Daptomycin Resistance and Tolerance Due to Loss of Function in Staphylococcus aureus dsp1 and asp23.

Lipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistant Staphylococcus aureus Alarmingly, daptomycin-resistant S. aureus isolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8) S. aureus HG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, the Δdsp1 mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in both dsp1 and asp23 resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to the S. aureus species that make previously uncharacterized contributions to antimicrobial resistance and tolerance in S. aureus.

Occurrence and Characteristics of Erythromycin-Resistant Streptococcus pneumoniae Strains Isolated in Three Major Brazilian States.

We investigated the occurrence and phenotypic and genotypic characteristics of erythromycin-resistant Streptococcus pneumoniae strains isolated in three major states in Brazil, from 1990 to 1999. Of the 931 pneumococcal strains evaluated, 40 (4.3%) were erythromycin-resistant (Ery-R). Among the 40 Ery-R strains, 90.0%, 80.0%, 27.5%, 5.0%, and 2.5% were resistant to tetracycline, trimethoprim-sulfamethoxazole, penicillin, chloramphenicol, and rifampin, respectively. None of the strains were resistant to ofloxacin or to vancomycin. Most [37 (92.5%)] of the 40 Ery-R isolates presented the MLS(B) phenotype and 3 (7.5%) strains showed the M phenotype. PCR testing indicated that all MLS(B) phenotype isolates harbored the erm(B) gene only, whereas the mef(A/E) gene was present in all isolates presenting the M phenotype. The tet(M) gene was the most frequent (86.1%) among Ery-R isolates that were also resistant to tetracycline. Pulsed-field gel electrophoresis (PFGE) analysis after SmaI digestion revealed the occurrence of clonal relationships within groups of strains belonging to serotypes 14, 19A, and 23F. All Ery-R isolates belonging to serotype 14 were susceptible to penicillin and were included in a single clonal group (named Ery(14)-A) related to the England(14-)9 internationally spread clone.

Carbapenem-resistant Acinetobacter baumannii in Brazil: susceptibility profile and diversity of oxacillinases / Acinetobacter baumannii resistente aos carbapenêmicos no Brasil: perfil de suscetibilidade e diversidade de oxacilinases

ABSTRACT Introduction: The Acinetobacter calcoaceticus-baumannii (ABC) complex includes five species, and the A. baumannii is the most important of them because it carries mechanisms of carbapenems resistance, especially the oxacillinases. Objectives: The objectives of this study were to identify the species of the ABC complex, to evaluate the susceptibility profile and to investigate the presence of oxacillinases in carbapenems-resistant isolates from four Brazilian States. Methods: In the study period, 92 isolates from Rio Grande do Sul (RS), Rio de Janeiro (RJ), Paraná (PR) and São Paulo (SP) were collected. The isolates were identified by matrix-assisted laser desorption ionization-time of fight mass spectrometry (MALDI-TOF MS) and sequencing of gyrB gene. Evaluation of susceptibility was performed by disk diffusion and broth microdilution. The presence of oxacillinases was performed by in-house multiplex polymerase chain reaction (PCR). Results: Ninety-one (99%) isolates were identified as A. baumannii by MALDI-TOF and sequencing. The majority of isolates (56; 61%) showed resistance to the six antimicrobial agents tested. Three isolates were resistant to polymyxin B [minimum inhibitory concentration (MIC) ≥ 4 µg/ml). Eighty (87%) isolates were positive to OXA-23-like, and twelve (13%) isolates to OXA-24-like. Conclusion: Our findings confirm the knowledge about the dissemination of the blaOXA-23 gene in Brazil and suggest the recent emergence and spread of blaOXA-24 gene, since it was identified in three of the four sampled states.

RESUMO Introdução: O complexo Acinetobacter calcoaceticus-baumannii (ABC) inclui cinco espécies, sendo A. baumannii a mais importante clinicamente por carrear muitos mecanismos de resistência aos carbapenêmicos, sobretudo as oxacilinases. Objetivos: Os objetivos deste estudo foram identificar as espécies do complexo ABC, avaliar o perfil de suscetibilidade e investigar a presença de oxacilinases em isolados resistentes aos carbapenêmicos provenientes de quatro estados brasileiros. Métodos: No período do estudo, foram coletados 92 isolados do Rio Grande do Sul (RS), do Rio de Janeiro (RJ), do Paraná (PR) e de São Paulo (SP). Os isolados foram identificados por matrix-assisted laser desorption ionization-time of fight mass spectrometry (MALDI-TOF MS) e sequenciamento do gene gyrB. A avaliação da suscetibilidade foi realizada por disco-difusão e microdiluição de caldo. A presença de oxacilinases foi realizada por reação em cadeia da polimerase (PCR) multiplex in house. Resultados: Noventa e um (99%) isolados foram identificados como A. baumannii por MALDI-TOF e pelo sequenciamento. A maioria dos isolados (56; 61%) apresentou resistência aos seis agentes antimicrobianos testados. Três isolados foram resistentes à polimixina B [concentração inibitória mínima (CIM) ≥ 4 µg/ml). Oitenta (87%) isolados foram positivos para OXA-23 e 12, (13%) para OXA-24. Conclusão: Nossos resultados confirmam a disseminação do gene blaOXA-23 no Brasil e sugerem a recente emergência e disseminação do gene blaOXA-24, uma vez que ele foi identificado em três dos quatro estados amostrados.