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.

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.

A comparison of virulence patterns and in vivo fitness between hospital- and community-acquired methicillin-resistant Staphylococcus aureus related to the USA400 clone.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates genetically related to the CA-MRSA clone MW2/USA400 (ST1-SCCmecIV lineage) from the United States have emerged in hospitals in Rio de Janeiro and are associated with nosocomial bloodstream infections. To understand the virulence mechanisms involved in the adaptability of ST1 isolates as a hospital pathogen in Rio de Janeiro, we compared the virulence traits and fitness properties of the Brazilian isolates with those displayed by the CA-MRSA isolates from the United States. Similar to the USA400 from the United States, all the Brazilian isolates tested carried the genes encoding SEH and LukDE. In contrast, none of the Brazilian isolates carried the lukSF PVL, sea, sec, and sek genes. Competition experiments in mice demonstrated a significant increase in the fitness for the CA-MRSA isolates MW2 and USA400-0051 from the United States compared to other isolates. In the foreign body animal model, 83 % more North-American bacterial cells were recovered compared to the Brazilian ST1 isolates. Differences in gene expression of important virulence factors were detected. Transcription of rnaIII and psmα3 was increased about two-fold in the isolates from the United States, and sasG about two-fold in the Brazilian isolates. Thus, it is possible that the virulence attenuation observed among the Brazilian hospital isolates, associated with the acquisition of multiple resistant determinants, are consequences of microevolutionary events that contributed to the necessary fitness adjustment of this lineage, allowing a typically community-acquired MRSA (MW2/USA400) to emerge as a successful hospital pathogen (Brazilian ST1-SCCmecIV).

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.

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.

Restriction modification (RM) tests associated to additional molecular markers for screening prevalent MRSA clones in Brazil.

In this study, we associated the restriction modification (RM) tests to the polymerase chain reaction (PCR) detection of molecular markers (SCCmec III, seh, agr II-SCCmec IV, and lukSF) for revealing the main methicillin-resistant Staphylococcus aureus (MRSA) clones circulating in Brazil. This simple and rapid approach allowed a precise classification of the MRSA analyzed when compared with pulsed-field gel electrophoresis (PFGE) data.

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.

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-beta-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.

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-beta-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.

Biofilm formation and prevalence of lukF-pv, seb, sec and tst genes among hospital- and community-acquired isolates of some international methicillin-resistant Staphylococcus aureus lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial agent of biopolymer-associated infections, and isolates of S. aureus can produce different virulence factors, including potent toxins. The biofilm formation and accumulation by certain international MRSA lineages were analysed, and the toxic shock syndrome-associated genes (tst, seb and sec) among these isolates were assessed. In addition, the presence of lukF-pv (encoding the F-subunit of Panton-Valentine leukocidin (PVL)) was investigated. Most of the MRSA isolates tested were capable of forming biofilm on polystyrene surfaces, but lacked the superantigen toxin genes that were tested. PVL was rarely detected among the hospital isolates analysed.

Emergence in Brazil of methicillin-resistant Staphylococcus aureus isolates carrying SCCmecIV that are related genetically to the USA800 clone.

An increasing incidence of nosocomial infections caused by non-multiresistant methicillin-resistant Staphylococcus aureus (nMMRSA) has been reported worldwide. The present study genotyped nMMRSA isolates obtained from hospitals in two cities in Brazil. The hospital isolates displayed pulsed-field gel electrophoresis (PFGE) patterns that were similar to those of the USA100 (ST5-SCCmecII) and USA 800 (ST5-SCCmecIV) strains, which are related to the New York/Japan and paediatric clones, respectively. Carriage of SCCmecIV and the classification by multilocus sequence typing (MLST) of a representative of this PFGE pattern in clonal complex 5 (CC5) confirmed the genetic relationship of the Brazilian isolates with USA800. The USA800-related Brazilian isolates were responsible for severe nosocomial infections in compromised adults and elderly patients in Brazil. A higher growth rate, an ability to form biofilm on inert polystyrene surfaces and the presence of the egc locus may have contributed, at least in part, to the fitness of these organisms as global nosocomial pathogens.

Molecular characterization of methicillin-resistant Staphylococcus aureus disseminated in a home care system.

OBJECTIVE: To study colonization with methicillin-resistant Staphylococcus aureus in a home care service during a 4-month period. DESIGN: Prospective study. SETTING: A home care service located in Rio de Janeiro, Brazil. PARTICIPANTS: Patients admitted to the home care service during this period, their household contacts, and health care workers (HCWs). METHODS: Swab specimens from the anterior nares were collected from each patient in the 3 groups at admission. Screening was repeated every 7 days. MRSA was detected using a mecA probe, and the clonality of isolates was evaluated by molecular methods, primarily pulsed-field gel electrophoresis. RESULTS: Of the 59 study patients, 9 (15.3%) had MRSA colonization detected; these cases of colonization were classified as imported. Only 1 (2.0%) of the 50 patients not colonized at admission became an MRSA carrier (this case of colonization was classified as autochthonous). Two (0.9%) of 224 household contacts and 16 (7.4%) of 217 HCWs had MRSA colonization. Cross-transmission from patient to HCW could be clearly demonstrated in 8 cases. The great majority of MRSA isolates belonged to the Brazilian epidemic clone. CONCLUSIONS: MRSA colonization was common in the home care service analyzed. The fact that the majority of MRSA isolates obtained were primarily of nosocomial origin (and belonged to the so-called Brazilian epidemic clone) substantiated our findings that all but 1 patient had already been colonized before admission to the home care service. Only cross-transmission from patients to healthcare workers could be verified. On the basis of these results, we believe that a control program built on admission screening of patients for detection of MRSA carriage could contribute to the overall quality of care.

Genetic relatedness between group B streptococci originating from bovine mastitis and a human group B Streptococcus type V cluster displaying an identical pulsed-field gel electrophoresis pattern.

Twenty isolates of group B streptococcus (GBS) were recovered from the milk of cows with bovine mastitis on three farms located in the south and south-east of Brazil between 1987 and 1988. These isolates were characterised by molecular methods and compared with a collection of 103 human GBS isolates from colonised and infected patients in the same region between 1980 and 2003. Some of the bovine isolates shared identical or similar pulsed-field gel electrophoresis (PFGE) patterns with a PFGE clone of human GBS type V. In addition, these bovine and human isolates also possessed the same ribotype. Multilocus sequence typing (MLST) of representative isolates confirmed the genetic relationship between the human and bovine GBS isolates with identical PFGE patterns, which clustered in the same ST-26 clonal complex. These data support the hypothesis that some bovine GBS strains are related closely to human isolates and may infect humans, or vice versa. Further comparative genomic analyses of GBS isolates from bovine and human origins are required to investigate this hypothesis further.

Detection and molecular characterization of a gentamicin-susceptible, methicillin-resistant Staphylococcus aureus (MRSA) clone in Rio de Janeiro that resembles the New York/Japanese clone.

Staphylococcus aureus is the leading cause of hospital-acquired infections in many countries, and multiple factors contribute to the ability of these bacteria to disseminate and spread in hospitals. In Brazil it has been demonstrated that a multiresistant methicillin-resistant S. aureus clone, the so-called Brazilian epidemic clone, is widespread geographically. This clone was first detected in 1992 in Brazil, and recently from many other countries within South America, Europe and Asia. The study describes the detection of a gentamicin-susceptible heterogeneous MRSA clone that resembles another MRSA clone widely spread in US and Japanese hospitals, and supports the premise that the detection of heterogeneous MRSA isolates by some recommended methods is a challenging task that may, occasionally, result in MRSA misidentification.

Clonal spread of methicillin-resistant Staphylococcus aureus in a large geographic area of the United States.

Methicillin resistance in Staphylococcus aureus has rapidly increased over the last two decades. This increase is paralleled by the emergence of unique multi-resistant MRSA clones. In Brazil, Argentina, Uruguay, Portugal and Czech Republic a specific MRSA clone is widely spread, the so-called Brazilian epidemic clone. Another epidemic clone, the Iberian clone, is disseminated in Spain, Portugal, Belgium, Scotland, Italy, Germany and New York. Thus, a large number of hospital-acquired infections have been caused by specific MRSA clones. Using different molecular techniques for MRSA typing, we verified that two unique epidemic clones are spread over large geographic area in the US. In addition, we showed that a previously described MRSA clone type, the New York clone (I::A:A), is widely spread beyond the New York frontiers.

Contamination of expressed human breast milk with an epidemic multiresistant Staphylococcus aureus clone.

Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major cause of outbreaks in intensive care units. Infants make up a sector of the population that presents a high risk for MRSA infections. Mother-to-infant transmission has been indicated as a possible cause of MRSA infections in neonates. The occurrence and characteristics of MRSA in samples of banked human milk were investigated by selective culture, antibiogram and pulsed-field gel electrophoresis. MRSA contamination was found in 11% of 500 samples of expressed, fresh-frozen milk from 500 different donors at five Brazilian milk banks. The great majority of the contaminated samples passed breast milk quality control criteria for dispensing as raw milk under Brazilian and American guidelines. Most of the MRSA isolates belonged to the Brazilian epidemic clone, which is reported to be widespread in several Brazilian states, in Argentina and in Portugal. It is concluded that expressed breast milk can be a reservoir of multiresistant S. aureus epidemic clones. Studies are necessary to assess the source of contamination and potential role of MRSA-contaminated milk in the transmission of MRSA to neonates.

Occurrence of methicillin-resistant and -susceptible Staphylococcus aureus within a single colony contributing to MRSA mis-identification.

Many methods have been described for the detection of methicillin-resistant Staphylococcus aureus (MRSA), but the homogeneous or heterogeneous expression of methicillin resistance affects the reliability of those methods. This study demonstrates that close association between methicillin-susceptible S. aureus (MSSA) and MRSA strains in the host colonisation site can present additional problems for the detection of MRSA in clinical laboratories, which may contribute to failure in the control of MRSA infection in hospital. Worse, this association may also account for the emergence of MRSA during antibiotic therapy.

Emergence of mupirocin resistance in multiresistant Staphylococcus aureus clinical isolates belonging to Brazilian epidemic clone III::B:A.

Mupirocin is a topical antimicrobial agent that has been successfully used to eradicate methicillin-resistant Staphylococcus aureus from the anterior nares and other sites of patients and health care personnel. This report describes the acquisition of a novel mupirocin resistance gene (ileS) by an epidemic MRSA clone that is geographically widespread in Brazil.