Pseudomonas aeruginosa 96 (PA96) was isolated during a multicenter surveillance study in Guangzhou, China, in 2000. Wholegenome sequencing of this outbreak strain facilitated analysis of its IncP-2 carbapenem-resistant plasmid, pOZ176. The plasmid had a length of 500,839 bp and an average percent G؉C content of 57%. Of the 618 predicted open reading frames, 65% encode hypothetical proteins. The pOZ176 backbone is not closely related to any plasmids thus far sequenced, but some similarity to pQBR103 of Pseudomonas fluorescens SBW25 was observed. Two multiresistant class 1 integrons and several insertion sequences were identified. The bla IMP-9 -carrying integron contained aacA4¡bla IMP-9 ¡aacA4, flanked upstream by Tn21 tnpMRA and downstream by a complete tni operon of Tn402 and a mer module, named Tn6016. The second integron carried aacA4¡catB8a¡bla OXA-10 and was flanked by Tn1403-like tnpRA and a sul1-type 3= conserved sequence (3=-CS), named Tn6217. Other features include three resistance genes similar to those of Tn5, a tellurite resistance operon, and two pil operons. The replication and maintenance systems exhibit similarity to a genomic island of Ralstonia solanacearum GM1000. Codon usage analysis suggests the recent acquisition of bla IMP-9 . The origins of the integrons on pOZ176 indicated separate horizontal gene transfer events driven by antibiotic selection. The novel mosaic structure of pOZ176 suggests that it is derived from environmental bacteria.

A ntimicrobial susceptibility testing is a key function of the mycobacteriology laboratory. Susceptibility results guide appropriate tuberculosis (TB) therapy and help prevent the emergence and spread of drug-resistant Mycobacterium tuberculosis strains. Pyrazinamide (PZA) is a front-line drug for the treatment of TB. Administered during the 2-month, intensive phase of the standard short-course regimen, PZA is effective primarily against slowly replicating bacilli and, thus, complements the activities of isoniazid (INH) and rifampin (RIF), which are bactericidal for rapidly replicating organisms.PZA is a prodrug. Conversion to the active form, pyrazinoic acid (POA), is mediated by the pyrazinamidase (PZase) encoded by the pncA gene. It is well established that mutations in pncA can mediate PZA resistance by disrupting PZase activity and the accumulation of POA (6). However, some PZA-resistant (PZA r ) strains have wild-type pncA (pncA WT ) alleles. In such strains, resistance has been proposed to result from altered PZA uptake, increased POA efflux, or impaired POA binding to drug targets (7,9). Recently, it has been demonstrated that POA binding to the 30S ribosomal protein S1 inhibits the trans-translation activity required for efficient protein synthesis (7). Mutations in rpsA, which encodes the S1 protein, result in altered POA binding and can mediate PZA resistance in pncA WT strains. Despite the in vivo efficacy of PZA, in vitro susceptibility testing is challenging (2). PZase activity and the intracellular accumulation of POA increase with decreasing pH, but Mycobacterium tuberculosis viability decreases with decreasing pH. The Clinical and Laboratory Standards Institute (CLSI) recommends the Bactec 460TB radiometric system with Bactec 460TB PZA test medium (BD Diagnostics Systems, Sparks, MD) as the reference method for phenotypic PZA susceptibility testing (1, 4). However, the 460TB system has been discontinued and the 460TB PZA test medium is no longer being manufactured. Many laboratories, including Public Health Ontario (PHO), have adopted the Bactec MGIT 960 (BT960) platform for PZA testing. At our large public health laboratory, the switch to BT960-based testing was accompanied by an elevated incidence of false-positive results, defined as strains that were PZA r by the BT960 method but PZA-susceptible (PZA s ) according to the reference 460TB method (3). To ensure accurate susceptibility results, confirmatory testing of potential PZA r isolates is necessary. A phenotypic strategy, involving a second round of BT960-based testing, can be effective but does not resolve all cases (3,8), and repeat testing requires an additional 5 to 7 days to complete (1). In contrast, confirmatory testing using molecular methods can be completed in less than 48 h. As such, we have investigated the utility of targeted gene sequencing for rapid verification of PZA r results.

The study of genetic and phenotypic variation is fundamental for understanding the dynamics of bacterial genome evolution and untangling the evolution and epidemiology of bacterial pathogens. Neisseria meningitidis (Nm) is among the most intriguing bacterial pathogens in genomic studies due to its dynamic population structure and complex forms of pathogenicity. Extensive genomic variation within identical clonal complexes (CCs) in Nm has been recently reported and suggested to be the result of homologous recombination, but the extent to which recombination contributes to genomic variation within identical CCs has remained unclear. In this study, we sequenced two Nm strains of identical serogroup (C) and multi-locus sequence type (ST60), and conducted a systematic analysis with an additional 34 Nm genomes. Our results revealed that all gene content variation between the two ST60 genomes was introduced by homologous recombination at the conserved flanking genes, and 94.25% or more of sequence divergence was caused by homologous recombination. Recombination was found in genes associated with virulence factors, antigenic outer membrane proteins, and vaccine targets, suggesting an important role of homologous recombination in rapidly altering the pathogenicity and antigenicity of Nm. Recombination was also evident in genes of the restriction and modification systems, which may undermine barriers to DNA exchange. In conclusion, homologous recombination can drive both gene content variation and sequence divergence in Nm. These findings shed new light on the understanding of the rapid pathoadaptive evolution of Nm and other recombinogenic bacterial pathogens.

Meningococcal disease is a widely distributed complex disease affecting all age categories. It can cause severe meningitis and septicemia, especially in unvaccinated infants and young children. The causative agent, Neisseria meningitidis (Nm), can be phenotypically and genetically differentiated into serogroups and sequence types (STs) and has a highly dynamic population structure. To obtain a deeper understanding of the epidemiology of Nm, we sequenced seven Nm genomes. Large-scale genomic analysis was conducted with these 7 Nm genomes, 27 additional Nm genomes from GenBank, and 4 other Neisseria genomes. We observed extensive homologous recombination in all gene functional categories among different Nm genomes. Homologous recombination is so frequent that it has resulted in numerous chimeric open reading frames, including genes in the capsule biosynthesis cluster and loci targeted by commercial vaccines. Our results reveal that, despite widespread use, evolutionary relationships inferred from the standard seven-gene multilocus sequence typing (MLST) method could not predict virulence gene content or strain phenotype. In fact, up to 28% of the virulence-associated genes could differ between strains of identical STs. Consistent with previous studies, we found that allelic recombination is also associated with alterations in antibiotic susceptibility. Overall, these findings emphasize the extensive genomic plasticity of Nm and the limitations of standard molecular methods to quantify this genotypic and phenotypic diversity.

Streptococcus pseudopneumoniae (SPPN) is a recently described species of the viridans group streptococci (VGS). Although the pathogenic potential of S. pseudopneumoniae remains uncertain, it is most commonly isolated from patients with underlying medical conditions, such as chronic obstructive pulmonary disease. S. pseudopneumoniae can be distinguished from the closely related species, S. pneumoniae and S. mitis, by phenotypic characteristics, including optochin resistance in the presence of 5% CO2, bile insolubility, and the lack of the pneumococcal capsule. Previously, we reported the draft genome sequence of S. pseudopneumoniae IS7493, a clinical isolate obtained from an immunocompromised patient with documented pneumonia. Here, we use comparative genomics approaches to identify similarities and key differences between S. pseudopneumoniae IS7493, S. pneumoniae and S. mitis. The genome structure of S. pseudopneumoniae IS7493 is most closely related to that of S. pneumoniae R6, but several recombination events are evident. Analysis of gene content reveals numerous unique features that distinguish S. pseudopneumoniae from other streptococci. The presence of loci for competence, iron transport, pneumolysin production and antimicrobial resistance reinforce the phylogenetic position of S. pseudopneumoniae as an intermediate species between S. pneumoniae and S. mitis. Additionally, the presence of several virulence factors and antibiotic resistance mechanisms suggest the potential of this commensal species to become pathogenic or to contribute to increasing antibiotic resistance levels seen among the VGS.

Streptococcus pseudopneumoniae is a member of the viridans group streptococci (VGS) whose pathogenic significance is unclear. We announce the complete genome sequence of S. pseudopneumoniae IS7493. The genome sequence will assist in the characterization of this new organism and facilitate the development of accurate diagnostic assays to distinguish it from Streptococcus pneumoniae and Streptococcus mitis.

We analyzed travel-associated clinical isolates of Escherichia coli O104:H4, including 1 from the 2011 German outbreak and 1 from a patient who returned from the Philippines in 2010, by genome sequencing and optical mapping. Despite extensive genomic similarity between these strains, key differences included the distribution of toxin and antimicrobial drug–resistance determinants.

Pseudomonas aeruginosa PA96 is a clinical isolate from Guangzhou, China, that is multiresistant to antibiotics. We previously described the 500-kb IncP-2 plasmid, pOZ176 that encodes many resistance genes including the IMP-9 carbapenemase. Whole-genome sequencing of PA96 enabled characterization of its genomic islands, virulence factors, and chromosomal resistance genes. We filled gaps using PCR and used optical mapping to confirm the correct contig order. We automatically annotated the core genome and manually annotated the genomic islands. The genome is 6 444 091 bp and encodes 5853 ORFs. From the whole-genome sequence, we constructed a physical map and constructed a phylogenetic tree for comparison with sequenced P. aeruginosa strains. Analysis of known core genome virulence factors and resistance genes revealed few differences with other strains, but the major virulence island is closer to that of DK2 than to PA14. PA96 most closely resembles the environmental strain M18, and notably shares a common serotype, pyoverdin type, flagellar operon, type IV pilin, and several genomic islands with M18.