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PubMed2004

Comparison between ultrafiltration and trichloroacetic acid precipitation method for concentration of Staphylococcus aureus enterotoxin in dairy samples.

Staphylococcus aureus enterotoxin that may be contained at low concentrations in milk and dairy products can cause food poisoning. To detect this enterotoxin at low concentrations, samples should be concentrated. We evaluated the performance of centrifugal ultrafiltration method (UF) in comparison with trichloroacetic acid precipitation method (TCA) for the concentration of S. aureus enterotoxin in milk and dairy products. S. aureus enterotoxin A (SEA) were added at various concentrations to ultra high-temperature heating process (UHT) milk, UHT concentrated skim milk, UHT skim milk powder, low heat-treated (LH) skim milk powder, and raw milk. SEA was concentrated by TCA and UF once a day on a total of 3 days by different researchers to prepare test solutions. The fluorescence value (TV) of test solutions was determined using an immunofluorescence autoanalyzer (miniVIDAS), and the linearity and slope of the regression line, relative standard deviation (RSD(RW)) at each added concentration, detection limit (DL), quantification limit (QL), and the recovery rate by each concentration method were obtained according to the guidelines of the International Conference on Harmonization (ICH). The slope of the regression line obtained by UF was steeper than that by TCA for all dairy samples excluding LH (74 degrees C, 20 s) skim milk powder. RSDRW, DL, and QL obtained by UF were comparable to or more excellent than those obtained by TCA. The procedure of UF was simpler than that of TCA. The recovery rate and rapidity were similar between the two methods. The DL and QL of enterotoxins other than SEA in dairy products by UF or TCA were estimated based on the DL and QL of SEA. In this estimation, consideration was given to reactions between each enterotoxin and its antibody, and also to the immunoactivity maintenance rate of each enterotoxin after addition of trichloroacetic acid in TCA. The estimated values were similar to those obtained by experiments using enterotoxin C1 (SEC1). UF using a centrifugal ultrafiltration membrane can be more readily performed and similar to or more reliable than TCA. UF combined with a miniVIDAS can be used for quantitative routine analysis.

Animals↗
PubMed2001

Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate.

Glycopeptide resistance in Staphylococcus aureus is poorly understood. The diversity of change documented in cell walls of clinical glycopeptide-intermediate S. aureus (GISA) isolates is evidence that a single genetic or biochemical change cannot account for resistance in all isolates described to date. Therefore, identification of new GISA clinical isolates provides an opportunity to gain insight into the range of adaptive strategies employed by staphylococci to survive in the presence of glycopeptides. In April 1999, a GISA isolate was obtained from the blood of a 63-year-old dialysis patient in Illinois. This isolate was one of six clonally identical MRSA isolates (A-F) serially obtained from the blood of this patient who was receiving vancomycin therapy. All isolates were resistant to oxacillin (MIC > 256 mg/L). The initial isolate had an MIC of vancomycin of 1 mg/L. However, the presence of a subpopulation that could grow in the presence of 5 mg/L of vancomycin indicated that this isolate was predisposed to the acquisition of the GISA phenotype (MIC of vancomycin 10-12 mg/L), which occurred 13 days later, associated with an increased MIC of the endopeptidase lysostaphin and slightly increased cell wall thickness. The first and last isolates in the series, A and F, resisted killing when incubated in vancomycin 2 mg/L, resisted autolysis when incubated in Triton X-100 and had a decreased expression of a c. 116 kDa autolytic band, properties that were different from glycopeptide-susceptible control isolates. Lysostaphin resistance was not accompanied by alterations in the peptidoglycan pentaglycine cross-bridge or a decrease in oxacillin MIC. These data, when taken together with the demonstration of increased cross-linking in isolate F compared with isolate A, demonstrate that vancomycin resistance in these isolates probably occurred by a mechanism different from that of other GISA isolates described to date.

Anti-Bacterial Agents↗
PubMed2002

[Evaluation of the efficacy of a multiresistant bacteria control programme in a teaching hospital, studying the evolution of methicillin-resistant Staphylococcus aureus incidence].

Methicillin-resistant Staphylococcus aureus (MRSA) constitute the most important multiresistant bacteria (MRB) recovered in French hospitals. Our objective was to measure these MRSA diffusion in our hospital to evaluate the MRB control programme which had been implemented in the beginning of 1999. This study was conducted in a teaching hospital containing 1800 beds, from February 1999 to January 2001. All MRSA isolated in clinical samples were included. Duplicates (same bacteria in the same patient) were excluded. The detection of methicillin-resistance was performed at 30 degrees C, by disk diffusion method. Incidence densities were determined with their 95% confidence interval (CI 95%). Their evolution by four-month period was evaluated with the chi-square test for trend. During the two-year period, 866 MRSA were isolated. The global incidence was 0,88 per 1000 patient-days (PD) (IC 95% = left open bracket 0,83-0,93 right open bracket ). For cases acquired in our hospital the incidence was 0,66 per 1000 PD, whereas it was 0,26 per 1000 PD for imported cases. Concerning the evolution of incidences, no significant trend was observed for global incidence. The incidence of acquired MRSA decreased during the first year, but increased thereafter. The incidence of imported MRSA increased with a significant trend (p < 10(-5)). The number of these imported MRSA isolated in our hospital was twice fold higher in 2000. This study emphasizes an important actual problem : the increase of patient colonization pressure at the time of admission in hospitals. This increase, which can be due in part to a community transmission, is responsible for a reduction of the efficacy of MRSA control programmes.

Chi-Square Distribution↗
PubMed1997

Transcriptional analysis of type 1 capsule genes in Staphylococcus aureus.

Serotype 1 capsule of Staphylococcus aureus plays an important role in staphylococcal infections. A 14.6 kb chromosomal DNA fragment containing 13 cap1 genes responsible for the synthesis of type 1 capsule in S. aureus strain M has been previously cloned and sequenced in our laboratory. These genes are closely linked and are transcribed in one orientation. To study the transcription of these genes, we employed genetic complementation using a set of plasmids with various deletions in the 14.6 kb region to complement mutants mapped in each of the 13 genes. We found that there were six transcriptional units. By Northern hybridization using the entire gene cluster as a probe, we identified a large 14 kb band and several smaller bands ranging from 0.3 to 6 kb. The 14 kb band was also identified by various individual gene probes, suggesting that all 13 genes are transcribed into a large transcript. The 14 kb transcript and the smaller bands were not detected when a 533 bp fragment containing the potential promoter region upstream of the first gene (cap1A) was deleted from the chromosome, suggesting that the small transcripts are the degradation (processing) products of the 14 kb transcript. The activities of the promoters identified by genetic complementation tests were quantitatively measured by transcriptional fusions using xylE as the reporter gene. The result showed that the promoter preceding the first cap1 gene was 45-198-fold higher than the downstream promoters. The result from gene-fusion studies is in agreement with the Northern analysis in that only the transcript transcribed from the first promoter was detected. Taken together, these results suggest that the cap1 genes are organized as a large operon with at least five weak internal promoters, and the long polycistronic transcript is processed (degraded) into several smaller transcripts. The transcription initiation site of the primary transcript was mapped by S1 nuclease mapping and the major promoter was defined by deletion analysis of the promoter-xylE fusion. In addition, we showed that the internal promoters, despite their weak activities, were sufficiently effective at expressing their downstream genes required for producing capsule, compared to the wild-type strain.

Bacterial Capsules↗
PubMed1996

Influence of agr on fibrinogen binding in Staphylococcus aureus Newman.

The ability of Staphylococcus aureus to bind fibrinogen is believed to be important in promoting bacterial adherence to both intravascular catheters and host tissues during infection. We investigated the influence of the global regulator agr on the fibrinogen binding capacity and its relationship to the expression of coagulase (encoded by coa) and clumping factor (encoded by clfA) in strain Newman. Strains were obtained by transducing site-specific mutations of clfA, coa, and agr into strain Newman to obtain single, double, and triple mutants of the respective genes. As expected, the clfA mutant bound less soluble 125I-labeled fibrinogen than the corresponding coa mutant in agr+ strains; however, with agr mutant strains, the upregulation in fibrinogen binding capacity correlated mostly with the increased expression and transcription of coagulase as shown by Western (immunoblot) and Northern (RNA) blot analysis. In particular, the coa agr double mutant resulted in a significant reduction in fibrinogen binding compared with that of the agr mutant. The contribution of clfA to fibrinogen binding in agr-negative strains was less than that of coa (32,740 +/- 1,189 versus 18,141 +/- 334 and 38,919 +/- 1,021 cpm for clfA agr, coa agr, and the single agr mutant, respectively). Thus, coagulase is a major binding protein for soluble fibrinogen in the agr-negative background. In in vitro microtiter and catheter adherence assays with solid-phase fibrinogen, clumping factor, but not coagulase, plays a major role in binding to immobilized fibrinogen. coa transcription was negatively modulated by agr and occurred mainly during the exponential growth phase. In contrast, clfA transcription was agr independent and was strongest during the postexponential phase. Although an agr coa clfA triple mutant bound less soluble fibrinogen than the agr coa double mutant (8,504 +/- 831 versus 18,141 +/- 334 cpm), significant residual fibrinogen binding capacity remained in the triple mutant, thus suggesting an additional fibrinogen binding component. By using direct ligand affinity blotting with 125I-fibrinogen, we could identify coagulase and an additional unidentified 52-kDa protein as a fibrinogen binding component in cell extracts. This band was absent in the extract of the coa clfA double mutant.

Bacterial Proteins↗
PubMed2004

Effect of granulocyte colony-stimulating factor in experimental methicillin resistant Staphylococcus aureus sepsis.

BACKGROUND: Methicillin resistant Staphylococcus aureus (MRSA) is the leading pathogenic cause of nosocomial infections, especially in bacteraemia and sepsis. The essential therapy for MRSA infection is glycopeptides. Therapeutic failure can be seen with this therapy and the mortality is still high. The aim of this study was to evaluate the additional effect of G-CSF on the traditional antibiotic treatment in an experimental MRSA sepsis. METHODS: Experimental sepsis was performed in mice by intraperitoneal injection of MRSA isolate. Inoculum dose was estimated as 6 x 10(9)ml. Mice were randomised for the study into four group; control group (not receive any therapy), G-CSF group (1000 ng/daily, subcutaneously for 3 d), antibiotic group (vancomycin 25 or 50 mg/kg intraperitoneally every 12 hours for 7 d), and vancomycin+G-CSF group (at the same concentrations and duration). Autopsy was done within one hour after mice died. If mice was still alive at the end of seventh day, they were sacrificed, and autopsy was done. In all groups, the effect of G-CSF therapy on the survival, the number of the MRSA colonies in the lung, liver, heart, spleen, and peritoneal cultures, the histopathology of the lung, liver, heart and spleen was investigated. RESULTS: One hundred and six mice were used. There were no significant differences in survival rates and bacterial eradication in G-CSF group compared with control group, and also in antibiotic +G-CSF group compared with antibiotic alone group. These parameters were all significantly different in antibiotic alone group compared with control group. Histopathologically, inflammation of the lung and liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF and vancomycin (50 mg/kg)+G-CSF subgroups, respectively (p < 0.01). The histopathological inflammation of the other organs was not significantly different in antibiotic+G-CSF group compared with antibiotic group and, also G-CSF group compared with control group. CONCLUSION: G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. G-CSF treatment is very expensive, likewise glycopeptides. The more interest in infection control measures, and prevent the spread of MRSA infections is more rational.

Animals↗
PubMed2006

Characteristics of patients with healthcare-associated infection due to SCCmec type IV methicillin-resistant Staphylococcus aureus.

OBJECTIVE: Methicillin-resistant Staphylococcus aureus (MRSA) with the staphylococcal cassette chromosome mec (SCCmec) type IV allele is most commonly associated with community-acquired MRSA (CA-MRSA) infection; however, such organisms have also been identified in the healthcare setting. The objective of the present study was to characterize the epidemiology of and clinical outcomes associated with SCCmec-IV MRSA infection acquired in the healthcare setting, compared with infection caused by MRSA of other SCCmec types. DESIGN: We evaluated a cohort of 100 inpatients with MRSA infection that met the Centers for Disease Control and Prevention definition for healthcare-associated infection and compared the patients' demographic characteristics, the antimicrobial susceptibilities of the MRSA isolates, the infection types, and the associated clinical and microbiological outcomes. For each MRSA isolate, the SCCmec type and the presence of Panton-Valentine leukocidin (PVL) were determined by polymerase chain reaction methods. RESULTS: SCCmec-IV MRSA isolates were isolated from 53 patients (42% of these isolates were positive for PVL), and SCCmec-II or SCCmec-III MRSA was isolated from 47 patients (3% of these isolates were positive for PVL). No differences were noted between the patients in the SCCmec-II/III group and the patients in the SCCmec-IV group with respect to age (median, 55 vs 50 years); sex (77% vs 64% of patients were male); medical service (surgical service, 60% in both groups; ICU admission, 55% vs 53%), Acute Physiology and Chronic Health Evaluation II score (median, 8 vs. 7); infection type; or underlying comorbidities, except for presence of a burn wound (13% vs 2%; P < .04). Patients in the SCCmec-II/III group were more likely to have multiple sites of infection (P = .006) and a longer length of stay (LOS) prior to detection of MRSA than were patients in the SCCmec-IV group (median, 4 vs 1 days; P < .001). Total LOS was significantly greater for patients in the SCCmec-II/III, compared with those in the SCCmec-IV group (P = .006). Multiple logistic regression identified liver disease and longer LOS prior to detection of MRSA as predictors of infection with SCCmec-II/III MRSA. Rates of susceptibility to clindamycin, gentamicin, ciprofloxacin, levofloxacin, and tetracycline was significantly greater among SCCmec-IV MRSA isolates, compared with type II/III isolates (P < or = .05). Compared with SCCmec-IV isolates acquired in the community, the susceptibility rates among healthcare-associated SCCmec-IV isolates was significantly less for clindamycin, gentamicin, and levofloxacin, indicating that these organisms may quickly acquire resistance to non- beta -lactam antibiotics, as do SCCmec-II/III strains. CONCLUSIONS: SCCmec-IV MRSA appears to have become established in hospitals. The onset of infection caused by SCCmec-IV strains is earlier than the onset of infection with SCCmec-II/III strains; however, associated types of infection are similar. Infection with SCCmec-II/III MRSA is currently associated with an adverse impact on outcome, compared with infection with SCCmec-IV MRSA. Further research is warranted to determine the impact of SCCmec type IV strains in hospital settings.

Adult↗
PubMed2005

BBL CHROMagar Staph aureus is superior to mannitol salt for detection of Staphylococcus aureus in complex mixed infections.

We used 200 sputum specimens from patients with cystic fibrosis to evaluate BBL CHROMagar Staph aureus medium (CSA; BD Diagnostics, Spark, MD). Samples were inoculated to CSA, trypticase soy blood agar (BA), and Mannitol Salt (MS; BD Diagnostics). After 18 hours of incubation, CSA detected 39 (78%) of 50 Staphylococcus aureus (SA) samples; BA detected 30 (60%); and MS detected 29 (58%). Sensitivity after overnight incubation (at least 18 hours) was 82% for CSA, 72% for BA, and 71% for MS. At 2 days, CSA detected 48 (96%) of the SA. There were 2 false-positive results on CSA (99% specificity). There were 4 (8%) minor and no major or very major discrepancies between minimum inhibitory concentration (MIC) results for isolates grown on CSA and those grown on BA. Even at early reading times, CSA was superior to conventional media for detection of SA in these very complex cultures. MICs from all SA samples can be reported 24 hours sooner, and an additional BA subculture is not needed.

Agar↗
PubMed2000

Antimicrobial resistance patterns and plasmid profiles of Staphylococcus aureus isolated from milk and meat.

OBJECTIVES: To determine the frequency of resistance of Staphylococcus aureus to various antimicrobial agents, and the relationship between antimicrobial resistance of the isolates and carriage of plasmids. DESIGN: A random sampling of milk and meat samples was carried out. SETTING: Milk was collected from various dairy co-operative societies in Nairobi and Kiambu districts. Minced meat samples were purchased from various outlets in the city of Nairobi. SUBJECTS: Ninety six Staphylococcus aureus isolates from milk (seventy five isolates) and minced meat (twenty one isolates) samples. MAIN OUTCOME MEASURES: Plasmid profiles and antimicrobial susceptibility tests to ampicillin, lincomycin, penicillin, erythromycin, methicillin, minocycline, cotrimoxazole and chloramphenicol. RESULTS: Seventy one per cent of the isolates carried between one and six plasmids of molecular sizes ranging from 0.1 to 14.5 kilobases. High frequency of resistance was observed with lincomycin (67.7%), penicillin (66.7%) and cotrimoxazole (51%). A high percentage (76%) of isolates were susceptible to minocycline followed by erythromycin (57.3%). Most (80.2%) of isolates were multiply resistant to between two and six antibiotics. CONCLUSION: Most Staphylococcus aureus isolates were multiply resistant to various antimicrobial agents, but there was no apparent relationship between carriage of plasmids and antimicrobial resistance. Milk and meat may contain resistant Staphylococcus aureus posing a potential risk to consumers.

Animals↗
PubMed2006

Evaluation of S. aureus ID, a chromogenic agar medium for the detection of Staphylococcus aureus.

BACKGROUND: Screening for staphylococci among various patient populations has become important for appropriate therapeutic management and for control of nosocomial infections. The purpose of this study is to evaluate the in vitro sensitivity and specificity of a chromogenic agar medium, S. aureus ID (bioMérieux, France), for the identification of Staphylococcus aureus. MATERIALS AND METHODS: A well-defined collection of S. aureus and coagulase-negative staphylococci (CNS) was used. The methicillin-resistant S. aureus (MRSA) isolates were collected in The Netherlands and all had a unique typing pattern. The methicillin-susceptible S. aureus (MSSA) and CNS were isolated from cultures of blood. The isolates were inoculated on Columbia agar plates with 5% sheep blood and incubated for 24 h at 35 degrees C. From the resulting cultures, a suspension of 0.5 McFarland was made and subsequently 10 mul was streaked on a S. aureus ID plate using a sterile loop. The results were read after 24 h and 48 h of incubation at 35 degrees C. Growth of colonies showing green coloration was considered to be positive (indicating S. aureus). RESULTS: A total of 519 S. aureus strains were tested (249 MSSA, 270 MRSA). The sensitivity to detect S. aureus was 96.5% (501/519) after 24 h and 97.5% (506/519) after 48 h. A total of 478 CNS were tested. The specificity was 98.5% (471/478) after 24 h and 98.3% (470/478) after 48 h. The differences between 24 h and 48 h incubation were not statistically significant. CONCLUSION: S. aureus ID is highly sensitive and specific to differentiate between S. aureus and CNS in vitro. Since the performance does not significantly differ between 24 h or 48 h of incubation, samples need only 1 day of incubation before optimal results can be obtained.

Agar↗
PubMed2002

Use of pulsed-field gel electrophoresis for detecting differences in Staphylococcus aureus strain populations between dairy herds with different cattle importation practices.

The hypothesis tested was that dairy herds which import cattle for replacement or expansion have a higher prevalence of Staphylococcus aureus mastitis and a greater number of new Staphylococcus aureus strains enter their herds than closed herds. Fifteen commercial dairy herds were divided into four groups based on cattle importation practices. Composite foremilk samples were collected at 4-monthly intervals for 1 year from all lactating cattle. Additionally, foremilk samples were collected from cattle at parturition and skin swabs were taken from the udder of primiparous heifers. All samples were cultured for Staphylococcus aureus and isolates were strain-typed using pulsed-field gel electrophoresis. Herds that purchased replacement heifers had a higher prevalence of Staphylococcus aureus mastitis than herds that purchased lactating cattle for expansion (P = 0.02). Herds that purchased replacement heifers had more total strains of Staphylococcus aureus (P = 0.01) and more new strains (P = 0.04) enter the herd than closed herds.

Animal Husbandry↗
PubMed1997

Simple enzymatic method for rapid identification of a Staphylococcus aureus subspecies aureus biovar.

In order to correctly identify a new biovar of Staphylococcus aureus subsp, aureus, (NBSA) a simple, rapid, and reliable enzymatic assay was developed. The assay was based on the detection of the production of three enzymes: alpha-glucosidase, beta-glucosidase and beta-N-acetyl-glucosaminidase. Of a total of 46 isolates of Staphylococcus aureus subsp. aureus from clinical specimens, the new assay correctly identified 19 as NBSA and 27 as typical Staphylococcus aureus subsp. aureus. Among the 19 NBSA isolates, 15 (79%) showed a clear biochemical profile while only four isolates (21%) showed a less well-defined enzymatic combination, due to a phenotypic alteration caused by subculturing. Since this assay is both simple to perform and inexpensive, it is potentially applicable in the laboratory.

Acetylglucosaminidase↗
PubMedDate not supplied

[Staphylococcus aureus septicemia in children: bacterial tolerance to vancomycin and serum bactericidal activity].

PURPOSE: To evaluate the susceptibility and of strains of Staphylococcus aureus isolated from children with septicemia, and to evaluate the importance of the serum bactericidal test. METHODS: Seventeen children with Staphylococcus aureus septicemia admitted to the Semi Intensive Care Unit of the Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo were studied. Twenty nine tests in the pico and 23 in the nadir of the antibiotics were made. RESULTS: Strains of Staphylococcus aureus from hospital origin were resistant to all the antibiotics but vancomycin and pefloxacin. The phenomenon of tolerance was seen in 5 (50%) of the strains that were tested for vancomycin, and 4 of the children had a bad evolution. The serum bactericidal tests showed titles in the pico > or = 1/8 in 55.5% of the observations; in this group the evolution was better. CONCLUSION: Strain of Staphylococcus aureus from hospital origin are multiresistant. The phenomenon of antimicrobial tolerance, as well as the serum bactericidal test may be related to a bad therapeutic evolution. The increasing value of the serum bactericidal test as a way to evaluate the therapeutic evolution in severe infections, and the role of the tolerance of the Staphylococcus aureus to vancomycin more studies.

Anti-Bacterial Agents↗
PubMed1992

Intracellular localization of Staphylococcus aureus within primary cultured mouse kidney cells.

Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity-tolerant (HOT) cells were obtained. The rate of cocci-ingesting cells varied from 16.9% to 93.4% among these of the HOT cells at the end of 3-hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK-1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK-1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast-like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast-like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30-min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.

Animals↗
PubMed2006

Diminished susceptibility to daptomycin accompanied by clinical failure in a patient with methicillin-resistant Staphylococcus aureus bacteremia.

We cared for a patient with methicillin-resistant Staphylococcus aureus bacteremia who experienced clinical failure with daptomycin. The failure was accompanied by progressive elevation of the daptomycin minimum inhibitory concentration during treatment. DNA fingerprinting confirmed that the minimum inhibitory concentration elevation occurred within the same strain of methicillin-resistant Staphylococcus aureus. This observation provides important new information to clinicians who adopt this promising drug for treatment of serious infections caused by methicillin-resistant Staphylococcus aureus.

Aged, 80 and over↗
PubMed1998

Role of Staphylococcus aureus surface-associated proteins in the attachment to cultured HaCaT keratinocytes in a new adhesion assay.

Colonization of human skin with Staphylococcus aureus is a common feature in a variety of dermatologic diseases. In order to reproducibly investigate the adherence of Staphylococcus aureus to human epidermal cells, an in vitro assay was established using the biotin/streptavidine labeling system and the HaCaT cell line. This assay was used to define the role of several Staphylococcus aureus surface proteins with regard to their function in the staphylococcal adhesion process. Our studies included the standard laboratory strain Newman as well as its genetically constructed mutants DU5873, DU5852, DU5854, and DU5886 generated by allele replacement or transposon mutagenesis, which are deficient in the elaboration of staphylococcal protein A (spa), clumping factor (clfA), coagulase (coa), and the fibronectin-binding proteins A and B (fnbA/B), respectively. In comparison with strain Newman all mutants showed remarkably reduced adherence to the HaCaT keratinocyte cell line in our assay, yielding only between 43% and 60% of the adherence capacity of strain Newman after 60 min. Bacterial adherence could be re-established by introducing the cloned wild-type genes for the surface proteins on shuttle plasmids into the chromosomally defective mutants, thus suggesting a pathogenetic role of these proteins in the attachment of Staphylococcus aureus to human keratinocytes. Bacterial adherence was additionally enhanced by alkaline pH-values that are characteristic for skin conditions with epidermal barrier dysfunction. The use of Staphylococcus aureus mutant strains, deficient in the elaboration of defined proteins, allows specific investigation of colonization and virulence factors of this dermatologic relevant microorganism.

Bacterial Adhesion↗
PubMed2003

High prevalence of superantigens associated with the egc locus in Staphylococcus aureus isolates from patients with atopic eczema.

The present study was aimed at identifying a possible correlation between disease severity and colonization with superantigen-producing Staphylococcus aureus strains in patients with atopic eczema. To this end, Staphylococcus aureus strains from 91 patients with atopic eczema were screened for various staphylococcal superantigens such as SEA, SEB, SEC, SED, TSST1, the recently described enterotoxin gene cluster egc (which encodes the enterotoxins SEG, SEI, SEK, SEM, and SEO), and the see, seh, and sej loci. Swabs were taken from seven different sites in each patient. The rate of colonization with Staphylococcus aureus was 87.9%. Of those patients colonized, 35% were colonized with more than one different strain. Of the 120 genetically different strains investigated, the egc locus was found in 48.3% and the sej locus in 7.5%. The see and seh loci were not found in any strain. The presence of the classical superantigens SEA-SED or TSST1 was found in 38.3%. Overall, 71.3% of the Staphylococcus aureus-positive patients harbored at least one superantigen-producing strain on their skin. There was no difference in the prevalence of superantigens between atopic eczema patients and healthy volunteers. Moreover, there was no difference in the extent of disease expression between patients colonized by superantigen-positive Staphylococcus aureus strains and those with superantigen-negative strains as measured by the SCORAD system. However, patients colonized with Staphylococcus aureus had a significantly higher SCORAD score than those not colonized.

Adolescent↗
PubMed2005

Blood neutrophil bactericidal activity against methicillin-resistant and methicillin-sensitive Staphylococcus aureus during cardiac surgery.

Whether methicillin-resistant Staphylococcus aureus (MRSA) constitutes per se an independent risk factor for morbidity and mortality after surgery as compared with methicillin-sensitive Staphylococcus aureus (MSSA) remains a subject of debate. The aim of this study was to assess whether innate defenses against MRSA and MSSA strains are similarly impaired after cardiac surgery. Both intracellular (isolated neutrophil functions) and extracellular (plasma) defenses of 12 patients undergoing scheduled cardiac surgery were evaluated preoperatively (day 0) and postoperatively (day 3) against two MSSA strains with a low level of catalase secretion and two MRSA strains with a high level of catalase secretion, inasmuch as SA killing by neutrophils relies on oxygen-dependent mechanisms. After surgery, an increase in plasma concentration of IL-10, an anti-inflammatory cytokine able to inhibit reactive oxygen species secretion and bactericidal activity of neutrophils, was evidenced. Despite the fact that univariate analysis suggested a specific impairment of neutrophil functions against MRSA strains, two-way repeated-measures ANOVA failed to demonstrate that the effect of S. aureus phenotype was significant. On the other hand, an increase in type-II secretory phospholipase A2 activity, a circulating enzyme involved in SA lysis, was evidenced and was associated with an enhancement of extracellular defenses (bactericidal activity of plasma) against MRSA. Overall, cardiac surgery and S. aureus phenotype had a significant effect on plasma bactericidal activity. Cardiac surgery was characterized by enhanced antibacterial defenses of plasma, whereas neutrophil killing properties were reduced. The overall effect of S. aureus phenotype on neutrophil functions did not seem significant.

Aged↗