Comparison of DNA-extraction methods and Selective Enrichment broths on the detection of Salmonella Typhimurium in swine feces by polymerase chain reaction (PCR)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A simple method for the selective enrichment of endohedral metallofullerenes

The concentration of a polar solvent DMF extract was found to be very effective for the selective enrichment of endohedral metallofullerenes against empty fullerenes. As the solvent evaporated, endohedral metallofullerenes were effectively enriched in the solution, while most of empty fullerenes (especially C-60 and C-70) were precipitated because of their scant solubility in DMF. Matrix-assisted laser-desorption-ionization time-of-fligh mass spectrometry analysis indicated that the purity of endohedral metallofullerenes increased dramatically after concentration of the DMF extract solution. Upon transferring the extract into toluene, a solution containing significantly enriched endohedral metallofullerenes was obtained. The different solubilities of endohedral metallofullerenes versus empty fullerenes are considered to account for this selective enrichment of endohedral metallofullerenes.

Comparação dos caldos selenito cistina, tetrationato Muller-Kauffmann e Rappaport-Vassiliadis no isolamento de Salmonella Typhimurium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da reação em cadeia da polimerase e do isolamento bacteriológico convencional na detecção de Salmonella Dublin em amostras de fezes de bezerros infectados experimentalmente

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparação do isolamento microbiológico e da reação em cadeia da polimerase no diagnóstico de salmonelose em bezerros infectados experimentalmente com Salmonella Typhimurium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of three enrichment broths and five plating media for Salmonella detection in poultry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparação de diferentes etapas de enriquecimento seletivo no isolamento de Salmonella sp. a partir de fezes de suínos de terminação

A detecção de Salmonella sp. é fundamental nos programas de controle de salmonelose. Métodos de detecção mais eficientes permitem uma melhor determinação do nível de infecção dos rebanhos, um melhor entendimento da epidemiologia da infecção por Salmonella sp. e o desenvolvimento de programas de controle do patógeno, que visem a segurança biológica do alimento. Nos métodos convencionais, o enriquecimento seletivo é uma etapa crítica, pois inibe a microbiota competitiva e permite a multiplicação de Salmonella sp. Vários caldos de enriquecimento seletivo têm sido comparados quanto à eficiência na recuperação de Salmonella sp. a partir de alimentos, contudo existem poucos estudos relativos a fezes de suínos. O objetivo deste trabalho foi comparar caldos de enriquecimento seletivo para o isolamento de Salmonella sp. a partir de fezes de suínos. Numa primeira fase, amostras de fezes foram contaminadas artificialmente e os caldos Rappaport-Vassiliadis incubado a 42°C (RV), Tetrationato Müller-Kauffmann a 37°C (TMK37) e 42°C (TMK42), e Selenito Cistina (SC) a 37°C foram testados, em associação com meios sólidos seletivos: Rambach (RA), Xilose Lisina Tergitol 4 (XLT4), e Verde Brilhante Vermelho de Fenol Lactose Sacarose (VB). Na segunda fase os caldos RV, TMK37 e TMK42, semeados nos meios XLT4 e VB, foram testados com amostras naturalmente contaminadas. Na primeira fase o RV, TMK42 e TMK37 foram mais eficientes que o SC. No isolamento de Salmonella sp. em amostras naturalmente contaminadas os caldos TMK42 e RV foram superiores ao TMK37. O desempenho destes influenciou diretamente a capacidade seletiva e indicadora dos meios sólidos seletivos. No presente estudo, a associação TMK42/XLT4 demonstrou ser mais sensível, e a RV/XLT4 mais específica. O ágar VB também é recomendado para aumentar a probabilidade de detecção do patógeno. Desta forma os caldos RV e TMK42 e o ágar XLT4 e o VB foram considerados os mais indicados para a implantação de protocolos de detecção de Salmonella sp. em fezes suínas.

Comparação da eficiência dos caldos de enriquecimento seletivo no isolamento de Salmonella Dublin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparação de Meios de Enriquecimento e de Plaqueamento Utilizados na Pesquisa de Salmonella em Carcaças de Frango e Fezes de Aves

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pituitary tumours : all silent on the epigenetics front

Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to 'uncover' or 'unmask' silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.

Salmonella Virchow and Salmonella Weltevreden in a Random Survey of the Asian House Gecko, Hemidactylus frenatus, in Houses in Northern Australia.

Background: Salmonella enterica serotype Virchow is the most common cause of invasive nontyphoid salmonellosis in North Queensland, particularly in infants, but the zoonotic source is unknown. This study aimed at determining (i) the prevalence of the introduced Asian house gecko, Hemidactylus frenatus, in houses in North Queensland and (ii) whether they were a potential source of Salmonella Virchow. Methods: Asian house geckos were collected in a random survey of houses in Townsville, North Queensland. Gut contents underwent microbiological analysis within 2 h of removal using both direct plating and enrichment broth methods. Any organism found to be a presumptive Salmonella spp. was then sent to a reference lab for confirmation of genus/species, serotyping, and phage typing if indicated. Results: One hundred Asian house geckos were collected from 57 houses. Geckos were present in 100% of houses surveyed, and prevalence of Salmonella in large intestinal contents was 7% (95% confidence interval 2, 12%). Three serotypes were found: Virchow (phage type 8), Weltevreden, and an untypable subspecies 1 serotype 11:-:1,7. Conclusion: Since Salmonella Virchow (phage type 8) is associated with invasive disease, the introduced Asian house gecko may play a significant role in the epidemiology of sporadic salmonellosis in places invaded by these peridomestic reptiles. These results justify more detailed epidemiological studies on the role of the Asian house gecko in sporadic salmonellosis and development of evidence-based strategies to decrease this potential zoonotic hazard.

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

Avaliação microbiológica, detecção e susceptibilidade a antimicrobianos de potenciais enteropatógenos das famílias Enterobacteriaceae e Vibrionaceae em mexilhões Perna perna da praia de Itaipu, Niterói-RJ

Por serem organismos filtradores, os mexilhões devem ser extraídos para consumo somente de águas com padrões microbiológicos regulamentados pelo Conselho Nacional do Meio Ambiente, uma vez que intoxicações alimentares de origem bacteriana são as consequências mais comuns relacionadas ao consumo destes moluscos. Após a retirada dos costões, estes organismos passam por processos de fervura, lavagem, acondicionamento em sacos plásticos e transporte até a chegada ao mercado onde são comercializados; etapas estas, realizadas sem cuidados assépticos. Objetivando avaliar a qualidade microbiológica de mexilhões Perna perna coletados na praia de Itaipu, Niterói, RJ; após a sua fervura; e comercializados no Mercado São Pedro, foram realizadas contagens de coliformes totais e termotolerantes e pesquisa de Escherichia coli, Salmonella spp. e Vibrio spp. por metodologia convencional e molecular em amostras obtidas destes locais. Pelo teste de disco-difusão foi observado o perfil de resistência das cepas isoladas. Do total de amostras analisadas (27) apenas 3 dos mexilhões que sofreram processo de aferventação, 1 do comercializado e 3 do in natura, se encontravam dentro dos padrões aceitáveis pela legislação. Não houve diferença significativa entre as contagens de coliformes termotolerantes dos diferentes mexilhões analisados, mas sim entre os períodos seco e chuvoso. Somente uma das nove coletas de água mostrou-se própria para o cultivo de mexilhões (até 14 CF/mL). Foram isoladas e caracterizadas fisiológicamente, 77 estirpes da espécie E. coli, sendo confirmadas molecularmente por PCR os sorotipos EPEC, STEC e EAEC; 4 cepas Salmonella spp. sendo apenas uma confirmada por PCR; e das 57 cepas caracterizadas como Vibrio spp., 51 foram confirmadas por PCR, sendo 46 Vibrio spp., 2 V. cholerae, 1 V. vulnificus, 1 V. parahaemolyticus e 1 V. mimicus. Entre as estirpes de E. coli, 13% apresentaram multirresistência e 15,6% apresentaram resistência múltipla. A estirpe de Salmonella spp. se mostrou sensível a todos os antimicrobiados testados. Das estirpes de Vibrio spp. testadas, 68,6% apresentaram multirresistência e 72,5% apresentaram resistência múltipla. A partir da pesquisa de genes direto do caldo de enriquecimento foi possível detectar todos os genes pesquisados, com exceção para os sorotipos de Salmonella e V. cholerae. Baseado nos resultados do presente trabalho pode-se inferir que os mexilhões, amplamente comercializados no município de Itaipú, podem se constituir em risco para a saúde pública dos consumidores no Rio de Janeiro, necessitando dos órgãos competentes uma eficiente fiscalização nos pontos de venda e cultivo destes moluscos.