A.M.S. Figueiredo

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.

Pulsed-Field Gel Electrophoresis of Human Group B Streptococci Isolated in Brazil

Abstract The present study addresses epidemiological aspects of Brazilian human group B streptococci (GBS). GBS (103 isolates) were serotyped with specific rabbit anticapsular antibodies by double diffusion in agarose gels. They represented 3 serotypes: 26 II, 41 III, and 36 V. Thereafter, the strains were characterized by pulsed-field gel electrophoresis (PFGE) of DNA treated with SmaI. DNA restriction band sizes were compared and displayed 54 PFGE profiles that were arranged into 18 patterns. Of the predominant patterns detected for the 41 type III isolates 4 were observed in 15 strains from individuals with infections whereas only 3 were identified in 22 streptococci from healthy carriers. Such differences did not separate types II and V streptococci from carriers and patients. The PFGE method is a sensitive, precise, and powerful tool for discriminating streptococcal strains for epidemiological purposes.

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.

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.

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 USA300 (ST8-SCCmec IV; PFGE-type B), USA800 (ST5-SCCmec IV; subtype D1), USA100 (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.

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.

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.

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.