Typing of Pseudomonas aeruginosa from hospitalized patients: a comparison of susceptibility and biochemical profiles with genotype

Typing techniques are essential for understanding hospital epidemiology, permitting the elucidation of the source of infection and routes of bacterial transmission. Although DNA-based techniques are the "gold standard" for the epidemiological study of Pseudomonas aeruginosa, antibiotic profiles and biochemical results are used because they are easy to perform and to interpret and relatively inexpensive. Antibiotypes (susceptibility profiles) and biotypes (biochemical profiles) were compared to genotypes established by DNA restriction enzyme analysis in 81 clinical isolates of P. aeruginosa from three hospitals in Porto Alegre, Brazil. The epidemiological relationship among patients was also evaluated. Susceptibility and restriction profiles were discrepant in more than 50% of the cases, and many antibiotypes were observed among isolates from the same genotype. Furthermore, susceptibility profiles did not allow the distinction of isolates from unrelated genotypes. Since a large number of isolates (63%) yielded the same biochemical results, only 10 biotypes were detected, showing that this typing method has a low discriminatory power. On the other hand, DNA restriction enzyme typing allowed us to establish 71 distinct types. Epidemiological data about the relation among P. aeruginosa isolates were not conclusive. The results of the present study indicate that the only method that can establish a clonal relation is DNA restriction enzyme typing, whereas the other methods may cause misleading interpretations and are inadequate to guide proper infection control measures.

Characterization of bacteriophages infecting clinical isolates of Pseudomonas aeruginosa stored in a culture collection

Some clinical isolates of Pseudomonas aeruginosa stored in our culture collection did not grow or grew poorly and showed lysis on the culture plates when removed from the collection and inoculated on MacConkey agar. One hypothesis was that bacteriophages had infected and killed those clinical isolates. To check the best storage conditions to maintain viable P. aeruginosa for a longer time, clinical isolates were stored at various temperatures and were grown monthly. We investigated the presence of phage in 10 clinical isolates of P. aeruginosa stored in our culture collection. Four strains of P. aeruginosa were infected by phages that were characterized by electron microscopy and isolated to assess their ability to infect. The best condition to maintain the viability of the strains during storage was in water at room temperature. Three Siphoviridae and two Myoviridae phages were visualized and characterized by morphology. We confirmed the presence of bacteriophages infecting clinical isolates, and their ability to infect and lyse alternative hosts. Strain PAO1, however, did not show lysis to any phage. Mucoid and multidrug resistant strains of P. aeruginosa showed lysis to 50% of the phages tested.

Variação da microbiota natural e de pseudomonas aeruginosa em água mineral não carbonatada embalada em diferentes materiais durante o armazenamento a 30°c ± 1°c

Garrafas de PVC, polipropileno e vidro adequadamente limpas e sanificadas foram enxaguadas e enchidas com água mineral de uma fonte localizada no município de Lindóia-SP, e a seguir armazenadas a 30°C ± 1°C. Foram enumerados os microrganismos heterotróficos totais nos meios Ágar Padrão para Contagem e Ágar R2A imediatamente após engarrafamento. A variação da contaminação foi seguida através de exames periódicos. A variação da população de Pseudomonas aeruginosa foi estudada inoculando garrafas contendo água mineral com uma suspensão de P. aeruginosa ATCC 10145 de maneira a se obter uma contaminação de aproximadamente 10² UFC/ml. A população da bactéria foi avaliada periodicamente durante o tempo de armazenamento usando o meio Ágar Pseudomonas P. Houve aumento da população dos microrganismos heterotróficos totais nos primeiros 30 dias de armazenamento para depois diminuir de maneira irregular e ficar aproximadamente constante até completar 6 meses de observação. As contagens de P. aeruginosa aumentaram sensivelmente nas 2 primeiras semanas de estocagem diminuindo ligeiramente a seguir até atingir níveis próximos ao inicial. Não foram constatadas diferenças entre os 3 tipos de materiais de embalagem comparados.

Resistência antimicrobiana de Pseudomonas aeruginosa isolados de pescado e de cortes e de miúdos de frango

Pseudomonas aeruginosa isolados de peixes de água doce e de frangos foram submetidos ao teste de suscetibilidade aos antimicrobianos utilizando quatorze drogas, com o objetivo de determinar e confrontar os padrões de suscetibilidade deste microrganismo. As cepas oriundas de peixes pertenciam à coleção do Laboratório de Bacterioses/IV/UFRRJ. Para o isolamento das cepas, foram selecionados miúdos (fígado) e cortes (coxa e sobrecoxa) de frangos adquiridos em estabelecimentos comerciais no município do Rio de Janeiro. A metodologia de isolamento incluiu o enriquecimento em água peptonada, seguido de semeadura em Agar EMB e Agar GSP. Para as cepas oriundas de peixes, procedeu-se à reativação em água peptonada, seguida de reisolamento em Agar EMB. Colônias sugestivas foram transferidas para Agar TSI e LIA para avaliação das características metabólicas. A capacidade de produção de pigmento verde-azulado foi avaliada em Agar Mueller-Hinton e a da enzima citocromo-oxidase, em Agar Nutriente. O teste de suscetibilidade a antimicrobianos realizado nas 63 cepas revelou maiores percentuais de resistência para NAL e NIT (96,8%), TCY (93,6%), AMC (92,1%), CHL (90,5%) e SXT (85,7%), destacando-se a multirresistência dos isolados. A totalidade das cepas oriundas de frangos apresentou sensibilidade a CAZ e IPM e nos isolados de peixes a ATM, CAZ, IPM e AMK.

The adherence of Pseudomonas fluorescens to marble, granite, synthetic polymers, and stainless steel

The adherence of Pseudomonas fluorescens cells to nine food-processing contact surfaces was evaluated using the plate-count method. The surfaces include marble, granite, stainless steel, polyvinyl chloride, polyurethane, and silicone-coated cloth, which have been used only in a few studies concerning bacterial adherence. The number of cells adhered to the surfaces increased with contact time reaching 5.0-6.1 log CDM.cm-2 after 10 hours, which can be considered a well established adherence process. The number of adhered cells doubled in 29.5 minutes and 23.5 minutes on stainless steel and thin polyvinyl chloride-coated cloth, respectively. For the other surfaces, this value was 9.8 minutes on average. Marble, granite, thick polyvinyl-coated cloth, double-faced rugous polyurethane, and silicone-coated cloth were not different (p < 0.05) in their ability to adhere cells (CFU/cm²) after 2 and 10 hours. The surfaces that had higher percentage of similarity in the adhesion level and higher log CFU/cm² of adhered cells were double-faced rugous polyurethane, silicone-coated cloth, and granite. The surfaces showed very different microtopography characteristics when viewed using scanning electron microscopy. This experiment showed the importance of using appropriate materials for food contact during processing, which will affect the cleaning and sanitation procedures.

Influência da velocidade de circulação do leite na adesão de Pseudomonas aeruginosa sobre aço inoxidável

A influência da velocidade de circulação do leite na adesão bacteriana de Pseudomonas aeruginosa foi avaliada em teste de uso simulado por meio de um modelo de circuito de processamento de leite. O circuito é composto por uma tubulação de aço inoxidável AISI 304, com 1,9 cm de diâmetro e 5,8 m de comprimento, além de um tanque de 25 L, utilizado como reservatório do produto e das soluções sanitizantes. O reservatório foi acoplado a uma bomba centrífuga de ½ HP, para impulsionar o alimento ou soluções de higienização pelo sistema equipado com cupons de prova em aço inoxidável nas formas cilíndrica, cotovelo 90º e T. As velocidades de circulação foram de 0,5, 1,0 e 1,5 m.s-1, correspondentes a fluxo turbulento com número de Reynolds de 14.000, 28.000 e 42.000, respectivamente. Quando se utilizou velocidade de 0,5 m.s-1, permaneceram aderidas à superfície 10,7% das células. Já nas velocidades e 1,0 e 1,5 m.s-1 as porcentagens de adesão foram de 5,36 e 4,9%, respectivamente, o que demonstra uma menor remoção de células aderidas à medida que o fluxo diminui, permitindo assim que mais células permaneçam aderidas na linha de produção, o que pode favorecer a formação de biofilmes.

Effectiveness of cleaning and sanitizing procedures in controlling the adherence of Pseudomonas fluorescens, Salmonella Enteritidis, and Staphylococcus aureus to domestic kitchen surfaces

The effectiveness of cleaning and sanitizing procedures in controlling Staphylococcus aureus, Salmonella Enteritidis, and Pseudomonasfluorescens adhered to granite and stainless steel was evaluated. There was no significant difference (p > 0.05) in the adherence of pure cultures of these microorganisms to stainless steel. The numbers of P. fluorescens and S. Enteritidis adhered to granite were greater (p < 0.05) than the numbers of S. aureus. Additionally, the adherence of P. fluorescens was similar to the adherence of S. Enteritidis on granite surface. In a mixed culture with P. fluorescens, S aureus adhered less (p < 0.05) to stainless steel surfaces (1.31 log CFU.cm-2) than when in a pure culture (6.10 log CFU.cm-2). These results suggest that P. fluorescens inhibited the adherence of S. aureus. However, this inhibition was not observed in the adherence process for granite. There was a significant difference (p < 0.05) between the number of adhered cells before and after pre-washing for S. aureus on stainless steel and granite surfaces, and after washing with detergent for all microorganisms and surfaces. The efficiency of the cleaning plus sanitizing procedures was not significantly different (p > 0.05) between the surfaces. However, a significant difference was observed (p < 0.05) between the sanitizer solutions. Sodium hypochlorite and peracetic acid were more bactericidal (p < 0.05) than a quaternary ammonium compound. With regard to microorganisms, S. aureus was the least resistant to the sanitizers. These results show the importance of good cleaning and sanitization procedures to prevent bacterial adherence and biofilm formation.

Avaliação da lipase extracelular de Pseudomonas cepacia para purificação em sistema bifásico aquoso

O presente trabalho teve como objetivo a produção de lipase a partir de Pseudomona cepacia por fermentação líquida em biorreator do tipo Bioflo III. As fermentações foram conduzidas a 150 rpm durante 96 horas a 30 °C. Analisou-se a atividade enzimática em diferentes condições de temperatura (20 a 50 °C) e pH (3,0 a 11,0), e obtiveram-se 37 °C e 8,0, as condições ótimas, respectivamente. Para avaliar a estabilidade térmica, a enzima foi incubada em temperaturas de 40, 50 e 60 °C durante 120 minutos. Em uma segunda etapa, foram realizados experimentos preliminares para verificar as condições adequadas de partição da enzima, bem como sua estabilidade e condições ótimas de hidrólise frente às modificações de temperatura e pH. Foram preparadas soluções de PEG 1500, 4000 e 6000 a 50% p/p e soluções tampão fosfato de pHs 6, 7 e 8. Foi feita a caracterização de um sistema bifásico aquoso (SBA) a partir da preparação de soluções estoques de PEG com massas molares de 1500, 4000 e 6000 (50% w/w) e tampão fosfato pH 6,7 e 8,0 (20% w/w de KH2PO4/K2HPO4). Esta caracterização do SBA posteriormente poderá ser utilizada para partição de lipases, bem como de biomoléculas que estejam dentro dessa faixa de pH.

Chemical sanitizers to control biofilms formed by two Pseudomonas species on stainless steel surface

The biofilm formation of Pseudomonas aeruginosa and Pseudomonas fluorescens on AISI 304 stainless steel in the presence of reconstituted skim milk under different temperatures was conducted, and the potential of three chemical sanitizers in removing the mono-species biofilms formed was compared. Pseudomonas aeruginosa cultivated in skim milk at 28 °C presented better growth rate (10.4 log CFU.mL-1) when compared with 3.7 and 4.2 log CFU.mL-1 for P. aeruginosa and P. fluorescens cultivated at 7 °C, respectively. Pseudomonas aeruginosa formed biofilm when cultivated at 28 °C. However, only the adhesion of P. aeruginosa and P. fluorescens was observed when incubated at 7 °C. The sodium dichloroisocyanurate was the most efficient sanitizer in the reduction of the adhered P. aeruginosa cells at 7 and 28 °C and those on the biofilm, respectively. The hydrogen peroxide was more effective in the reduction of adhered cells of P. fluorescens at 7 °C.

Production of medium-chain-length polyhydroxyalkanoate by Pseudomonas oleovorans grown in sugary cassava extract supplemented with andiroba oil

Pseudomonas oleovorans were grown on sugary cassava extracts supplemented with andiroba oil for the synthesis of a mediumchain- length polyhydroxyalkanoate (PHA MCL). The concentration of total sugars in the extract was approximately: 40 g/L in culture 1, 15 g/L in cultures 2 and 3, and 10 g/L in culture 4. Supplementation with 1% andiroba oil and 0.2 g/L of (NH4)2HPO4 was performed 6.5 hours after growth in culture 3, and supplementation with the same amount of andiroba oil and 2.4 g/L of (NH4)2HPO4 was performed at the beginning of growth in culture 4. The synthesis resulted mainly in 3-hydroxy-decanoate and 3-hydroxy-dodecanoate units; 3-hydroxy-butyrate, 3-hydroxy-hexanoate; and 3-hydroxy-octanoate monomers were also produced but in smaller proportions. P. oleovorans significantly accumulated PHA MCL in the deceleration phase of growth with an oxygen limitation but with sufficient nitrogen concentration to maintain cell growth. The sugary cassava extract supplemented with andiroba oil proved to be a potential substrate for PHA MCL production.

An unusual postharvest spotting disease of the comercial mushroom, Agaricus bisporus, caused by a novel pathovar of Pseudomonas tolaasii

An unusual postharvest spotting disease of the commercial mushroom, Agaricus bisporus, which was observed on a commercial mushroom farm in Ontario, was found to be caused by a novel pathovar of Pseudomonas tolaasii. Isolations from the discoloured lesions, on the mushroom pilei, revealed the presence of several different bacterial and fungal genera. The most frequently isolated genus being Pseudomonas bacteria. The most frequently isolated fungal genus was Penicillium. Of the bacteria and fungi assayed for pathogenicity to mushrooms, only Pseudomonas tolaasii was able to reproduce the postharvest spotting symptom. This symptom was typically reproduced 1 to 7 days postharvest, when mushroom pilei were inoculated with 101 to 105 cfu. Of the fungi tested for pathogenicity only a Penicillium sp. and Verticillium fungicola were shown to be pathogenic, however, neither produced the postharvest spotting symptom. The Pseudomonas tolaasii strain isolated from the postharvest lesions differed from a type culture (Pseudomonas tolaasii ATCC 33618) in the symptoms it produced on Agaricus bisporus pilei under the same conditions and at the same inoculum concentration. It was therefore designated a pathovar. This strain also differed from the type culture in its cellular protein profile. Neither the type culture, nor the mushroom pathogen was found to contain plasmid DNA. The presence of plasmid DNA is therefore not responsible for the difference in pathogenicity between the two strains.

Identificación de antígenos proteicos inmunocloneinantes de Pseudomonas aeruginosa, reconocidas por sueros de pacientes infectados (por) María del Rocío Galván García

Tesis (Maestría en Ciencias, con especialidad en Microbiología Médica) U.A.N.L.

Detección por PCR multiplexde pseudomonas aeruginosa, proteus mirabilis y malassezia SPP. en caninos con otitis externa.

Tesis (Maestría en Ciencia Animal) UANL, 2011.

Detección por PCR multiplex de pseudomonas aeruginosa, proteus mirabilis y malassezia SPP en caninos con otitis externa.

Tesis (Maestría en Ciencia Animal) UANL, 2011.

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

Affiliation: André Dagenais: Centre hospitalier de l'Université de Montréal/ Hôtel-Dieu, Département de médecine, Université de Montréal. Yves Berthiaume: Médecine et spécialités médicales, Faculté de médecine