Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.

Emergence of clonal complex 5 (CC5) methicillin-resistant Staphylococcus aureus (MRSA) isolates susceptible to trimethoprim-sulfamethoxazole in a Brazilian hospital

In this study, genotyping techniques including staphylococcal chromosomal cassette mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and restriction-modification tests were used to compare the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered at two times within a 10-year interval (1998 and 2008) from a tertiary Brazilian hospital. In addition, the antimicrobial susceptibility profiles were analyzed. All 48 MRSA isolates from 1998 and 85.7% from 2008 (48/56 isolates) displayed multidrug-resistance phenotypes and SCCmec III. All but one of the 13 representative SCCmec III isolates belonged to CC8 and had PFGE patterns similar to that of the BMB9393 strain (Brazilian epidemic clone of MRSA; BEC). In 2008, we found an increased susceptibility to rifampicin and chloramphenicol among the SCCmec III isolates. In addition, we detected the entrance of diverse international MRSA lineages susceptible to trimethoprim-sulfamethoxazole (SXT), almost all belonging to CC5. These non-SCCmec III isolates were related to the USA 300 (ST8-SCCmec IV; PFGE-type B), USA 800 (ST5-SCCmec IV; subtype D1), USA 100 (ST5-SCCmec II; subtype D2), and EMRSA-3/Cordobes (ST5-SCCmec I, type C) clones. To the best of our knowledge, this is the first report of the emergence of isolates genetically related to the EMRSA-3/Cordobes clone in southeast Brazil. In this regard, these isolates were the most common non-SCCmec III MRSA in our institution, accounting for 8.9% of all isolates recovered in 2008. Thus, despite the supremacy of BEC isolates in our country, significant changes may occur in local MRSA epidemiology, with possible consequences for the rationality of MRSA empiric therapy.

Genotyping of methicillin-resistant Staphylococcus aureus isolates obtained in the Northeast region of Brazil

Methicillin-resistant Staphylococcus aureus (MRSA) is a major agent of hospital infections worldwide. In Brazil, a multiresistant MRSA lineage (ST239-SCCmecIIIA), the so-called Brazilian epidemic clone (BEC), has predominated in all regions. However, an increase in nosocomial infections caused by non-multiresistant MRSA clones has recently been observed. In the present study, 45 clinical isolates of MRSA obtained from a university hospital located in Natal city, Brazil, were identified by standard laboratory methods and molecularly characterized using staphylococcal chromosome cassette mec (SCCmec) typing and pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was carried out using CLSI methods. The MRSA isolates studied displayed a total of 8 different pulsed-field gel electrophoresis patterns (types A to H) with predominance (73 percent) of pattern A (BEC-related). However, MRSA harboring SCCmec type IV were also identified, 3 (7 percent) of which were genetically related to the pediatric clone - USA800 (ST5-SCCmecIV). In addition, we found a considerable genetic diversity within BEC isolates. MRSA displaying SCCmecIV are frequently susceptible to the majority of non-β-lactam antibiotics. However, emergence of multiresistant variants of USA800 was detected.

The first report in Brazil of severe infection caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA)

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emergent pathogen in Brazil. However, there are no data on the prevalence of CA-MRSA. We report here the first well-characterized case of severe life-threatening CA-MRSA infection in a child living in Rio de Janeiro city. The patient had many complications including hematogenous osteomyelitis and involvement of multiple sites requiring drainage of soft-tissue abscess, and pleural and pericardial empyema. The MRSA isolates recovered were genotyped using PFGE, SCCmec typing and multilocus sequence typing. Disk diffusion tests were performed following Clinical and Laboratory Standards Institute recommendations. In addition, the presence of Panton-Valentine leukocidin (PVL) was assessed by PCR amplification, using specific primers for lukF-pv (encoding for the F subunit of the PVL). The bacterial isolates were related to the ST30-SCCmecIV lineage (Oceania Southwest Pacific clone), a PVL producer CA-MRSA previously detected in Porto Alegre, RS, Brazil. Also, the isolates analyzed were susceptible to all non-â-lactam antibiotics tested. The present report demonstrates that disseminated CA-MRSA disease is also occurring in Rio de Janeiro. Thus, the empirical treatment of moderate or severe infections suspected of being associated with CA-MRSA needs to be reviewed in order to allow prompt initiation of an effective therapy that also covers these microorganisms.

Adenocarcinoma do apendice cecal. Relato de um caso do tipo colico. / Adenocarcinoma of the cecal appendix.Report of a case of the colic type

A proposito de um caso de adenocarcinoma do tipo colico do apendice cecal, operado no Hospital de Ensino da Faculdade de Medicina do ABC, e feita uma revisao do assundo, assinalando que, dentre os carcinomas do apendice, este tem tendencia a metastizacao por via linfatica e venosa, alem de se propagar por contiguidade. Praticamente destituido os sintomatologia propria, revela-se, na maioria das vezes, por manifestacoes de apendicite aguda. Sao ressaltadas as dificuldades de diagnostico macroscopico, o que faz com que a sua real natureza seja estabelecida, em geral, apos o exame histopatologico da peca cirurgica.A hemicolectomia direita e a operacao indicada e cujos melhores resultados sao obtidos como primeira intervencao ou dentro de 30 dias desta. No caso estudado, o paciente foi operado com o diagnostico pre e intra-operatorio de apendicite aguda e o exame histopatologico alem de confirma-lo, demonstrou a presenca da neoplasia. Indicada a reintervencao, a rapida propagacao da doenca neoplasica impediu a realizacao de qualquer procedimento desejado, demonstrando o grande potencial maligno da afeccao

Lisogenia em estreptococos do grupo A e avaliacao de meios de cultura para deteccao de bacteriofagos temperados obtidos desses microrganismos. / Lysogeny in group A streptococci and evaluation of culture media for detection of temperated bacteriophages obtained from these microorganisms

Cento e trinta e quatro amostras de estreptococos do grupo A foram examinadas quanto a ocorrencia de lisogenia. Utilizando-se quatro meios de cultura diferentes para o preparo do crescimento confluente da amostra indicadora em placas de agar, observamos uma porcentagem de deteccao de lisogenia de 14,2%. Dos 19 bacteriofagos obtidos, apenas 10 produziram lises na amostra indicadora preparada em quaisquer dos meios. Os restantes falharam para a producao de "plaque" em um ou mais meios. A taxa de lisogenia das amostras isoladas da orofaringe foi de 16,7% e 13,5% para as amostras obtidas de lesoes na pele. As porcentagens de amostras lisogenicas nos estreptococos M-tipaveis e nos Mnao tipaveis, para os microrganismos isolados da orofaringe, foram 16,7% e 20% respectivamente. Entretanto, para os estreptococos isolados de lesoes na pele, esta taxa foi de 24,2% para as amostras M-tipaveis e, 9,1% para as amostras M-nao tipaveis

Isolation of bacteriophages from Streptococcus pyogenes.

Tecnicas sao descritas para o isolamento de bacteriofagos, a partir de Streptococcus pyogenes e para a sua propagacao.Fagos temperados foram isolados a partir de 15 das 90 amostras bacterianas testadas.Mitomicina C foi empregada como agente induto

Hyaluronidase associated with temperate bacteriophages from Streptococcus pyogenes.

Utilizando uma microdeterminacao espectrofotometrica da hialuronidase, atividades hialuronidasicas foram encontradas em associacao com bacteriofagos temperados purificados, isolados, a partir de duas cepas de Streptococcus pyogenes. A enzima associada ao fago representou 4 a 8% da atividade enzimatica presente nos lisados de fago. A maior parte da atividade hialuronidasica presente nos lisados, consistia de enzima livre. Os 2 fagos nao necessitaram da adicao de hialuronidase para a penetracao na capsula de acido hialuronico da cepa indicadora