Use of molecular epidemiology to monitor the nosocomial dissemination of methicillin-resistant Staphylococcus aureus in a University Hospital from 1991 to 2001

Methicillin-resistant Staphylococcus aureus (MRSA) has been the cause of major outbreaks and epidemics among hospitalized patients, with high mortality and morbidity rates. We studied the genomic diversity of MRSA strains isolated from patients with nosocomial infection in a University Hospital from 1991 to 2001. The study consisted of two periods: period I, from 1991 to 1993 and period II from 1995 to 2001. DNA was typed by pulsed-field gel electrophoresis and the similarity among the MRSA strains was determined by cluster analysis. During period I, 73 strains presented five distinctive DNA profiles: A, B, C, D, and E. Profile A was the most frequent DNA pattern and was identified in 55 (75.3%) strains; three closely related and four possibly related profiles were also identified. During period II, 80 (68.8%) of 117 strains showed the same endemic profile A identified during period I, 18 (13.7%) closely related profiles and 18 (13.7%) possibly related profiles and, only one strain presented an unrelated profile. Cluster analysis showed a 96% coefficient of similarity between profile A from period I and profile A from period II, which were considered to be from the same clone. The molecular monitoring of MRSA strains permitted the determination of the clonal dissemination and the maintenance of a dominant endemic strain during a 10-year period and the presence of closely and possibly related patterns for endemic profile A. However, further studies are necessary to improve the understanding of the dissemination of the endemic profile in this hospital.

Inactivation of Staphylococcus aureus and Salmonella enteritidis in tryptic soy broth and caviar samples by high pressure processing

We studied the action of high pressure processing on the inactivation of two foodborne pathogens, Staphylococcus aureus ATCC 6538 and Salmonella enteritidis ATCC 13076, suspended in a culture medium and inoculated into caviar samples. The baroresistance of the two pathogens in a tryptic soy broth suspension at a concentration of 10(8)-10(9) colony-forming units/ml was tested for continuous and cycled pressurization in the 150- to 550-MPa range and for 15-min treatments at room temperature. The increase of cycle number permitted the reduction of the pressure level able to totally inactivate both microorganisms in the tryptic soy broth suspension, whereas the effect of different procedure times on complete inactivation of the microorganisms inoculated into caviar was similar.

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-β-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.

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%) of pattern A (BEC-related). However, MRSA harboring SCCmec type IV were also identified, 3 (7%) 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.

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.

High prevalence of methicillin resistance and PVL genes amongStaphylococcus aureus isolates from the nares and skin lesions of pediatric patients with atopic dermatitis

Staphylococcus aureus is highly prevalent among patients with atopic dermatitis (AD), and this pathogen may trigger and aggravate AD lesions. The aim of this study was to determine the prevalence of S. aureus in the nares of pediatric subjects and verify the phenotypic and molecular characteristics of the isolates in pediatric patients with AD. Isolates were tested for antimicrobial susceptibility, SCCmectyping, and Panton-Valentine Leukocidin (PVL) genes. Lineages were determined by pulsed-field gel electrophoresis and multilocus sequence typing (MLST). AD severity was assessed with the Scoring Atopic Dermatitis (SCORAD) index. Among 106 patients, 90 (85%) presented S. aureus isolates in their nares, and 8 also presented the pathogen in their skin infections. Two patients had two positive lesions, making a total of 10 S. aureusisolates from skin infections. Methicillin-resistant S. aureus(MRSA) was detected in 24 (26.6%) patients, and PVL genes were identified in 21 (23.3%), including 6 (75%) of the 8 patients with skin lesions but mainly in patients with severe and moderate SCORAD values (P=0.0095). All 24 MRSA isolates were susceptible to trimethoprim/sulfamethoxazole, while 8 isolates had a minimum inhibitory concentration (MIC) to mupirocin >1024 μg/mL. High lineage diversity was found among the isolates including USA1100/ST30, USA400/ST1, USA800/ST5, ST83, ST188, ST718, ST1635, and ST2791. There was a high prevalence of MRSA and PVL genes among the isolates recovered in this study. PVL genes were found mostly among patients with severe and moderate SCORAD values. These findings can help clinicians improve the therapies and strategies for the management of pediatric patients with AD.

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.

CONTROLE DE Staphylococcus aureus EM CHARQUES (JERKED BEEF) POR CULTURAS INICIADORAS

Jerked beef - JB - é um produto cárneo curado, salgado e seco ao sol, derivado de um típico produto cárneo brasileiro - o charque. Ambos carecem de estudos que orientem seu aprimoramento. Staphylococcus spp. têm sido reportado como o gênero predominante na microbiota do produto, o que evidencia o risco de desenvolvimento de linhagens enterotoxigênicas de S. aureus. Foram empregadas duas linhagens inócuas de estafilococos na elaboração de JB, a fim de avaliar sua influência sobre o desenvolvimento de S. aureus por mecanismo competitivo ou pela produção de bacteriocinas. Os resultados demonstraram que ambas inibiram o desenvolvimento do patógeno tanto in vitro como durante o processamento do produto. Não se observou produção de compostos inibitórios pelas linhagens iniciadoras, ficando a explicação para a inibição observada restrita ao mecanismo competitivo. Este trabalho permitiu demonstrar a possibilidade de aumentar a segurança e padronização de JB pelo emprego de culturas bacterianas selecionadas.

Quantificação de coliformes, Staphylococcus aureus e mesófilos presentes em diferentes etapas da produção de queijo frescal de leite de cabra em laticínios

O objetivo do presente estudo foi acompanhar a produção de queijo frescal de leite de cabra, avaliando a qualidade higiênica do processamento pela quantificação de coliformes, S. aureus e mesófilos totais. A produção de três diferentes lotes de queijo foi acompanhada, sendo coletadas amostras de várias etapas do processamento. Além disso, amostras de queijo pertencente ao lote acompanhado foram coletadas na prateleira de um estabelecimento comercial durante seu período de validade. Os suabes coletados foram semeados em ágar sangue ovino; as amostras de água foram submetidas à Colimetria; as demais amostras foram submetidas à contagem de S. aureus, coliformes e mesófilos totais por meio de protocolos de rotina. Verificou-se que, apesar da pasteurização ter diminuído consideravelmente as populações microbianas presentes no leite cru, a falta de sanificação adequada de um equipamento que entrava em contato com o leite cru e que dá acesso ao tanque de coagulação, resultou na recontaminação da matéria-prima. Ao final do processamento, o queijo encontrava-se dentro dos padrões exigidos pela legislação, contudo a elevada contagem de mesófilos totais sugere que sejam melhoradas as medidas de sanitização durante o processamento, a fim de garantir a qualidade higiênica e uma maior vida de prateleira ao queijo produzido.

Quality of mussels cultivated and commercialized in Ubatuba, SP, Brazil: monitoration Bacillus cereus and Staphylococcus aureus growth after post-harvest processing

Aiming at improving the quality of Perna perna mussels cultivated and commercialized in Ubatuba, SP, Brazil, the growth and elimination of Staphylococcus aureus and Bacillus cereus artificially inoculated in mussels were studied. The inoculation was carried out in "in natura" and pre-cooked mussels for 30 min, and after that the mussels were kept for 10 hours at room temperature (25 ± 1 °C) and under refrigeration (7 ± 1 °C). Six thermal treatments were evaluated: three using steam (5, 10 and 15 minutes) and three in boiling water (5, 10 and 15 minutes), in order to find the best time/temperature binomial to provide pathogenic control. Yield and physical-chemical and sensory characteristics were evaluated. All thermal treatments were efficient to eliminate microorganisms in 2 logarithmic cycles. However, the boiling water treatments presented better results than the steam treatments. The physical-chemical and sensory analyses did not show statistical differences among the thermal treatments studied. The best performances were reached in the shortest times of heat exposure. Overall, the treatments in boiling water presented better results than the steam treatments.

Theoretical modelling of Staphylococcus aureus growth in a cooked meat product kept at ambient temperature using temperature profiles of selected Mexican cities

A theoretical model is used to predict the growth of Staphylococcus aureus in a pasteurized meat product kept at ambient temperatures for several hours. For this purpose, the temperature profiles of some cities of Mexico were combined with literature data on the kinetics of S. aureus growth. As shown by theoretical predictions, if the food is kept at ambient temperature, the average daily temperature may not give accurate predictions.

Formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo

O objetivo desta pesquisa consistiu em avaliar a formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo. As variáveis em estudo consistem no cultivo monoespécie e combinado, dos referidos microrganismos e nas temperaturas de 4, 7 e 18 °C. Recipientes contendo 1000 mL de leite, densidade populacional de 10(5) UFC.mL-1 de cada microrganismo e 10 cupons de aço inoxidável (10 × 20 mm) foram lacrados e armazenados, sob agitação de 60 rpm, por um período de 10 dias. As análises ocorreram a cada 48 horas. Células sésseis de A.hydrophila e S. aureus foram enumeradas através do plaqueamento seletivo em ágar m-Aeromonas selective e Baird-Parker, respectivamente. Estudos sobre o tempo de geração, enumeração de células planctônicas e observação dos cupons através da microscopia eletrônica de varredura foram conduzidos. S. aureus, em monocultivo, formou biofilme a 18 °C e a 7 °C. Para 4 °C, foi observado um processo de adesão. A presença de A. hydrophila reduziu o desempenho de S. aureus. Nesta condição de cultivo multiespécie houve formação de biofilme a 18 °C. A. hydrophila, tanto em monocultivo quanto em presença de S. aureus, formou biofilme em todas as condições pesquisadas.

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.

Enterotoxigenic potential of Staphylococcus aureus isolated from Artisan Minas cheese from the Serra da Canastra - MG, Brazil

This study aimed to evaluate the presence of enterotoxigenic S. aureus in the endogenous starter and in Artisan Minas cheeses from the Serra da Canastra. Sixteen samples of endogenous starters and cheese were collected during the rainy and dry seasons. The isolation and enumeration of S. aureus were performed using the PetrifilmTM-Rapid S. aureus Plate Count method. The presence of enterotoxin in the cheese samples was analyzed by the Optimal Sensitivity Plate (OSP) method and the ELFA-VIDAS®-Staph enterotoxin-II assay. S. aureus strains were tested for their ability to produce enterotoxins using the Optimal Sensitivity Plate (OSP) method and the polymerase chain reaction (PCR) assay for the classical enterotoxin genes. The Optimal Sensitivity Plate (OSP) method data showed that staphylococcal enterotoxin A (SEA) was detected in 75% of the cheese samples, but no toxin was detected with the ELFA-VIDAS method. It was found that 12.5% of the isolated strains produced staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin C (SEC). When using the the polymerase chain reaction (PCR) assay, only one isolate was found to harbor an enterotoxin gene, contrary our expectations. However, discrepancies between the immunological and molecular assays are not uncommon. Despite the fact that most isolates did not produce classical enterotoxins, high S. aureus counts in the cheese samples causes concern since there is a risk of the presence of non-classical enterotoxins.

Inactivation of Staphylococcus aureus in raw salmon with supercritical CO2 using experimental design

Abstract Considering the microbial safety of consumption of raw foods (Asian food), this study aimed to explore the inactivation S. aureus in raw salmon by supercritical CO2 treatment (SC-CO2). For this purpose, experimental design methodology was employed as a tool to evaluate the effects of pressure (120-220 bar), the depressurization rate (10 to 100 bar.min–1) and the salmon:CO2 mass relation (1:0.2 to 1:1.0). It was observed that the pressure and the depressurization rate was statistically significant, i.e. the higher the system pressure and depressurization rate, the greater the microbial inactivation. The salmon: CO2 mass relation did not influence the S. aureus inactivation in raw salmon. There was a total reduction in S. aureus with 225 bar, a depressurizing rate of 100 bar.min–1, a salmon: CO2 mass relation of 1:0.6, for 2 hours at 33 °C.