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.