative prevalence of CC22, or of any sub-lineages within in.ConclusionsAncestral state reconstruction coupled with temporal trend analysis demonstrated that epidemic MRSA CC22 has an evolving MGE composition, and indicates that this important MRSA lineage has continued to adapt to changing selective pressure since its emergence.