Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

Two outbreaks of mixed etiology associated with central venous catheters inserted by phlebotomy in critical neonates

Staphylococcus aureus and coagulase-negative staphylococci are the main cause of sepsis in Neonatal Intensive Care Unit (NICU). Central venous catheters (CVCs) are an important part of critical neonates' treatment and are associated with sepsis. The aim of this study was to investigate two outbreaks caused by Staphylococcus aureus and Staphylococcus epidermidis associated with CVC inserted by phlebotomy in critical neonates. The surveillance was performed from January 2001 to December 2005 at the Brazilian NICU. The genotypic analysis of oxacillin susceptible S. aureus (OSSA) and oxacillin resistant S. epidermidis (ORSE) was performed based on pulsed-field gel electrophoresis (PFGE). Staphylococcus was the most frequent pathogen (65.8 percent) with highest incidence of CoNS (59.9 percent) followed by S. aureus (40.1 percent). During the five years of surveillance, there were two outbreaks detected, occurred in January-February/02 and August/02 and confirmed by PFGE analysis. The predisposing factors for infection corresponding to both outbreaks were: age <7 days, hospitalization > 7 days, and use of polyethylene CVC through dissection of vein (phlebotomy). This is the first relate of staphylococcal outbreaks associated with CVC inserted by phlebotomy in NICU. PFGE showed polyclonal spread of OSSA during both epidemic and endemic period, and two monoclonal outbreaks of ORSE in the same epidemic period of OSSA.

Correlation between API 20 STREP and multiplex PCR for identification of Enterococcus spp. isolated from Brazilian foods

We evaluated the suitability of API 20 STREP and multiplex PCR to speciate 52 Enterococcus spp. obtained from Brazilian foods. A high percentage of isolates (78.9 percent) presented discrepant results between evaluated tests. Similar results were obtained for six E. faecalis and five E. faecium. The PCR multiplex was more effective than API 20 STREP for complete identification of the isolates.

A identificação das espécies de 52 Enterococcus spp. isolados de amostras de alimentos foi realizada empregando-se duas metodologias: sistema API 20 STREP e PCR multiplex. Os resultados obtidos revelaram que 78,9 por cento dos isolados apresentaram resultados diferentes nos dois testes utilizados. Apenas seis E. faecalis e cinco E. faecium apresentaram resultados concordantes pelos dois métodos. PCR multiplex permitiu a identificação completa de um número significantemente maior de enterococos do que o sistema API 20 STREP.

Eletroforese em campo pulsante em bacteriologia - uma revisão técnica / Pulsed field gel electrophoresis use in bacteriology - a technical review

PFGE (pulsed field gel eletrophoresis) é a sigla usada para indicar qualquer técnica de eletroforese apropriada para separar grandes fragmentos de DNA, por meio da reorientação do DNA em gel pela ação de campos elétricos alternados. Esta técnica é reconhecida como padrão ouro para identificação de linhagens bacterianas, fúngicas e de protozoários. O principal objetivo deste estudo de revisão é o da elucidação dos fundamentos da técnica de PFGE ou eletroforese de campo pulsante aplicada em estudo com bactéria. Sua resolução depende de uma série de fatores como: voltagem, concentração de agarose, temperatura, solução tamponante, tempo de pulso e de corrida eletroforética. A compreensão dos mecanismos moleculares envolvidos nesse tipo de eletroforese é fundamental para a otimização e obtenção dos resultados apropriados.

Aplicaçöes da ribotipagem na epidemiologia molecular de infecçöes bacterianas / Ribotyping on molecular epidemiology of bacterial infections

A tipagem molecular do genoma bacteriano, na maioria das vezes, envolve a análise de fragmentos de restriçäo do DNA cromossômico. Desde que a ribotipagem foi descrita, em 1986, tem sido amplamente utilizada para analisar relaçöes taxonômicas e/ou epidemiológicas entre os diferentes grupos de organismos. A ribotipagem usa o padräo de restriçäo do opéron de RNA ribossômico (rrn) como ferramenta epidemiológica e tem fornecido ótimos resultados para a detecçäo de polimorfismo do comprimento dos fragmentos de restriçäo (RFLPs). O número de opérons rrn da bactéria está diretamente relacionado ao potencial discriminatório da técnica, fornecendo um maior ou menor número de bandas.