Comparative Genomic Analysis of a Clinical Isolate of Klebsiella quasipneumoniae subsp. similipneumoniae, a KPC-2 and OKP-B-6 Beta-Lactamases Producer Harboring Two Drug-Resistance Plasmids from Southeast Brazil.

The aim of this study was to unravel the genetic determinants responsible for multidrug (including carbapenems) resistance and virulence in a clinical isolate of Klebsiella quasipneumoniae subsp. similipneumoniae by whole-genome sequencing and comparative analyses. Eighty-three clinical isolates initially identified as carbapenem-resistant K. pneumoniae were collected from nosocomial infections in southeast Brazil. After RAPD screening, the KPC-142 isolate, showing the most divergent DNA pattern, was selected for complete genome sequencing in an Illumina HiSeq 2500 instrument. Reads were assembled into scaffolds, gaps between scaffolds were resolved by in silico gap filling and extensive bioinformatics analyses were performed, using multiple comparative analysis tools and databases. Genome sequencing allowed to correct the classification of the KPC-142 isolate as K. quasipneumoniae subsp. similipneumoniae. To the best of our knowledge this is the first complete genome reported to date of a clinical isolate of this subspecies harboring both class A beta-lactamases KPC-2 and OKP-B-6 from South America. KPC-142 has one 5.2 Mbp chromosome (57.8% G+C) and two plasmids: 190 Kbp pKQPS142a (50.7% G+C) and 11 Kbp pKQPS142b (57.3% G+C). The 3 Kbp region in pKQPS142b containing the blaKPC-2 was found highly similar to that of pKp13d of K. pneumoniae Kp13 isolated in Southern Brazil in 2009, suggesting the horizontal transfer of this resistance gene between different species of Klebsiella. KPC-142 additionally harbors an integrative conjugative element ICEPm1 that could be involved in the mobilization of pKQPS142b and determinants of resistance to other classes of antimicrobials, including aminoglycoside and silver. We present the completely assembled genome sequence of a clinical isolate of K. quasipneumoniae subsp. similipneumoniae, a KPC-2 and OKP-B-6 beta-lactamases producer and discuss the most relevant genomic features of this important resistant pathogen in comparison to several strains belonging to K. quasipneumoniae subsp. similipneumoniae (phylogroup II-B), K. quasipneumoniae subsp. quasipneumoniae (phylogroup II-A), K. pneumoniae (phylogroup I), and K. variicola (phylogroup III). Our study contributes to the description of the characteristics of a novel K. quasipneumoniae subsp. similipneumoniae strain circulating in South America that currently represent a serious potential risk for nosocomial settings.

The invasive MenC cc103 lineage with penicillin reduced susceptibility persisting in Brazil.

Penicillin is the antibiotic of choice for the treatment of meningococcal infections, and mutations in penA gene are involved with reduced susceptibility (penI) emergence to this antibiotic. This study aimed to characterize the penA allelic diversity, their association with penI phenotype and distribution among prevalent meningococci serogroups in Brazil. The entire penA from 49 invasive strains of distinct serogroups circulating in Brazil for more than two decades were obtained by PCR and sequencing. Additionally, the penA from 22 publicly available complete Neisseria meningitidis genomes from Brazil were included in the study. The allelic diversity was determined and a genetic tree was built using the penA sequence alignment. The penicillin MIC was obtained by the E-Test method. In general, the identified penA alleles correlated with the observed penI phenotype. The canonical penA1 was the most prevalent allele, however, several altered penA were also identified in strains presenting increased penicillin MICs. It was identified a new penA amino acid position (residue 480) that possibly influence the penicillin MIC in some strains. Interestingly, the altered penA14 was found in penI invasive MenC cc103 strains spread in Brazil and persisting since 2011, indicating that the biological cost imposed by penI phenotype can be ameliorated by particular features present in this lineage, which represents an additional public health threat.

Revisiting molecular serotyping of Streptococcus pneumoniae.

BACKGROUND: Ninety-two Streptococcus pneumoniae serotypes have been described so far, but the pneumococcal conjugate vaccine introduced in the Brazilian basic vaccination schedule in 2010 covers only the ten most prevalent in the country. Pneumococcal serotype-shifting after massive immunization is a major concern and monitoring this phenomenon requires efficient and accessible serotyping methods. Pneumococcal serotyping based on antisera produced in animals is laborious and restricted to a few reference laboratories. Alternatively, molecular serotyping methods assess polymorphisms in the cps gene cluster, which encodes key enzymes for capsular polysaccharides synthesis in pneumococci. In one such approach, cps-RFLP, the PCR amplified cps loci are digested with an endonuclease, generating serotype-specific fingerprints on agarose gel electrophoresis. METHODS: In this work, in silico and in vitro approaches were combined to demonstrate that XhoII is the most discriminating endonuclease for cps-RFLP, and to build a database of serotype-specific fingerprints that accommodates the genetic diversity within the cps locus of 92 known pneumococci serotypes. RESULTS: The expected specificity of cps-RFLP using XhoII was 76% for serotyping and 100% for serogrouping. The database of cps-RFLP fingerprints was integrated to Molecular Serotyping Tool (MST), a previously published web-based software for molecular serotyping. In addition, 43 isolates representing 29 serotypes prevalent in the state of Minas Gerais, Brazil, from 2007 to 2013, were examined in vitro; 11 serotypes (nine serogroups) matched the respective in silico patterns calculated for reference strains. The remaining experimental patterns, despite their resemblance to their expected in silico patterns, did not reach the threshold of similarity score to be considered a match and were then added to the database. CONCLUSION: The cps-RFLP method with XhoII outperformed the antisera-based and other molecular serotyping methods in regard of the expected specificity. In order to accommodate the genetic variability of the pneumococci cps loci, the database of cps-RFLP patterns will be progressively expanded to include new variant in vitro patterns. The cps-RFLP method with endonuclease XhoII coupled with MST for computer-assisted interpretation of results may represent a relevant contribution to the real time detection of changes in regional pneumococci population diversity in response to mass immunization programs.