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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.

Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA) has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR). Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

Background: Methicillin-resistant staphylococci can colonize and cause diseases in companion animals. Unfortunately, few molecular studies have been carried out in Brazil and other countries with the aim of characterizing these isolates. Consequently, little is known about the potential role of companion animals in transmitting these resistant bacteria to humans. In this work we searched for mecA gene among Staphylococcus isolates obtained from nasal microbiota of 130 healthy dogs and cats attended in a veterinary clinic located in the west region of Rio de Janeiro. The isolates recovered were identified to the species level and characterized using molecular tools., Results: A community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolate related to USA1100 (Southwest Pacific clone) and susceptible to all non-β-lactams was detected in a cat (1.7%, 1/60). Another coagulase-positive isolate harboring mecA was recovered from a dog (1.4%, 1/70) and identified as Staphylococcus pseudintermedius (MRSP) related to the European clone (ST71). The two isolates of Staphylococcus conhii subsp. urealyticus (1.4%, 1/70 dogs and 1.7%, 1/60 cats), similarly to the MRSP isolate, also presented high-level multiresistance. The majority of the methicillin-resistant coagulase-negative staphylococci recovered were Staphylococcus saprophyticus (5.7%, 4/70 dogs and 6.7%, 4/60 cats) and all clustered into the same PFGE type., Conclusions: This work demonstrates that mecA-harboring Staphylococcus isolates are common members of the nasal microbiota of the healthy companion animals studied (9.2%, 12/130 animals), including some high-level multiresistant isolates of S. pseudintermedius and S. conhii subsp. urealyticus. The detection, for the first time in South America, of USA1100-related CA-MRSA and of ST71 MRSP (European clone), colonizing companion animals, is of concern. Both S. pseudintermedius and S. aureus are important agents of infections for animals. The USA1100 CA-MRSA is a causative of severe and disseminated diseases in healthy children and adults. Additionally, MRSP is a nosocomial pathogen in veterinarian settings. It had already been demonstrated that the virulent ST71 MRSP is geographically spread over Europe and USA, with potential for zoonotic infections.

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.

Community-acquired infections by methicillin-resistant Staphylococcus aureus (CA-MRSA) in the absence of classic risk factors for MRSA diseases have been reported in different continents. In the article presented here, using molecular typing methods as pulsed-field gel electrophoresis, staphylococcal cassette chromosome mec typing, and multilocus sequence typing, we characterized CA-MRSA isolates from Rio Janeiro and Porto Alegre. The results indicated the presence of international CA-MRSA clones in these 2 Brazilian cities. In addition, Panton-Valentine leukocidin and a number of staphylococcal enterotoxin encoding genes were accessed in these MRSA isolates by polymerase chain reaction detection.

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.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a therapeutic problem. In the present study, the molecular characterization by pulsed-field gel electrophoresis of MRSA isolates collected from a university hospital revealed that the predominant variant of the Brazilian epidemic clonal complex (BECC) was responsible for the increase in the incidence of MRSA strains, which reached 28% in 1998. It was verified that this predominant variant of the BECC displayed an enhanced ability to produce biofilm on inert polystyrene surfaces and to adhere to and invade epithelial airway cells. These results indicate that MRSA strains belonging to the BECC have evolved advantageous properties that might play a role in their predominance as international nosocomial pathogens.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged in the southwestern Pacific, North America, and Europe. These S. aureus isolates frequently shared some genetic characteristics, including the SCCmec type IV and lukS-lukF genes. In this paper we show that typical CA-MRSA isolates have spread to South America (Brazil).

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.

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., (Copyright 2003 The Hospital Infection Society)

The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.

Methicillin-resistant Staphylococcus aureus (MRSA) infections have been increasing at an alarming rate world-wide. MRSA epidemics due to the clonal spread of multi-resistant isolates have been described. In this paper we show the absolute predominance of MRSA strains from the Brazilian epidemic clone in a hospital in the Northeast region of Brazil and the emergence of a vancomycin and teicoplanin heterogeneous resistant subpopulation among these isolates.

Staphylococcus aureus is a major bacterial pathogen involved in a wide range of diseases varying from infections to toxaemia. Staphylococcal food-poisoning syndrome is caused by ingestion of bacterial enterotoxins. These toxins are microbial superantigens and may also be virulence factors involved in staphylococcal infection. This study determined the enterotoxin types and pulsed-field gel patterns found among S. aureus isolates obtained from food handlers in community or hospital-located kitchens. Staphylococcal enterotoxin C was the most frequent enterotoxin produced. The data also suggested horizontal spread of ent genes among isolates belonging to the Brazilian epidemic MRSA clone III::B:A. A subclone of MRSA clone III::B:A was isolated from two hospital kitchen workers. This was the first report of this clone from a hospital in Teresina, Piaui State, although the presence of this MRSA clone has already been reported in six other Brazilian cities.

Methicillin-resistant Staphylococcus aureus (MRSA) are important nosocomial pathogens Diseases caused by these resistant bacteria frequently are serious and there is a need to control the spread of epidemic MRSA clones in hospitals. However, detection is complicated by the fact that expression of the resistance is variable and, commonly, heterogeneous within strains. The reliability of several tests recommended to discriminate heterogeneous MRSA isolates from borderline-resistant and susceptible strains was evaluated. Screening for growth on agar with methicillin 25 mg/L was the only method that detected all MRSA strains tested, but screening on agar with methicillin 10 mg/L or oxacillin 6 mg/L detected all but one of 10 heterogeneously resistant strains tested. None of the borderline-resistant nor any truly susceptible staphylococci tested grew on any of these screening plates.

In a previous study we described the extensive geographic spread of a multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) clone in hospitals located in the southern, southeastern, and northern parts of Brazil. In this study we used a set of molecular markers to demonstrate the emergence of a novel MRSA clone distinct from but closely related to the widely spread Brazilian epidemic clone. The new MRSA clone caused an outbreak among acquired immunodeficiency syndrome patients in a Brazilian hospital specializing in tropical diseases and human immunodeficiency virus- and human T-cell leukemia virus (HLTV)-related infections.

Staphylococcus aureus isolates from five large teaching hospitals and one medium-size community hospital located in geographically distant parts of Brazil, in the south and southeast (Rio de Janeiro, Niteroi, Sao Paulo, Porto Alegre) and in the north (Manaus), were tested for their antibiotic resistance patterns and genetic backgrounds. Eighty-five of the 152 isolates were identified as methicillin-resistant S. aureus (MRSA) by using a combination of an agar dilution screen and a mecA gene-specific DNA probe. All MRSA isolates were resistant to penicillin, erythromycin, gentamicin, oxacillin, and cephalothin, and the majority of isolates (74%) were also resistant to chloramphenicol, sulfamethoxazole-trimethoprim, ciprofloxacin, and clindamycin as well and were susceptible only to vancomycin. Isolates obtained from hospitals in Sao Paulo, Rio de Janeiro, Niteroi, and Porto Alegre (1,600 km from one another) and Manaus (3,700 km from Rio de Janeiro) were examined by a variety of molecular fingerprinting techniques: the nature of the mecA polymorph and Tn554 attachment sites and restriction fragment length polymorphism of genomic DNAs after SmaI restriction and separation of the digested DNA by pulsed-field gel electrophoresis. The overwhelming majority of the isolates shared a common pulsed-field gel electrophoresis pattern and carried mecA polymorph III in combination with Tn554 pattern B, indicating the presence of a single, epidemic MRSA clone spread over large geographic distances of Brazil.

Cultures of heterogeneous strains of methicillin resistant Staphylococcus aureus are composed of several subpopulations of cells that differ in their level of antibiotic resistance and frequencies. Experimental evidence is presented indicating that the number of these subpopulations, their resistance level and the frequency with which they are represented in a culture are strain-specific and reproducible with a surprising degree of precision from one culture to another. This implies a genetic control of population structure in these bacteria.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infection. Outbreaks of infection caused by these pathogens are generally considered to be traceable to introduction of single strains into a hospital population. A large outbreak of bacteremic disease that recently occurred in our neonatal intensive care unit (11 episodes in 10 patients) involved 9 low birth weight infants and was associated with serious infection (4 episodes of meningitis). To determine the role of a single point source in this outbreak, isolates were characterized based on phenotypic and genotypic analyses. Phenotypic analysis included assessing hemolytic activity, phage typing, antimicrobial susceptibility testing and methicillin resistance population analysis. Genotypic analysis included assessment of plasmid profiles, dot-blot hybridization, restriction enzyme fragment pattern analysis and hybridization analysis of chromosomal DNA using a panel of staphylococcal gene probes. This analysis established that at least two distinct strains of MRSA were responsible for disease during this outbreak. This experience demonstrates the potential for MRSA to cause severe disease in the neonatal intensive care unit and indicates that the epidemiology of MRSA outbreaks is more complex than the spread of a single strain of bacteria.

To define the stability of methicillin-resistant Staphylococcus aureus (MRSA) in vivo, 22 isolates collected at one New York institution in 1989 and 1990 were studied. All 22 belonged to one of two distinct methicillin-resistant phenotypes (class 3 or 2), which were precisely identified as belonging to two distinct genotypes. Genotypic classification was based on restriction analysis of chromosomal DNA with EcoRI and HindIII and Southern analysis of ClaI digests using two DNA probes. One was specific for the mec gene; the other was specific for transposon Tn554. The findings suggest that the MRSA isolates studied were representative of two genetically distinct MRSA "clones," each with a unique strain-specific methicillin-resistant phenotype that is stable under the conditions of invasive disease, carriage, and spread from patient to patient.

The mec gene of a number of clinical methicillin-resistant Staphylococcus aureus isolates exhibiting a variety of heterogeneous expression modes was selectively inactivated by allelic replacement mutagenesis. While the resistance level of each of the transformants was reduced, the methicillin MIC for these transformants was well above the MIC for susceptible laboratory strains of S. aureus and was similar to the methicillin MIC for many contemporary clinical isolates which did not react with the mec-specific DNA probe but which showed a low or borderline level of resistance to methicillin. A number of those strains had no detectable beta-lactamase, and for about half of the isolates that did carry plasmid-borne beta-lactamase, elimination of the plasmid caused only partial reduction of the methicillin MIC or no reduction at all. The findings suggest that many contemporary strains of staphylococci harbor a combination of at least three distinct beta-lactam resistance mechanisms: (i) the mechanism related to the acquisition of the foreign mec gene and (ii) a beta-lactamase-dependent and (iii) a beta-lactamase-independent mechanism, each one of which can provide a certain degree of resistance against penicillinase-resistant beta-lactam antibiotics.