Nosocomial and community infections due to class A extended-spectrum β-lactamase (ESBLA)-producing Escherichia coli and Klebsiella spp. in southern Brazil

OBJECTIVES: To determine the prevalence of class A extended spectrum β-lactamases (ESBL)-producing Escherichia coli and Klebsiella spp., and to investigate clonality among ESBL-producing isolates of nosocomial and community infections. METHODS: The study involved 354 nosocomial infections samples and 992 community infections samples, obtained between 2003 and 2006 at Caxias do Sul, RS. The detection of ESBL was performed by the disk-diffusion test. Presence of blaCTX-M, blaSHV and blaTEM β-lactamase genes was evaluated by PCR, and genomic typing was determined by pulsed-field gel electrophoresis analysis. RESULTS: Higher frequency of ESBL-producing isolates were detected among nosocomial samples of E. coli (6.7 percent) and Klebsiella (43.7 percent), than those obtained from community infections (0.4 percent and 2.6 percent). blaTEM and blaCTX were the most prevalent ESBL gene families in both E. coli and Klebsiella isolates. Different pulsotypes were obtained among ESBL-producing E. coli and 11 clones for Klebsiella spp., which occurred over the years and in different hospital wards. Among ESBL-producing K. pneumoniae, 74.3 percent transferred ESBL genes by conjugation and exhibited concomitant decreased aminoglycosides susceptibility. CONCLUSION: ESBL-producing E. coli, and especially K. pneumoniae are essentially a nosocomial problem, and their dissemination to the community is relatively limited. The great genetic variability observed among ESBL-producing bacteria indicates polyclonal spread and high transference of ESBL genes between bacteria in the hospital environment. This information is of paramount importance for nosocomial infection control.

Aeromonas associated diarrhoeal disease in south Brazil: prevalence, virulence factors and antimicrobial resistance

Aeromonas were isolated from 27 (6.6 percent) of 408 patients admitted with acute gastroenteritis in two hospitals at Rio Grande do Sul, Brazil. Isolates were classified as A. hydrophila (51.8 percent), A. caviae (40.8 percent), and A. veronii biotype sobria (7.4 percent). The highest prevalence of Aeromonas associated infections occurred in lactants and children. Virulence genes (aerA -aerolysin/hemolysin, ahpA -serine-protease, satA - glycerophospholipid-cholesterol acyltransferase, lipA -lipase, and ahyB -elastase) and virulence factors (hemolytic, proteolitic, lipolitic activities, and biofilm formation) were identified in most A. hydrophila and A. veronii biotype sobria isolates, with lower frequencies on A. caviae. All Aeromonas isolates were resistant to ampicillin, ticarcillin/clavulanic acid, cephalotin, and cephazolin, and most of them (>70 percent) exhibited resistance to imipenem, carbenicillin, amoxillin/sulbactan, and piperacillin. Multiple-resistance, more than four antibiotics, was evidenced in 29.6 percent of the isolates. The most efficient antibiotics were the quinolones (ciprofloxacin and norfloxacin), and the aminoglycosides (amikacin and netilmicin).

Aeromonas foram isoladas de 27 (6.6 por cento) dos 408 pacientes admitidos com gastroenterite aguda em dois hospitais do Rio Grande do Sul, Brasil. Os isolados foram classificados com A. hydrophila (51.8 por cento), A. caviae (40.8 por cento), e A. veronii biotype sobria (7.4 por cento). A maior prevalência de Aeromonas ocorreu em lactantes e crianças. Genes (aerA -aerolisina/hemolisina, ahpA -serina-protease, satA - glicerofosfolipidio-colesterol aciltransferase, lipA -lipase, e ahyB -elastase) e factores (atividade hemolítica, proteolítica, lipolítica, e formação de biofilme) de virulência foram identificados na maioria dos isolados de A. hydrophila e A. veronii biotype sobria, com freqüências menores em A. caviae. Todos os isolados de Aeromonas apresentaram resistência a ampicilina, ticarcilina/ácido clavulânico, cefalotina e cefazolina, e a maior parte (>70 por cento) exibiram resistência a imipenem, carbenicilina, amoxacilina/sulbactam e piperacilina. Resistência múltipla foi evidenciada em 29,6 por cento dos isolados. Os antibióticos mais eficientes foram as quinolonas (ciprofloxacina e norfloxacina) e os aminoglicosídicos (amicacina e netilmicina).

Intracellular polyamine pools, oligopeptide-binding protein A expression, and resistance to aminoglycosides in Escherichia coli

The role of intracellular free polyamine (putrescine and spermidine) pools in multiple resistance to aminoglycoside antibiotics was investigated among in vitro selected kanamycin-resistant Escherichia coli J53 mutants expressing diminished oligopeptide-binding protein (OppA) levels and/or defective ornithine decarboxylase (ODC) activity. The results suggest that diminished OppA content, but not defective ODC activity expression, increased the relative concentration of free spermidine as compared to the wild type strain. Moreover, by adding exogenous polyamines or polyamine synthesis inhibitors to cultures with different mutant strains, a direct relationship between the intracellular OppA levels and resistance to kanamycin was revealed. Collectively these results further suggest a complex relation among OppA expression, aminoglycoside resistance and polyamine metabolism.

High frequency of auxotrophy in clinical isolates of Proteus mirabilis harboring an R plasmid

We report a novel phenomenon of high genetic instability, related to auxotrophy, in strains of Proteus mirabilis. Among P. mirabilis strains harboring the R plasmid Kept in our laboratory collection, and some freshly isolated strians from clinical material, 54 per cent of the samples presented auxotrophy at frequencies higher than 10(-3). Prototrophic closes gave rise to auxotrophic ones at frequencies not explainable by the usual mutation mechanisms. The instability mainly affected the carbamoyl phosphate synthetase gene (car), which leads to a double requirement for arginine and uracil for growth in minimal medium. Other genes were also affected, at a lower frequency. The car mutation does not revert to prototrophy. A similar phenomenon of instability was induced in Escherichia coli strain HB 101 upon introduction of a drug-resistance plasmid from P. mirabilis. We have ruled out the hypothesis of a transposon in the generation of auxotrophy.

A review of protease secretion instability in Proteus mirabilis

Proteus mirabilis secreta uma única protease que é uma metalo-enzima alcalina. Descrevemos aqui alguns fatores fisiológicos que afetam a secreçäo de protease em clones isolados. Mais de cem diferentes isolados clínicos foram examinados quanto a capacidade de secretar protease. Verificou-se que algumas linhagens herdam o determinante de protease como um caráter estável (säo denominadas linhagens estáveis); outras linhagens exigem um fenótipo muito instável (denominadas linhagens instáveis) com 10% das colônias-filhas perdendo o caráter de produçäo de protease. Nunca foi observada a reversäo do fenótipo protease negativa. Por outro lado, a introduçäo de fatores R em células produtoras de protease reduziu a atividade proteásica em cerca de 50%, o que sugere uma interferência na expressäo e/ou secreçäo de protease. Embora haja evidências de que um locus da protease tenha localizaçäo extracromossômica, näo foi demosntrada a presença de plasmídios nas linhagens instáveis. Um modo que pode explicar os vários dados é apresentado

Behavior of transposon Tn1 IN Proteus mirabilis

A freqüência de transposiçäo do Tn1 do plasmídio pTH10 para o cromossomo da célula hospedeira foi maior em Proteus mirabilis que em Escherichia coli. Contudo pode-se supor que os valores obtidos em P. mirabilis foram subestimados desde que o plamídio doador do transposoma é instavél na linhagem PG1342. Experimentos de hibridaçäo sugerem que existem mais do que uma inserçäo por cromossomo de P. mirabilis, mas análises adicionais säo necessárias para a localizaçäo dessas inserçöes. Se o transposon T n1 se insere em um ponto quente do cromossomo da célula hospedeira, esse alvo näo causou auxotrofia em nenhum dos clones testados de PG1342::Tn1. Transposiçöes entre plasmídios albergados por P. mirabilis parecem näo ocorrer em níveis maiores do que para o cromossomo, confrome relatados para E. coli. Outrossim, as freqüências de inserçäo do Tn1 variam de acordo com o DNA alvo. A seleçäo de um sítio de inserçäo ocorre diferentemente nos dois gêneros, sugerindo que fatores dos hospedeiros têm uma participaçäo importante nessa seleçäo. Por outro lado, Tn1 mostrou inserçäo preferencial em outrom transposon (Tn402), independentemente do hospedeiro. Neste caso a inserçäo do Tn1 näo é estável, levando à excisäo do transposon ampicilina