Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: epidemiological and molecular aspects

Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase-negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCC mec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin-resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin-resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil.     

Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.     

The effects of subinhibitory concentrations of costus oil on virulence factor production in Staphylococcus aureus

Aim: To determine the antimicrobial activity of costus (Saussurea lappa) oil against Staphylococcus aureus, and to evaluate the influence of subinhibitory concentrations of costus oil on virulence‐related exoprotein production in staph. aureus. Methods and Results: Minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of costus oil against 32 Staph. aureus strains ranged from 0.15 to 0.6 μl ml^?1. The MIC₅₀ and MIC₉₀ were 0.3 and 0.6 μl ml^?1, respectively. Western blot, haemolytic, tumour necrosis factor (TNF) release and real‐time RT‐PCR assays were performed to evaluate the effects of subinhibitory concentrations of costus oil on virulence‐associated exoprotein production in Staph. aureus. The data presented here show that costus oil dose dependently decreased the production of α‐toxin, toxic shock syndrome toxin 1 (TSST‐1) and enterotoxins A and B in both methicillin‐sensitive Staph. aureus (MSSA) and methicillin‐resistant Staph. aureus (MRSA). Conclusion: Costus oil has potent antimicrobial activity against Staph. aureus, and the production of α‐toxin, TSST‐1 and enterotoxins A and B in Staph. aureus was decreased by costus oil. Significance and Impact of the Study: The data suggest that costus oil may deserve further investigation for its potential therapeutic value in treating Staph. aureus infections. Furthermore, costus oil could be rationally applied in food products as a novel food preservative both to inhibit the growth of Staph. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

凝固酶阴性葡萄球菌耐药基因和粘附基因检测及耐药性分析

目的了解凝固酶阴性葡萄球菌（CoNS）耐药基因和粘附基因的分布及其耐药性情况。方法收集45株凝固酶阴性葡萄球菌临床分离株,采用PCR检测其耐药基因mecA和粘附基因c弘知如B,并分析其耐药性。结果（1）凝固酶阴性葡萄球菌中mecA、cva、fn6pB的阳性率依次为86．7％（39／45）、11．1％（5／45）、71．1％（32／45）。（2）凝固酶阴性葡萄球菌中耐甲氧西林凝固酶阴性葡萄球菌（MRCoNS）的检出率为88．9％,对β-内酰胺类抗生素的耐药率都在80％以上,对利福平、左氧氟沙星、林可霉素的耐药率较低,未发现对万古霉素、利奈唑胺耐药菌株。结论CoNS中MRCoNS检出率很高,对常用抗生素耐药率高,且有一定的粘附能力,临床上应该根据药敏试验结果慎重及合理的使用抗生素。     

Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

Presence of enterotoxin C and toxic shock syndrome toxin--1 (TSST-1) genes in population of Staphylococcus aureus phage type 187

The aim of this study was to examine whether Staphylococcus aureus of phage type 187 possess the genes of enterotoxins and toxic shock syndrom toxin. Sixteen phage type 187 strains were isolated from the hospital patients (12) and the carriers (4) in twelve medical centres in Poland during 1991 and 2005. Biotyping, phage typing, antibiotic susceptibility, detection of the genes of enterotoxins (sea--sed) and toxic shock syndrome toxin (tst) was tested. The results of this study showed that all staphylococci of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Almost all of them (93.8%) had the enterotoxin C gene and TSST-1 gene. This fact allows to consider them the strains of potentially high virulence.     

Staphylococcal enterotoxin genes are common in Staphylococcus aureus intestinal flora in Sudden Infant Death Syndrome (SIDS) and live comparison infants

Pathological and epidemiological findings in sudden infant death syndrome (SIDS) suggest an infectious aetiology with indications of involvement of staphylococcal enterotoxins (SEs). While SEA, SEB and SEC have been found in the sera and tissues of SIDS cases, little is known about the role of intestinal Staphylococcus aureus or the roles of later-described toxins SEE, SEG, SEH, SEI and SEJ in SIDS. We used a molecular-based approach to define whether the intestinal tract could be a source of SEs to support the staphylococcal toxic shock hypothesis for SIDS. Intestinal contents from 57 SIDS infants and faeces from 79 age- and gender-matched live comparison infants were cultured and tested for S. aureus and sea-b-c-e-g-h-j and TSST using PCR. High proportions of infants in both groups carried toxigenic and nontoxigenic S. aureus . Significantly greater proportions of SIDS compared with comparison babies were positive for S. aureus (68.4% vs. 40.5%) and for SE genes (43.8% vs. 21.5%), suggesting a possible role in SIDS. The results indicate that colonization by S. aureus with SE genes is common in infants; however, their detection is unlikely to be a strong predictive tool for SIDS. Other factors (including immune response) may reveal a specific susceptibility to SEs in SIDS infants.     

Intranasal immunization of mutant toxic shock syndrome toxin 1 elicits systemic and mucosal immune response against Staphylococcus aureus infection

We investigated whether an intranasal immunization with mutant toxic shock syndrome toxin 1 (TSST-1) could elicit a protective effect against nasal colonization as well as systemic infection of Staphyloccoccus aureus in a mouse model. Anti-TSST-1 antibody production in the mucosal exudates and in sera was efficiently induced. Bacterial numbers were reduced in spleen, liver and also nasal cavities in the early stage of nasal colonization, and the survival rate was significantly improved in the immunized mice. It was suggested that the neutralizing activity of antibodies and the enhanced bactericidal activity of neutrophils were involved in the protection against systemic S. aureus infection, and the secreted antibodies could be involved in reduction of S. aureus bacterial counts in the nasal cavity.     

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

Aims: The objective of this study was to investigate the detection of SEE, SEG, SEH and SEI in strains of Staphylococcus aureus and coagulase‐negative staphylococci (CNS) using RT‐PCR. Methods and Results: In this study, 90 Staph. aureus strains and 90 CNS strains were analysed by PCR for the detection of genes encoding staphylococcal enterotoxins (SE) E, G, H and I. One or more genes were detected in 54 (60%) Staph. aureus isolates and in 29 (32·2%) CNS isolates. Staphylococcus epidermidis was the most frequently isolated CNS species (n = 64, 71·1%), followed by Staphylococcus warneri (n = 8, 8·9%) and other species (Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus saprophyticus and Staphylococcus xylosus: n = 18, 20%). The genes studied were detected in Staph. epidermidis, Staph. warneri, Staph. haemolyticus, Staph. hominis, Staph. simulans and Staph. lugdunensis. The highest frequency of genes was observed in Staph. epidermidis and Staph. warneri, a finding indicating differences in the pathogenic potential between CNS species and highlighting the importance of the correct identification of these micro‐organisms. RT‐PCR used for the detection of mRNA revealed the expression of SEG, SEH and/or SEI in 32 (59·3%) of the 90 Staph. aureus isolates, whereas expression of some of these genes was observed in 10 (34·5%) of the 90 CNS isolates. Conclusions: Staphylococcus epidermidis was the most toxigenic CNS species. Among the other species, only Staph. warneri and Staph. lugdunensis presented a positive RT‐PCR result. PCR was efficient in confirming the toxigenic capacity of Staph. aureus and CNS. Significance and Impact of the Study: This study permitted to confirm the toxigenic capacity of CNS to better characterize the pathogenic potential of this group of micro‐organisms. In addition, it permitted the detection of SEG, SEH and SEI, enterotoxins that cannot be detected by commercially available immunological methods.     

Immunization with glutathione S-transferase and mutant toxic shock syndrome toxin 1 fusion protein protects against Staphylococcus aureus infection

To investigate whether immunization with glutathione S-transferase (GST) and mutant toxic shock syndrome toxin 1 (mTSST-1) fusion protein can protect against Staphylococcus aureus infection, we purified a non-toxic mutant GST-mTSST-1 fusion protein. Mice were immunized with the GST-mTSST-1 plus alum adjuvant and then challenged with viable S. aureus. The results showed that the survival rate of GST-mTSST-1-immunized group was higher and the bacteria counts in the organs were significantly lower than those of the non-immunized mice. Immunization with GST-mTSST-1 induced strongly the production of TSST-1 specific antibodies, especially immunoglobulin G1 and immunoglobulin G2b. Furthermore, the serum samples from GST-mTSST-1-immunized mice also significantly inhibited interferon-gamma and tumor necrosis factor-alpha production from murine spleen cells by TSST-1. These results suggest that vaccination with GST-mTSST-1 provides protection against S. aureus infection and that the protection might be mediated by TSST-1-neutralizing antibody.     

Staphylococcus aureus isogenic mutant, deficient in toxic shock syndrome toxin-1 but not staphylococcal enterotoxin A production, exhibits attenuated virulence in a tampon-associated vaginal infection model of toxic shock syndrome.

Since menstrual toxic shock syndrome (MTSS) is associated with a predominant clone of Staphylococcus aureus which produces both toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA), we sought to clarify the role of TSST-1 in a tampon-associated vaginal infection model in New Zealand White (NZW) rabbits, using isogenic tst+/sea+ S. aureus mutants in which tst was inactivated by allelic replacement. Rabbits infected with the tst-/sea+ strain became ill within 3 days, with fever, weight loss, conjunctival hyperemia, and lethargy. Mortality was significantly higher with the tst+/sea+ strain compared to its tst-/sea+ isogenic derivative (4/13 vs. 0/14; p < 0.05, Fisher's exact test, 2-tailed). Mean fever index was higher (p < 0.005; t test, 2-tailed) and weight loss more sustained among survivors in the tst+/sea+ group. Furthermore, culture filtrates from the tst+/sea+ strain induced a significantly greater response in mitogenesis and TNF alpha secretion from rabbit splenocytes in vitro compared to the tst-/sea+ isogenic derivative. Thus, regardless of the role of SEA, TSST-1 significantly contributed to both morbidity and mortality in this tampon-associated vaginal infection model in NZW rabbits. This is the first demonstration of the potential role of TSST-1 and SEA in the pathogenesis of MTSS with a MTSS-associated clinical S. aureus strain in a relevant animal model.     

Incidence of antibodies reactive with toxic shock syndrome toxin 1 in bovine milk

Fourteen samples of bovine milk, purchased in six states, were found to contain significant antibody titers to toxic shock syndrome toxin 1. This reactivity seems to have been due primarily to antibodies of the immunoglobulin A class. Some of these antibodies survived the cheese-making process.     

Comparison of two commercial methods with PCR restriction fragment length polymorphism of the tuf gene in the identification of coagulase-negative staphylococci

AIMS: Two commercial methods for the identification of coagulase-negative staphylococci (CNS) were compared with the restriction fragment length polymorphism (RFLP) of the amplified tuf gene, which served as the reference method. METHODS AND RESULTS: One hundred and forty-five CNS were evaluated using the API 32 Staph ID and the Crystal GP/ID BBL systems. The PCR-RFLP of the tuf gene served as the reference method. The APIStaph and the GP/ID BBL had an overall rate of agreement with the molecular method of 58.6% and 46.2% respectively, with the inability of the GP/ID BBL to characterize 11.7% of the isolates. The APIStaph showed higher sensitivity and better agreement than the GP/ID BBL with the PCR-RFLP, except for Staphylococcus hominis and Staphylococcus capitis. CONCLUSIONS: Neither of the commercial systems was as reliable as the PCR-RFLP method for identifying isolates of CNS. Overall the APIStaph had better agreement with the PCR-RFLP than the GP/ID system. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the PCR-RFLP method is more reliable than the two commercial systems tested, suggesting that it is more reliable for routinely identifying CNS.     

Growth and analysis of crystal forms of toxic shock syndrome toxin 1

Native toxic shock syndrom toxin 1 (TSST-1) purified from Staphylococcus aurius has been crystallized in four different forms. The highest resolution data (2.05 Å) was collected from orthorhombic crystals belonging to the space group C222₁. The unit cell dimension are a = 108.7 Å, b = 177.5 Å, c = 97.6 Å. Rotation function analysis of this from indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 Å. The space group is P2₁ with unit cell parameters of a = 44.4 Å, b = 34.0 Å, c = 55.2 Å, β = 93.0°. © 1993 Wiley-Liss, Inc.     

Detection of antibiotic resistance genes associated with methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci in hospital air filter dust by PCR

Dust from the pre-filters of stand-alone hospital isolation room air cleaners was tested by PCR for the presence of antibiotic resistance genes associated with methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci. Resistance genes for three classes of antibiotics (aac(6′)-aph(2″), ermA, and mecA) were detected in multiple samples, indicating the presence of genetic material and likely cells of multidrug-resistant MRSA and other staphylococci in hospital air and that stand-alone air cleaners can reduce airborne levels of these contaminants. Screening for vanA was negative. Our results further suggest that dust may serve as an important reservoir of genetic elements that can confer drug resistance.