Biofilm production by clinical isolates of Pseudomonas aeruginosa and structural changes in LasR protein of isolates non biofilm-producing.

INTRODUCTION: Biofilm production is an important mechanism for the survival of Pseudomonas aeruginosa and its relationship with antimicrobial resistance represents a challenge for patient therapeutics. P. aeruginosa is an opportunistic pathogen frequently associated to nosocomial infections, especially in imunocompromised hosts. OBJECTIVES: Analyze the phenotypic biofilm production in P. aeruginosa isolates, describe clonal profiles, and analyze quorum sensing (QS) genes and the occurrence of mutations in the LasR protein of non-biofilm producing isolates. METHODS: Isolates were tested for biofilm production by measuring cells adherence to the microtiter plates. Clonal profile analysis was carried out through ERIC-PCR, QS genes were by specific PCR. RESULTS: The results showed that 77.5% of the isolates were considered biofilm producers. The results of genotyping showed 38 distinct genetic profiles. As for the occurrence of the genes, 100% of the isolates presented the lasR, rhlI and rhlR genes, and 97.5%, presented the lasI gene. In this study nine isolates were not biofilm producers. However, all presented the QS genes. Amplicons related to genes were sequenced in three of the nine non-biofilm-producing isolates (all presenting different genetic similarity profile) and aligned to the sequences of those genes in P. aeruginosa strain PAO1 (standard biofilm-producing strain). Alignment analysis showed an insertion of three nucleotides (T, C and G) causing the addition of an amino acid valine in the sequence of the LasR protein, in position 53. CONCLUSION: The modeling of the resulting LasR protein showed a conformational change in its structure, suggesting that this might be the reason why these isolates are unable to produce biofilm.

Biofilm production by clinical isolates of Pseudomonas aeruginosa and structural changes in LasR protein of isolates non biofilm-producing

ABSTRACT Introduction: Biofilm production is an important mechanism for the survival of Pseudomonas aeruginosa and its relationship with antimicrobial resistance represents a challenge for patient therapeutics. P. aeruginosa is an opportunistic pathogen frequently associated to nosocomial infections, especially in imunocompromised hosts. Objectives: Analyze the phenotypic biofilm production in P. aeruginosa isolates, describe clonal profiles, and analyze quorum sensing (QS) genes and the occurrence of mutations in the LasR protein of non-biofilm producing isolates. Methods: Isolates were tested for biofilm production by measuring cells adherence to the microtiter plates. Clonal profile analysis was carried out through ERIC-PCR, QS genes were by specific PCR. Results: The results showed that 77.5% of the isolates were considered biofilm producers. The results of genotyping showed 38 distinct genetic profiles. As for the occurrence of the genes, 100% of the isolates presented the lasR, rhlI and rhlR genes, and 97.5%, presented the lasI gene. In this study nine isolates were not biofilm producers. However, all presented the QS genes. Amplicons related to genes were sequenced in three of the nine non-biofilm-producing isolates (all presenting different genetic similarity profile) and aligned to the sequences of those genes in P. aeruginosa strain PAO1 (standard biofilm-producing strain). Alignment analysis showed an insertion of three nucleotides (T, C and G) causing the addition of an amino acid valine in the sequence of the LasR protein, in position 53. Conclusion: The modeling of the resulting LasR protein showed a conformational change in its structure, suggesting that this might be the reason why these isolates are unable to produce biofilm.

Analysis of biofilm production by clinical isolates of Pseudomonas aeruginosa from patients with ventilator-associated pneumonia. / Análise da produção de biofilme por isolados clínicos de Pseudomonas aeruginosa de pacientes com pneumonia associada à ventilação mecânica.

OBJECTIVE: To phenotypically evaluate biofilm production by Pseudomonas aeruginosa clinically isolated from patients with ventilator-associated pneumonia. METHODS: Twenty clinical isolates of P. aeruginosa were analyzed, 19 of which were from clinical samples of tracheal aspirate, and one was from a bronchoalveolar lavage sample. The evaluation of the capacity of P. aeruginosa to produce biofilm was verified using two techniques, one qualitative and the other quantitative. RESULTS: The qualitative technique showed that only 15% of the isolates were considered biofilm producers, while the quantitative technique showed that 75% of the isolates were biofilm producers. The biofilm isolates presented the following susceptibility profile: 53.3% were multidrug-resistant, and 46.7% were multidrug-sensitive. CONCLUSION: The quantitative technique was more effective than the qualitative technique for the detection of biofilm production. For the bacterial population analyzed, biofilm production was independent of the susceptibility profile of the bacteria, demonstrating that the therapeutic failure could be related to biofilm production, as it prevented the destruction of the bacteria present in this structure, causing complications of pneumonia associated with mechanical ventilation, including extrapulmonary infections, and making it difficult to treat the infection.

OBJETIVO: Avaliar fenotipicamente a produção de biofilme por isolados clínicos de Pseudomonas aeruginosa de pacientes com pneumonia associada à ventilação mecânica. MÉTODOS: Foram analisados 20 isolados clínicos de P. aeruginosa, sendo 19 provenientes de amostras clínicas de aspirado traqueal e uma de lavado broncoalveolar. A avaliação da capacidade de P. aeruginosa em produzir biofilme foi verificada por duas técnicas, sendo uma qualitativa e outra quantitativa. RESULTADOS: A técnica qualitativa mostrou que apenas 15% dos isolados foram considerados produtores de biofilme, enquanto que a quantitativa demonstrou que 75% dos isolados foram produtores de biofilme. Os isolados produtores de biofilme apresentaram o seguinte perfil de suscetibilidade: 53,3% eram multidroga-resistentes e 46,7% eram multidroga-sensíveis. CONCLUSÃO: A técnica quantitativa foi mais eficaz para detecção da produção de biofilme em comparação com a qualitativa. Para a população bacteriana analisada, a produção de biofilme independeu do perfil de suscetibilidade das bactérias, demonstrando que a falha terapêutica pode estar relacionada com a produção de biofilme, por impedir a destruição das bactérias presentes nesta estrutura, ocasionando complicações da pneumonia associada à ventilação mecânica, incluindo infecções extrapulmonares, e dificultando o tratamento da infecção.

Análise da produção de biofilme por isolados clínicos de Pseudomonas aeruginosa de pacientes com pneumonia associada à ventilação mecânica / Analysis of biofilm production by clinical isolates of Pseudomonas aeruginosa from patients with ventilator-associated pneumonia

RESUMO Objetivo: Avaliar fenotipicamente a produção de biofilme por isolados clínicos de Pseudomonas aeruginosa de pacientes com pneumonia associada à ventilação mecânica. Métodos: Foram analisados 20 isolados clínicos de P. aeruginosa, sendo 19 provenientes de amostras clínicas de aspirado traqueal e uma de lavado broncoalveolar. A avaliação da capacidade de P. aeruginosa em produzir biofilme foi verificada por duas técnicas, sendo uma qualitativa e outra quantitativa. Resultados: A técnica qualitativa mostrou que apenas 15% dos isolados foram considerados produtores de biofilme, enquanto que a quantitativa demonstrou que 75% dos isolados foram produtores de biofilme. Os isolados produtores de biofilme apresentaram o seguinte perfil de suscetibilidade: 53,3% eram multidroga-resistentes e 46,7% eram multidroga-sensíveis. Conclusão: A técnica quantitativa foi mais eficaz para detecção da produção de biofilme em comparação com a qualitativa. Para a população bacteriana analisada, a produção de biofilme independeu do perfil de suscetibilidade das bactérias, demonstrando que a falha terapêutica pode estar relacionada com a produção de biofilme, por impedir a destruição das bactérias presentes nesta estrutura, ocasionando complicações da pneumonia associada à ventilação mecânica, incluindo infecções extrapulmonares, e dificultando o tratamento da infecção.

ABSTRACT Objective: To phenotypically evaluate biofilm production by Pseudomonas aeruginosa clinically isolated from patients with ventilator-associated pneumonia. Methods: Twenty clinical isolates of P. aeruginosa were analyzed, 19 of which were from clinical samples of tracheal aspirate, and one was from a bronchoalveolar lavage sample. The evaluation of the capacity of P. aeruginosa to produce biofilm was verified using two techniques, one qualitative and the other quantitative. Results: The qualitative technique showed that only 15% of the isolates were considered biofilm producers, while the quantitative technique showed that 75% of the isolates were biofilm producers. The biofilm isolates presented the following susceptibility profile: 53.3% were multidrug-resistant, and 46.7% were multidrug-sensitive. Conclusion: The quantitative technique was more effective than the qualitative technique for the detection of biofilm production. For the bacterial population analyzed, biofilm production was independent of the susceptibility profile of the bacteria, demonstrating that the therapeutic failure could be related to biofilm production, as it prevented the destruction of the bacteria present in this structure, causing complications of pneumonia associated with mechanical ventilation, including extrapulmonary infections, and making it difficult to treat the infection.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil.

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosa isolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding ß-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosa isolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and ß-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-β-lactamase-producing isolates

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL) genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-ß-lactamase-producing isolates.

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-ß-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-ß-lactamase (MBL) genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil.

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopeptide resistance by conjugative transfer of VanA elements.

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopetide resistance by conjugative transfer of VanA elements.

Polymorphism of the rDNA and tDNA loci in clinical isolates of Pseudomonas aeruginosa: a perspective for molecular epidemiology surveillance

The Gram-negative bacterium Pseudomonas aeruginosa has a wide environmental and ecological distribution. It is an opportunistic pathogen that acquires resistance to multiple antimicrobial agents and can infect plants, animals and humans. We used rDNA and tDNA PCR markers to characterize the bacterial diversity of P. aeruginosa strains isolated at a Brazilian teaching hospital (Oswaldo Cruz University Hospital, Recife, Brazil) between March 2003 and February 2004. Clonal groups of P. aeruginosa clinical isolates were identified from different patients in different hospital units using either rDNA or tDNA markers, or a combination of both in a duplex PCR. These PCR-typing methods together with drug-resistance profiles were used to trace the distribution of antibiotic resistant P. aeruginosa clones and to identify cross-infection of the same patient with a different bacterial clone after being moved to a different hospital unit. The data presented here demonstrates a rapid, reliable and useful method for epidemiological surveillance that can contribute to the control of P aeruginosa infections in hospital environments.

Prevalência de microorganismos em infecções urinárias nosocomiais e de comunidade durante um período de cinco anos / Prevalence of microorganisms in nosocomial and community-acquired urinary tract infections during a five years period

O objetivo deste estudo foi descrever a distribuição de microorganismos em infecções do Trato Urinário (ITU) em pacientes internados no Hospital Universitário Oswaldo Cruz (HUOC). Foram analisadas 1908 culturas de pacientes com ITU, internados e atendidos no HUOC, 899 casos de origem hospitalar e 1009 da comunidade. As infecções por leveduras do gênero Candida spp corresponderam a 9,4por cento. Os mais frequentes Gram-negativos, que corresponderm a 78,4por cento do total de infecções, foram Escherichia coli e Klebsiella pneumoniae. Dentre os gram-positivos, as espécies mais freqüentes foram Stapylococus aureus, Streptococcus agalactiae e Staphylococcus sapropyticcus. Na comunidade, E. coli (53,2por cento), K. pneumoniae (15,9por cento) e S.agalactiae (7,7por cento) foram as espécies bacterianas mais representativas. O percentual de infecções por Candida spp de origem hospitalar foi aproximadamente quatro vezes maior que o percentual da Candida spp proveniente da comunida. As freqüências de ITUs nosocomiais em homens e mulheres foram semelhantes 46,8por cento e 53,2por cento, respectivamente), ao contrario da diferença caracteristica observada em ITUs de comunidade (71.1por cento em mulheres e 28,9por cento em homens)

Immobilization of lipase from "Fusarium solani" FS1

Lipase from "Fusarium solani" FS1 was immobilized by covalent attachment to polyacrylamide beads and onto magnetized Dacron, retaining 12(per cent) and 97(per cent) of activity, respectively. Lipase was also entrapped within polyacrylamide beads, retaining 53(per cent) of activity. Investigations of the kinetic characteristics of the immobilized derivates using triolein as substrate showed that lipase immobilized onto polyacrilamide beads and Dracon did not follow Michaelis-Menten kinetics.

Production of extracellular lipase by the phytopathogenic fungus "Fusarium solani" FS1

A Brazilian strain of "Fusarium solani" was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.L(E-1) of lipase after 72 hours of cultivation at 25ºC in shake-flask at 120rpm in a medium containing 3(per cent) (w/v) peptone plus 0.5(per cent)(v/v) olive oil. Glucose (1(per cent) w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3(per cent)(w/v) resulted in a reduced lipase production while increased olive oil concentration (above o.5(per cent)) did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30ºC and a good enzyme stability (80(per cent) activity retention) was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50ºC. Lipase activity was stimulated by addition of n-hexane to the culture medium supernatatnts, in contrast to incubation with water-soluble solvents