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PubMed2006

Selective growth of Staphylococcus aureus from flushed dairy manure wastewater using acriflavine-supplemented mannitol salt agar.

AIMS: To investigate the use of mannitol salt agar (MSA) supplemented with acriflavine for selective growth and quantification of Staphylococcus aureus from flushed dairy manure wastewater (FDMW). METHODS AND RESULTS: Minimal inhibitory concentrations of acriflavine in MSA were determined by comparing the growth of S. aureus subsp. aureus (ATCC 33591) and Staphylococcus epidermidis (ATCC 155) in pure culture. Acriflavine concentrations of 1.3, 1.4 and 1.5 mg l(-1) reduced CFU of S. epidermidis by 43%, 55% and 87%, respectively, while CFU of S. aureus subsp. aureus were only reduced by 15%, 20% and 26% at the respective concentrations of acriflavine. MSA supplemented with 1.5 mg l(-1) acriflavine was tested for selective growth of indigenous S. aureus from three grab samples of FDMW. Acriflavine concentrations of 1.5 mg l(-1) reduced background flora without significantly reducing (P < 0.05) indigenous S. aureus counts. CONCLUSIONS: Acriflavine-supplemented MSA provides an effective media for selective growth and quantification of indigenous S. aureus from FDMW in the presence of high levels of background microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: S. aureus is implicated for mastitis infections in dairy cows. Therefore, a reliable means for monitoring and detecting the organism in FDMW provides a tool for measuring the effectiveness of treatment for reducing S. aureus levels and implementing flushwater recycling without affecting herd health.

Acriflavine↗

PubMed2006

Induced thermotolerance under nonisothermal treatments of a heat sensitive and a resistant strain of Staphylococcus aureus in media of different pH.

AIMS: The aim was to assess the induced thermotolerance under nonisothermal treatments of two strains of Staphylococcus aureus in media of different pH. METHODS AND RESULTS: Staphylococcus aureus ATCC 25923 was more heat resistant than S. aureus ATCC 13565 at any pH investigated under isothermal conditions. At pH 7.4, the D58 value of the resistant strain was approx. 30 times greater. Both strains showed a higher heat resistance at pH 4.0 than at pH 7.4. In contrast, under nonisothermal treatments (0.5-2 degrees C min(-1)), both strains were more heat resistant when treated at pH 7.4 than at pH 4.0 due to heat adaptation at the higher pH. At the slowest heating up rate tested at pH 7.4, the initially heat-sensitive strain nearly reached the thermotolerance of the heat-resistant strain. CONCLUSIONS: The induced thermotolerance under nonisothermal treatments depended on the treatment medium pH and the microbial strain tested. The induced thermotolerance in a sensitive strain can be greater than in a heat-resistant strain, showing similar resistance under nonisothermal conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows data of interest about mechanisms of microbial resistance and adaptation to heat. Moreover, it contributes to the development of more adequate combined processes for food preservation.

Adaptation, Physiological↗

PubMed2002

PEG site infections: the emergence of methicillin resistant Staphylococcus aureus as a major pathogen.

OBJECTIVE: We reviewed the records of 126 patients who underwent PEG insertion during a 36-month period to determine the etiology of an observed increase in PEG-related infections. METHODS: Charts were reviewed to determine predictive factors of infection, the occurrence of infection, and culture results of infected sites. Insertion was performed in all cases using a standard sterile, pull-through technique. Infections were defined as having at least two of the following conditions: peristomal erythema, induration, and purulent discharge. RESULTS: PEG infections occurred in 22 patients. During the first 12-month interval, 0 of 25 patients (0%) had PEG-related infections; during the second 12-month interval, four of 37 patients (10.8%); and during the third 12-month interval, 18 of 64 patients (28.1%) (p < 0.05). Cultures from 14 of 22 peristomal infections grew methicillin resistant Staphylococcus aureus (nine), pseudomonads (three), and other organisms (two). One hundred twenty-four of 126 patients received prophylactic antibiotics or were receiving concomitant antibiotics. Of the infected patients, 21 of 22 (95.5%) received prophylaxis, and 11 of 22 (50%) were receiving concomitant antibiotics before PEG. In the noninfected group, 78 of 104 (75%) received prophylaxis, and 47 of 104 (45.2%) received concomitant antibiotics. CONCLUSIONS: Methicillin resistant Staphylococcus aureus is emerging as a major pathogen in PEG site infections. Further prospective studies are needed to establish whether current prophylactic antibiotic recommendations are adequate.

Female↗

PubMed2000

Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus.

The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA.

Amino Acid Sequence↗

PubMed1989

Competition between Neisseria gonorrhoeae and Staphylococcus epidermidis during iron-limited or replete continuous culture.

Neisseria gonorrhoeae strain P9-2 was grown in iron-limited or replete continuous culture at a dilution rate of 0.05 h-1, in the presence and absence of Staphylococcus epidermidis. Gonococci maintained expression of pili (P+) and the transparent colony phenotype in pure culture during transitions of iron- and cystine-limited growth. They competed well with staphylococci during iron-limited co-culture and comprised greater than 95% of the population. Transition to cystine-limited growth allowed the staphylococcus to predominate but the gonococcus did not wash out. Furthermore, the gonococcal opaque colony phenotype (O+), indicating synthesis of outer membrane proteins II, was now expressed. Restoration of iron limitation returned the co-culture to its original composition but with P+O+ gonococci dominating. These results suggest that environments might exist in Man where gonococci can compete successfully with normal indigenous bacteria during infection.

Iron↗

PubMed1990

Isolation and characterization of staphyloferrin A, a compound with siderophore activity from Staphylococcus hyicus DSM 20459.

A highly hydrophilic compound was isolated from low iron culture broth of Staphylococcus hyicus DSM 20459 which exhibits siderophore activity to the producer and 37 other staphylococci. The previously unknown metabolite was designated staphyloferrin A and consists of two molecules of citric acid, each linked to D-ornitine by an amide bond. Using an ion-pair HPLC-system we detected staphyloferrin A and a second iron regulated compound (staphyloferrin B) in the culture fluid of several Staphylococcus strains. We found no evidence that staphylococci synthesize catecholor hydroxamate-type siderophores.

Chromatography, High Pressure Liquid↗

PubMed1992

Enzyme-linked immunosorbent assay for the detection of antibodies to exocellular proteins of Staphylococcus aureus in bone infection.

An enzyme-linked immunosorbent assay (ELISA) was developed for measurement of the antibody response to exocellular protein antigens of Staphylococcus aureus. The wells of a microtitre plate were coated with the exocellular proteins present in the tryptic soya broth growth medium from a stationary phase culture of a bone infection strain of S. aureus. The wells were then reacted sequentially with patient sera, protein A-peroxidase conjugate and chromogenic substrate. Serum from patients with S. aureus bone infection gave a significantly higher IgG titre than sera from patients with Staphylococcus epidermidis or Streptococcus sanguis bone infection or healthy uninfected individuals. The assay therefore appears to have potential in the serodiagnosis of S. aureus bone infection.

Antibodies, Bacterial↗

PubMed1993

Using fusions with luxAB from Vibrio harveyi MAV to quantify induction and catabolite repression of the xyl operon in Staphylococcus carnosus TM300.

The luxA,B genes from the Gram-negative marine bacterium Vibrio harveyi MAV were used in Staphylococcus carnosus TM300 as a reporter system for regulated expression of xylose utilization. The luciferase genes were fused to the xyl operon from Staphylococcus xylosus C2a. Expression of bioluminescence was induced through addition of xylose and repressed in the presence of glucose. A method to quantitate bioluminescence directly from the culture is described.

Bacterial Proteins↗

PubMed1993

Construction and overexpression in Escherichia coli of genetically engineered derivatives of penicillin-binding protein 2' of Staphylococcus epidermidis.

Removal of the putative amino-terminal membrane spanning region of penicillin-binding protein 2' (PBP-2') of Staphylococcus epidermidis WT55 was carried out by truncating the amino terminus-coding end of the mecA gene. PCR and site directed mutagenesis were used to introduce unique restriction sites at position 68 (HindIII) and at position 80 (NcoI) of the mecA gene, respectively. The coupling of the shortened coding regions to the trc promoter and gene fusion to the lacZ gene, aimed to facilitate subsequent protein purifications, resulted in strong expression in the cytoplasm of Escherichia coli and partial sequestration into insoluble protein granules. The truncated PBP-2' retained its penicillin-binding ability and also bound the monoclonal antibody directed against PBP-2' of Staphylococcus aureus.

Bacterial Proteins↗

PubMed1994

Evidence for importance of the Staphylococcus hyicus lipase pro-peptide in lipase secretion, stability and activity.

To investigate the function of the pro-peptide (PP) region of the Staphylococcus hyicus exolipase, restriction sites were created in the lipase gene to facilitate the construction of deletions in this region. Lipase gene expression was carried out in Staphylococcus carnosus. In the presence of the entire PP region, the 86-kDa pro-lipase was efficiently exported, had high lipolytic activity, and hardly any degradation products were seen in Western blot analysis. In addition to the 86-kDa pro-lipase, the membrane fraction contained a 106-kDa immunoreactive form. If the PP was completely or partially deleted, signal peptide processing, lipase secretion, lipase activity and/or lipase stability were impaired. The results obtained with lipase PP deletion mutants indicate that the PP region may have two functional domains. The N-terminal region of the lipase PP appears to be more important for lipase activity and the C-terminal portion for lipase secretion and proteolytic stability. In the presence of only the C-terminal part of the PP lipase, secretion was hardly affected. However, the activity of the extracellular lipase was markedly reduced. If only a small portion of the C-terminal part of the PP was present, lipase secretion was again markedly reduced and no lipase activity was detectable. In the presence of the N-terminal half of the PP region, lipase secretion was affected to a lesser extent. However, the resulting 60-kDa form, which showed comparably good specific lipase activity, suffered severe proteolytic degradation.

Amino Acid Sequence↗

PubMed1995

Cloning and nucleotide sequence of the signal peptidase II (lsp)-gene from Staphylococcus carnosus.

Staphylococcus carnosus TM300 is able to synthesize at least seven lipoproteins with molecular masses between 15 and 45 kDa; the proteins are located in the membrane fraction. It can be concluded that this strain also posesses the enzymes involved in lipoprotein modification and prolipoprotein signal peptidase (signal peptidase II) processing. The gene encoding the prolipoprotein signal peptidase, lsp, from Staphylococcus carnosus TM300 was cloned in Escherichia coli and sequenced. The deduced amino acid sequence of the Lsp showed amino acid similarities with the Lsp's of S. aureus, Enterobacter aerogenes, E. coli, and Pseudomonas fluorescens. The hydropathy profile reveals four hydrophobic segments which are homologous to the putative transmembrane regions of the E. coli signal peptidase II. E. coli strains carrying lsp of S. carnosus exhibited an increased globomycin resistance.

Amino Acid Sequence↗

PubMed1998

Photobactericidal activity of phenothiazinium dyes against methicillin-resistant strains of Staphylococcus aureus.

The photodynamic antibacterial properties of a closely related series of phenothiazinium dyes were tested against several pathogenic strains of Staphylococcus aureus, four of which were methicillin-resistant. Illumination of the photosensitisers at a fluence rate of 1.75 mW cm-2 generally resulted in the enhancement of antibacterial activity in liquid culture and in greater efficacy than the methicillin analogue flucloxacillin. For methylene blue, dimethyl methylene blue and new methylene blue illumination led to increases in bactericidal activity < or = 16-fold, typically 4-fold. In addition dimethyl methylene blue and new methylene blue were active against epidemic strains of methicillin-resistant Staphylococcus aureus at concentrations lower than that of vancomycin (> or = 0.5 microM).

Anti-Bacterial Agents↗

PubMed2000

Restoration of effectiveness of beta-lactams on methicillin-resistant Staphylococcus aureus by tellimagrandin I from rose red.

We found that extract from petals of Rosa canina L. (rose red) strikingly reduced the minimum inhibitory concentration of beta-lactams in methicillin-resistant Staphylococcus aureus. We isolated two compounds that reduced the minimum inhibitory concentrations of beta-lactams from the extract, tellimagrandin I and rugosin B. Tellimagrandin I was very effective regarding the reduction of the minimum inhibitory concentration, and rugosin B showed some effect. Tellimagrandin I showed a weak bactericidal action when added together with oxacillin. Judging from the fractional inhibitory concentration index, the effect of tellimagrandin I and oxacillin was synergistic. Tellimagrandin I also significantly reduced the minimum inhibitory concentration of tetracycline in some strains of methicillin-resistant Staphylococcus aureus.

Anti-Bacterial Agents↗

PubMed2001

Catecholamine inotropes as growth factors for Staphylococcus epidermidis and other coagulase-negative staphylococci.

Drugs commonly used in intensive care settings were assayed for their ability to affect the growth of Staphylococcus epidermidis in a minimal salts medium containing 30% serum. Of 28 compounds tested, the inotropic catecholamines adrenaline, dobutamine, dopamine, isoprenaline and noradrenaline significantly stimulated bacterial growth. These drugs, but not structurally similar compounds lacking a dihydroxybenzoyl moiety (such as tyramine, phenylephrine and salbutamol), were able to remove iron from iron-saturated transferrin and to supply transferrin-bound 55Fe to S. epidermidis cells. Similar results were observed with a range of coagulase-negative staphylococci associated with line infections, but not with Staphylococcus aureus (including MRSA).

Catecholamines↗

PubMed2006

Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin.

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. The capacity of S. epidermidis to form biofilms, allowing it to evade host immune defence mechanisms and antibiotic therapy, is considered to be crucial in colonizing the surfaces of medical implants and dissemination of infection. It has previously been demonstrated that the biofilm of a model strain S. epidermidis RP62A comprises two carbohydrate-containing moieties, a polysaccharide having a structure of a linear poly-N-acetyl-(1-->6)-beta-D-glucosamine and teichoic acid. In the present paper we show that, unlike this model strain, certain clinical isolates of coagulase-negative staphylococci produce biofilms that do not contain detectable amounts of poly-N-acetyl-(1-->6)-beta-D-glucosamine. In contrast to that of S. epidermidis RP62A, these biofilms are not detached with metaperiodate, while proteinase K causes their partial dispersal.

Biofilms↗

PubMed2006

Occurance of Staphylococcus nepalensis strains in different sources including human clinical material.

Five isolates of coagulase-negative staphylococci were obtained from human urine, the gastrointestinal tract of squirrel monkeys, pig skin and from the environment. All key biochemical characteristics of the tested strains corresponded with the description of Staphylococcus xylosus species. However, partial 16S rRNA gene sequences obtained from analysed strains corresponded with those of Staphylococcus nepalensis reference strains, except for two strains which differed in one residue. Ribotyping with EcoRI and HindIII restriction enzymes, whole cell protein profile analysis performed by SDS-PAGE and SmaI macrorestriction analysis were used for more precise characterization and identification of the analysed strains. Obtained results showed that EcoRI and HindIII ribotyping and whole cell protein fingerprinting are suitable and reliable methods for the differentiation of S. nepalensis strains from the other novobiocin resistant staphylococci, whereas macrorestriction analysis was found to be a good tool for strain typing. The isolation of S. nepalensis is sporadic, and according to our best knowledge this study is the first report of the occurrence of this species in human clinical material as well as in other sources.

Bacterial Typing Techniques↗

PubMed2005

Adherence of Staphylococcus epidermidis to extracellular matrix proteins and effects of fibrinogen-bound bacteria on oxidase activity and apoptosis in neutrophils.

Staphylococcus epidermidis often causes foreign-body infections such as those associated with hip prostheses, but the underlying pathogenic mechanisms are not fully understood. We performed spectrophotometry to study the ability of S. epidermidis to bind to immobilised fibrinogen, fibronectin, vitronectin, and collagen. The strains were isolated from infected hip prostheses or from normal flora and the well-known protein-binding strain Staphylococcus aureus Cowan was used as positive control. We also analysed the interaction between neutrophils and a fibrinogen-bound prosthesis-derived strain of S. epidermidisby measuring chemiluminescence to determine the neutrophil oxidative response and binding of annexin V to indicate neutrophil apoptosis. We found that binding of S. epidermidis to extracellular matrix proteins varied under different growth conditions, and that prosthesis isolates adhered better to vitronectin than did strains from normal flora. The oxidative response caused by fibrinogen-bound S. epidermidis was not above the background level, which was in marked contrast to the distinct response induced by fibrinogen-associated S. aureus Cowan. Furthermore, fibrinogen-adhering S. epidermidis retarded neutrophil apoptosis. We conclude that surface-bound S. epidermidis induces only a weak inflammatory response, which in combination with the ability of the adherent bacteria to retard neutrophil apoptosis may contribute to low-grade inflammation and loosening of prostheses.

Apoptosis↗

PubMed1980

Immunochemical analysis of the teichoic acid from Staphylococcus simulans.

The wall teichoic acid of Staphylococcus simulans has been characterized as a glycerol phosphate polymer with glycosidically linked N-acetylglucosamine. Susceptibility to beta-N-acetylglucosaminidase and serological similarity to poly C beta from Staphylococcus saprophyticus, showed that the amino sugar is in the beta-configuration.

Immunologic Techniques↗