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1.
Diagn Microbiol Infect Dis ; 90(2): 148-150, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29150370

RESUMO

ST252 Enterobacter cloacae, producing GES-5 carbapenemase, was isolated in a Czech hospital. blaGES-5 was part of a novel class 1 integron, In1406, which also included a new allele of the aadA15 gene cassette. In1406 was located on a ColE2-like plasmid, pEcl-35771cz (6953bp).


Assuntos
Proteínas de Bactérias/genética , Enterobacter cloacae/genética , Infecções por Enterobacteriaceae/microbiologia , beta-Lactamases/genética , República Tcheca , Enterobacter cloacae/enzimologia , Hospitais , Humanos , Integrons/genética , Tipagem de Sequências Multilocus
2.
PLoS One ; 12(12): e0190038, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29287094

RESUMO

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) -based identification of bacteria and fungi significantly changed the diagnostic process in clinical microbiology. We describe here a novel technique for bacterial and yeast deposition on MALDI target using an automated workflow resulting in an increase of the microbes' score of MALDI identification. We also provide a comparison of four different sample preparation methods. In the first step of the study, 100 Gram-negative bacteria, 100 Gram-positive bacteria, 20 anaerobic bacteria and 20 yeasts were spotted on the MALDI target using manual deposition, semi-extraction, wet deposition onto 70% formic acid and by automatic deposition using MALDI Colonyst. The lowest scores were obtained by manual toothpick spotting which significantly differ from other methods. Identification score of semi-extraction, wet deposition and automatic wet deposition did not significantly differ using calculated relative standard deviation (RSD). Nevertheless, the best results with low error rate have been observed using MALDI Colonyst robot. The second step of validation included processing of 542 clinical isolates in routine microbiological laboratory by a toothpick direct spotting, on-plate formic acid extraction (for yeasts) and automatic deposition using MALDI Colonyst. Validation in routine laboratory process showed significantly higher identification scores obtained using automated process compared with standard manual deposition in all tested microbial groups (Gram-positive, Gram-negative, anaerobes, and yeasts). As shown by our data, automatic colony deposition on MALDI target results in an increase of MALDI-TOF MS identification scores and reproducibility.


Assuntos
Bactérias/isolamento & purificação , Robótica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Leveduras/isolamento & purificação , Automação
4.
Artigo em Inglês | MEDLINE | ID: mdl-28993328

RESUMO

The objective of this study was to perform molecular surveillance for assessing the spread of carbapenemase-producing Pseudomonas aeruginosa in Czech hospitals. One hundred thirty-six carbapenemase-producing isolates were recovered from 22 hospitals located throughout the country. Sequence type 357 (ST357) dominated (n = 120) among carbapenemase producers. One hundred seventeen isolates produced IMP-type (IMP-7 [n = 116] and IMP-1 [n = 1]) metallo-ß-lactamases (MßLs), 15 produced the VIM-2 MßL, and the remaining isolates expressed the GES-5 enzyme. The blaIMP-like genes were located in three main integron types, with In-p110-like being the most prevalent (n = 115). The two other IMP-encoding integrons (In1392 and In1393) have not been described previously. blaVIM-2-carrying integrons included In59-like, In56, and a novel element (In1391). blaGES-5 was carried by In717. Sequencing data showed that In-p110-like was associated with a Tn4380-like transposon inserted in genomic island LESGI-3 in the P. aeruginosa chromosome. The other integrons were also integrated into the P. aeruginosa chromosome. These findings indicated the clonal spread of ST357 P. aeruginosa, carrying the IMP-7-encoding integron In-p110, in Czech hospitals. Additionally, the sporadic emergence of P. aeruginosa producing different carbapenemase types, associated with divergent or novel integrons, punctuated the ongoing evolution of these bacteria.


Assuntos
Cromossomos Bacterianos/química , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , República Tcheca/epidemiologia , Monitoramento Epidemiológico , Expressão Gênica , Ilhas Genômicas , Genótipo , Hospitais , Humanos , Incidência , Integrons , Isoenzimas/genética , Isoenzimas/metabolismo , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-27855076

RESUMO

The aim of this study was to characterize the first cases and outbreaks of OXA-48-like-producing Enterobacteriaceae recovered from hospital settings in the Czech Republic. From 2013 to 2015, 22 Klebsiella pneumoniae isolates, 3 Escherichia coli isolates, and 1 Enterobacter cloacae isolate producing OXA-48-like carbapenemases were isolated from 20 patients. Four of the patients were colonized or infected by two or three different OXA-48-like producers. The K. pneumoniae isolates were classified into nine sequence types (STs), with ST101 being predominant (n = 8). The E. coli isolates were of different STs, while the E. cloacae isolate belonged to ST109. Twenty-four isolates carried blaOXA-48, while two isolates carried blaOXA-181 or blaOXA-232 Almost all isolates (n = 22) carried blaOXA-48-positive plasmids of a similar size (∼60 kb), except the two isolates producing OXA-181 or OXA-232. In an ST45 K. pneumoniae isolate and an ST38 E. coli isolate, S1 nuclease profiling plus hybridization indicated a chromosomal location of blaOXA-48 Sequencing showed that the majority of blaOXA-48-carrying plasmids exhibited high degrees of identity with the pOXA-48-like plasmid pE71T. Additionally, two novel pE71T derivatives, pOXA-48_30715 and pOXA-48_30891, were observed. The blaOXA-181-carrying plasmid was identical to the IncX3 plasmid pOXA181_EC14828, while the blaOXA-232-carrying plasmid was a ColE2-type plasmid, being a novel derivative of pOXA-232. Finally, sequencing data showed that the ST45 K. pneumoniae and ST38 E. coli isolates harbored the IS1R-based composite transposon Tn6237 containing blaOXA-48 integrated into their chromosomes. These findings underlined that the horizontal transfer of pOXA-48-like plasmids has played a major role in the dissemination of blaOXA-48 in the Czech Republic. In combination with the difficulties with their detection, OXA-48 producers constitute an important public threat.


Assuntos
Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , República Tcheca , Enterobacteriaceae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Transferência Genética Horizontal/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
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