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Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.

Staphylococcal chromosome cassette mec (SCCmec) elements within major lineages of healthcare- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) clones were characterised using intra-SCCmec multilocus sequencing. A strong correlation was observed between sequence- and PCR-based typing methods (p

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in many countries is increasing and, in hospitals in some areas, more than half of all S. aureus disease isolates are MRSA. MRSA strains are becoming increasingly multiresistant, and have recently developed resistance to vancomycin, used successfully to treat MRSA for more than 30 years. This review summarises recent studies that have elucidated the evolutionary history of MRSA. The first MRSA isolate evolved from a sensitive, epidemic strain prevalent in Europe, and its progeny—the first MRSA clone—quickly spread to other continents. Analyses of epidemic MRSA isolates from hospitals in different countries by molecular methods, including multilocus sequence typing (MLST) and DNA microarray analysis, reveal that MRSA strains have evolved separately within five distinct epidemic, sensitive lineages. However, resistance has been transferred to S. aureus on many more than five occasions, as some lineages have acquired different structural types of the element carrying the methicillin resistance gene. The emergence of MRSA as a community pathogen has been noted in several countries, and MLST and SCCmec typing have been used to demonstrate that community-acquired MRSA strains are typically related only distantly to hospital MRSA strains, and thus represent novel acquisitions of SCCmec.

In 1996 a new epidemic MRSA emerged in three hospitals North of Berlin. This strain, Barnim epidemic MRSA, was isolated in 15 hospitals in Northern Germany in 1997 and 29 hospitals throughout Germany in 1998. Isolates of this clone are non-typeable by phages, its resistance phenotype is PEN, OXA, ERY, CLI, CIP (genotype: mecA, ermC , mutations in grlA and gyrA ). The Sma I macrorestriction pattern corresponds to particular phage group II strains which is confirmed by the 16S-23S rRNA gene spacer pattern. Isolates of this clone differ by less than three Sma I macrorestriction fragments from isolates of the EMRSA15 clone from the United Kingdom, the most common epidemic MRSA isolates in the United Kingdom in recent years. Both epidemic strains produce enterotoxin C and possess the sec determinant for this toxin, the configuration of the mec regulon is mecI - , mecRB + , mecRC + . Both share the same Alu I pattern of PCR amplimers of the 3′ end region of the coagulase gene. EMRSA 15 and Barnim EMRSA share a common multilocus sequence type indicating a recent, shared evolutionary origin.

Both Neisseria meningitidis and Streptococcus pneumoniae are naturally transformable species and are known to be freely recombining in the wild. Large multilocus sequence typing (MLST) datasets have been generated for these species. Here we outline an approach which exploits these data sets in order to quantify the extent of recombination, thus enabling meaningful comparisons between the two species. Two parameters are estimated; the rate at which recombination changes alleles, compared to point mutation, and the rate at which recombination changes individual nucleotide sites, compared to point mutation. Estimates for the former parameter are 4:1 in the meningococcus (i.e. alleles are changed four-fold more frequently by recombination than by mutation), and 10:1 in the pneumococcus. However, estimates for the latter parameter are at least 80:1 in the meningococcus (i.e. an individual nucleotide site is at least 80-fold more likely to change by recombination than by mutation) and 50:1 in the pneumococcus. These data imply that recombination events, compared to mutational events, may be more common in the pneumococcus than in the meningococcus. However, because it is a more diverse species, each recombinational exchange in the meningococcus results in more nucleotide changes on average.

Multilocus sequence typing (MLST) provides a new approach to molecular epidemiology that can identify and track the global spread of virulent or antibiotic-resistant isolates of bacterial pathogens using the Internet. MLST databases, together with interrogation software, are available for Neisseria meningitidis and Streptococcus pneumoniae and databases for Streptococcus pyogenes and Staphylococcus aureus will be released shortly.

During the 1950s, the notorious penicillin-resistant clone of Staphylococcus aureus known as phage type 80/81 emerged and caused serious hospital-acquired and community-acquired infections worldwide. This clone was largely eliminated in the 1960s, concurrent with the widespread use of penicillinase-resistant beta lactams. We investigated whether early 80/81 isolates had the genes for Panton-Valentine leucocidin, a toxin associated with virulence in healthy young people. Multilocus sequence analysis suggested that descendants of 80/81 have acquired meticillin resistance, are re-emerging as a community-acquired meticillin-resistant S aureus (MRSA) clone, and represent a sister lineage to pandemic hospital-acquired MRSA.

P19.03 Evaluation of Molecular Typing Methods in Characterizing the European “Harmony” Collection of Epidemic Methicillin – Resistant Staphylococcus aureus B. Cookson1 *, S. Murchan1, D. Ashley Robinson2, A. Monk2, M. Enright3. 1Laboratory of Healthcare Associated Infection, HPA, UK, 2Department of Infectious Disease Epidemiology, Imperial College, London, UK, 3Dept of Infectious Disease Epidemiology, Imperial College London UK and the HARMONY IUMS Group, EU