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Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain.

AIMS: Control of the enterotoxigenic Staphylococcus aureus CECT 976 strain by enterocin AS-48 in laboratory cultures, and behaviour of the AS-48 activity in the presence of food preservatives. METHODS AND RESULTS: Enterocin AS-48 shows inhibitory activity on the majority of the Staphylococcus species tested. This enterocin has a bactericidal and bacteriolytic mode of action on S. aureus CECT 976, a strain selected for this study by its enterotoxigenic character (SEA production). The inhibitory effect of AS-48 was pH and temperature dependent, and enterocin activity was higher at pH 5. The minimum bactericidal concentration (MBC) of AS-48, decreased from 15 microg ml(-1) at 37 degrees C to 10 microg ml(-1) at 15 degrees C. Sublethally injured cells showed an increased sensitivity with a MBC of 5 microg ml(-1). In this way, the highest effectiveness of Ent AS-48 against S. aureus CECT 976 was obtained at 4 degrees C in combination with high concentrations of NaCl (6 and 7%). Interestingly, enterotoxin SEA production by strain CECT 976 was markedly inhibited by subinhibitory concentrations of Ent AS-48. These low concentrations also provoked a delay of bacterial growth. CONCLUSION: The results presented indicated that Ent AS-48 has a potential for application as a protective agent against S. aureus in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have established the conditions for an efficient inhibition of growth and enterotoxin production by S. aureus CECT 976 in culture media by a combination of environmental factors and Ent AS-48.

Anti-Bacterial Agents↗

Variation in resistance of natural isolates of Staphylococcus aureus to heat, pulsed electric field and ultrasound under pressure.

AIMS: To study and compare the resistance of 15 Staphylococcus aureus isolates to heat, pulsed electric field (PEF) and ultrasound (UW) under pressure (manosonication, MS). METHODS AND RESULTS: Survival curves to heat (58 degrees C), to PEF (22 kV cm(-1), 2 micros square wave pulses) and to UW under pressure (117 microm, 20 kHz, 200 kPa) were obtained and inactivation parameters (decimal reduction times for heat and UW under pressure, and b-values for PEF) were calculated. A wide resistance variation to heat treatment, but not to PEF and MS, was observed amongst the 15 strains. CONCLUSIONS: There was no relationship between the resistances to the three physical agents studied. Staphylococcus aureus was relatively resistant to MS but sensitive to PEF. Heat resistance varied with strain and was positively correlated to carotenoid pigment content. SIGNIFICANCE AND IMPACT OF THE STUDY: Results would help in defining safe food preservation processes. Care should be taken to choose the most adequate strain of S. aureus to model food preservation processing.

Bacteriological Techniques↗

Staphylococcus aureus: a review of the literature.

Staphylococcus aureus has a long association with nosocomial infection. Problems date from the 1950s, although methicillin-resistant Staphylococcus aureus (MRSA) did not emerge until the following decade. Initially the pathogenicity of antibiotic-resistant strains was underestimated, and is still sometimes questioned, but today most authorities consider MRSA a serious threat, especially given current preoccupation with cost-effectiveness within the health service: nosocomial infection is associated with increased expenditure and may be regarded as a hallmark of indifferent nursing and medical care. This review documents the emergence of MRSA and recognition of the ensuing problems throughout the 1980s and early 1990s, with suggestions for nursing activities which could contribute towards improved control. Lessons learnt during outbreaks are seen to be of value, but there is also a need for staff at ward level to review routine practice continually so that awareness of activities likely to result in cross-infection is maintained. The use of protective clothing emerges as less important than handwashing, which may be periodically audited to maintain standards.

Cross Infection↗

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis.

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear beta-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyranosyl residues of which 80-85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the icaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped icaA promoter. IcaA contains four potential transmembrane helices, indicative of a membrane location. The deduced IcaA sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that IcaA has N-acetylglucosaminyltransferase activity in the formation of the beta-1, 6-linked N-acetyl-D-glucosaminyl polymer. IcaB is secreted into the medium and contains a typical signal peptide. IcaC is hydrophobic and contains six predicted transmembrane helices distributed over its entire length, typical for an integral membrane protein. Neither IcaB nor IcaC shares similarity with known proteins, and their function is unknown. Inactivation of icaA, icaB, or icaC in pCN27 led to the complete loss of the intercellular adhesion phenotype in S. carnosus, suggesting that all three genes are involved in intercellular adhesion, PIA expression, and translocation.

Bacterial Adhesion↗

Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus.

Staphylococcus species belong to one of the few bacterial genera that are completely lysozyme resistant, which greatly contributes to their persistence and success in colonizing the skin and mucosal areas of humans and animals. In an attempt to discover the cause of lysozyme resistance, we identified a gene, oatA, in Staphylococcus aureus. The corresponding oatA deletion mutant had an increased sensitivity to lysozyme. HPLC and electrospray ionization tandem mass spectrometry analyses of the cell wall revealed that the muramic acid of peptidoglycan of the wild-type strain was O-acetylated at C6-OH, whereas the muramic acid of the oatA mutant lacked this modification. The complemented oatA mutant was lysozyme resistant. We identified the first bacterial peptidoglycan-specific O-acetyltransferase in S. aureus and showed that OatA, an integral membrane protein, is the molecular basis for the high lysozyme resistance in staphylococci.

Acetylation↗

Cloning of swine desmoglein 1 and its direct proteolysis by Staphylococcus hyicus exfoliative toxins isolated from pigs with exudative epidermitis.

Exudative epidermitis (EE) is an acute, often fatal skin disease of piglets caused by Staphylococcus hyicus. Clinical and histopathological manifestations of EE are similar to those of staphylococcal scalded skin syndrome (SSSS), a human blistering skin disease, in which exfoliative toxins produced by Staphylococcus aureus digest the extracellular domains of desmoglein (Dsg) 1 and cause loss of epidermal cell-cell adhesion. The aims of this study were to isolate and characterize cDNA for full length of swine Dsg1, and to determine whether the extracellular domains of swine Dsg1 produced by baculovirus (sDsg1-His) could be digested by four isoforms of exfoliative toxin produced by S. hyicus (ExhA, ExhB, ExhC and ExhD). Nucleotide sequencing revealed that swine Dsg1 cDNA consisted of an open reading frame of 3138 bp, encoding a precursor protein of 1045 amino acids. Deduced amino acid sequence of the swine Dsg1 precursor were highly homologous to corresponding bovine, canine, human and murine sequences. Immunoadsorption assay with a secreted form of sDsg1-His revealed that sDsg1-His specifically absorbs the immunoreactivity of 10 human pemphigus foliaceus sera against swine keratinocyte cell surfaces, suggesting its proper conformation. When sDsg1-His was incubated in vitro with Exhs, all four isoforms of Exh directly digested sDsg1-His into smaller peptides, whereas removal of calcium from sDsg1-His completely inhibited its proteolysis by these four Exhs. Recognition and digestion of calcium-stabilized structure on the extracellular domains of swine Dsg1 by Exhs indicated that EE shares similar molecular pathophysiological mechanisms of intra-epidermal splitting with SSSS in humans.

Amino Acid Sequence↗

Staphylococcus aureus in atopic dermatitis and in nonatopic dermatitis.

Skin colonization with Staphylococcus aureus (S. aureus) was examined in 30 patients with atopic dermatitis (AD), in 25 patients with nonatopic eczema (NAE) and in 30 individuals as healthy controls (HC). Bacteria growth was examined in aerobic cultures and the population densities per dish were estimated; S. aureus colonization was found in the eczematous skin of 24 of 30 (80%) AD patients and in 13 of 25 (52%) NAE patients (NS, p greater than 0.1). In nonaffected skin S. aureus colonization was found in 19 of 30 (63%) of all AD patients compared with 6 of 25 (24%) in NAE patients and 1 of 30 (3%) in HC, respectively (p less than 0.05). In nonaffected skin, coagulase negative strains of staphylococcus were found in 25 of 30 (84%) controls and in 18 of 25 (72%) NAE patients compared with 12 of 30 (40%) patients with AD. It seems that colonization with S. aureus is not a characteristic feature for atopic dermatitis but is a frequent event in damaged skin; significantly elevated values were also observed in nonatopic eczema. The degree of colonization may depend on the severity and duration of the eczematous lesions.

Dermatitis, Atopic↗

Methicillin resistance in Staphylococcus epidermidis. Relationship between the additional penicillin-binding protein and an attachment transpeptidase.

The penicillin-binding proteins (PBP) of a methicillin-resistant strain of Staphylococcus epidermidis, 100,604 p+m+ and a non-isogenic sensitive strain, p-m- were characterised. The presence of a novel PBP, produced by the methicillin-resistant strain of S. epidermidis, with an Mr identical to that of PBP2' in Staphylococcus aureus 13,136 p-m+, was revealed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and subsequent fluorography of solubilised membrane proteins isolated from cells labelled with [3H]benzylpenicillin. This novel PBP was only detected in cells which had been grown at 30 degrees C, in media containing beta-lactam antibiotic and 5% NaCl. The sensitivity of an attachment transpeptidation reaction measured under non-growing conditions in the sensitive and resistant strains indicated that the novel PBP catalysed this reaction. The similarity of radiolabelled peptides resulting from partial proteolytic digestion of the novel PBP in S. epidermidis 100,604 p+m+ and from PBP2' in S. aureus 13,136 p+m+ lends support to the theory that the additional DNA encoding PBP2' in S. aureus and the same protein in S. epidermidis has been passed to both species from an unknown source. Studies of the development and loss of resistance of attachment transpeptidase activity, and the appearance and disappearance of the novel protein when cultures of the resistant strain were transferred from conditions allowing the expression of resistance to those not allowing such expression and vice-versa, indicated that there was a strong correlation between the presence of PBP2' and the degree of resistance of the attachment transpeptidation reaction and that the production of this protein was affected by temperature at a regulatory or genetic level. Studies on the induction and loss of beta-lactamase activity and of the novel PBP when the resistant strain was grown in the presence or absence of beta-lactam antibiotics at either 40 degrees C or 30 degrees C suggests that there is little relationship between the production of this enzyme and of PBP2' other than the fact that beta-lactam antibiotics are common inducers of both.

Acyltransferases↗

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and protein sequencing of the Staphylococcus carnosus histidine-containing protein, and cloning and DNA sequencing of the ptsH gene.

The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. The protein sequence was determined by Edman degradation of peptides obtained by proteolytic digestion with proteases V8, trypsin and chemical cleavage with BrCN. Furthermore, immunological screening of a chromosomal S. carnosus DNA gene library in pUC19 vector enabled us to isolate S. carnosus HPr-expressing colonies. The nucleotide sequence of this ptsH gene and its flanking regions was determined by the dideoxy-chain-termination technique. Upstream, the 264-bp open reading frame of the ptsH gene is flanked by a putative S. carnosus promoter structure and a putative ptsI gene downstream suggesting that ptsH gene is the first gene in the PTS operon of S. carnosus. Comparison of the amino acid sequence of S. carnosus HPr with the HPr sequence of Staphylococcus aureus (derived from peptide sequencing) showed a high degree of similarity.

Amino Acid Sequence↗

Glycopeptide resistant Staphylococcus.

Insensitivity of Staphylococcus aureus to glycopeptides became known at the end of the 1990s. To distinguish intermediate (VISA and GISA) and full resistance (vancomycin-resistant Staphylococcus aureus, VRSA) is of both epidemiological and clinical importance. The VISA (GISA) phenotype is obviously selected by glycopeptide usage in individual patients and can be disseminated by clonal spread of particularly affected staphylococcal isolates, preferentially methicillin-resistant S. aureus (MRSA). In the meantime emergence of VISA became known worldwide. VRSA evolve by acquisition of the vanA gene from enterococci, which are of significance as reservoir of resistance genes among gram-positive bacterial pathogens. Until now three independent single cases of emergence of VRSA became known from the USA, in two of them an association with vanA containing Enterococcus spp. at the same site of the affected patients could be established. The frequency of VISA (GISA) among MRSA in Central Europe is still low (<0.1%); no VRSA have been detected until now.

Animals↗

Microbiological features of Staphylococcus schleiferi subsp. coagulans, isolated from dogs and possible misidentification with other canine coagulase-positive staphylococci.

Staphylococcus schleiferi subsp. coagulans has only rarely been isolated and identified from the external auditory meatus of dogs suffering from external otitis. Its morphological and basic biochemical characteristics are of relatively little value for identification, as it phenotypically resembles another coagulase-positive staphylococci (CPS) and, consequently, may be easily misidentified as S. intermedius or even as S. aureus. In the present work, differentiation of S. schleiferi ssp. coagulans was therefore based on specific biochemical and genetic methods. All the strains were evaluated with the following commercial methods: Api Staph System (bioMérieux, Marcy l'Etoil, France), BBL Crystal Identification Systems (Gram-Positive ID Kit and Rapid Gram-Positive ID Kit; Becton Dickinson), and GEN-PROBE AccuProbe, Staphylococcus aureus identification test (bioMérieux). Gram-Positive ID System/GP database includes the broadest range of staphylococcal species and correctly identifies the majority of strains important in veterinary medicine. Therefore, it is an acceptable alternative to conventional methods for identification of canine staphylococcal isolates. Reliable differentiation of S. aureus from S. schleiferi ssp. coagulans and S. intermedius was feasible with AccuProbe for S. aureus, which gave positive results only for S. aureus; all other CPS tested were negative.

Animals↗

Comparative assessment of the antimicrobial susceptibility of Staphylococcus aureus isolates from bovine mastitis in biofilm versus planktonic culture.

Mastitis is one of the most important diseases in dairy cattle of which Staphylococcus aureus is a major pathogen. Despite an apparently good antimicrobial susceptibility in vitro, the cure of diseased animals from this bacteriological infection is often disappointing, which results in cases of recurrent clinical- and chronic subclinical infections. It has been suggested that these recurrent and chronic Staphylococcus infections can be attributed to the growth of bacteria in biofilm. The objective of this study was to compare the susceptibility for antimicrobial agents of S. aureus isolates obtained from bovine mastitis growing under different conditions. These conditions include a conventional conventional microbroth dilution assay in which minimal inhibitory concentration values are determined, the MBEC assay which measures both the susceptibility in biofilm and the susceptibility of sequester cells released from the biofilm. A comparison of the susceptibility for antimicrobial agents of a number of representative S. aureus isolates grown in broth (representing in vitro growth conditions) or milk (representing in vivo growth conditions) is also made. The results indicate that S. aureus isolates obtained from bovine mastitis are highly resistant to antimicrobial agents when growing in biofilms.

Animals↗

A Staphylococcus aureus mouse keratitis topical infection model: cytokine balance in different strains of mice.

Staphylococcus is a leading cause of the potentially blinding disease microbial keratitis. Even with the use of antibiotic therapy, the host inflammatory response continues to damage the cornea, which may lead to blindness. Manipulation of the host response may help improve patient outcome from this devastating disease. We aim to understand the contribution of the host response to Staphylococcus aureus infection. A S. aureus keratitis mouse model was developed in both C57BL/6 and BALB/c mice using two different strains of S. aureus (8325-4 and Staph 38). Twenty-four hours postinfection, mice were killed and eyes were harvested for enumeration of bacteria, polymorphonuclear leucocytes, chemokines and cytokines. The laboratory strain 8325-4 was not as virulent as the clinical isolate Staph 38. In vitro data showed a 250-fold increase in invasion of human corneal epithelial cells by Staph 38 compared to 8325-4. BALB/c mice were susceptible to S. aureus infection whereas C57BL/6 mice were resistant. The resistant C57BL/6 mice were polarized towards a Th2 response, which may be protective for these mice. IL-4, IL-10 and IL-6 were elevated significantly in C57BL/6 mice infected with Staph 38 (P < 0.05). Macrophage inflammatory peptide (MIP)-2 was also significantly elevated in C57BL/6 mice (P < 0.001). The susceptible BALB/c mice had a muted cytokine response, which suggests that S. aureus might be 'walled off' during infection and might avoid host defences. IL-4, IL-10 and IL-6 cytokines may be protective during Gram-positive corneal infection and therefore may be useful for adjunct therapies in the treatment of this disease.

Animals↗

Evaluation of the Vitek 2 system for identification and antimicrobial susceptibility testing of Staphylococcus spp.

The Vitek 2 system was assessed against reference methods with 197 methicillin-resistant Staphylococcus aureus from Belgian hospitals and 121 clinically significant blood culture isolates of Staphylococcus spp. Vitek 2 identified 95% of staphylococcal isolates correctly, detected oxacillin resistance with a sensitivity/specificity of 99/96%, and showed acceptable accuracy for susceptibility testing of five of eight other evaluable antibiotics. The median time for reporting results was 2 h 45 min for identification and 7 h for susceptibility tests.

Methicillin Resistance↗

Use of broth enrichment and real-time PCR to exclude the presence of methicillin-resistant Staphylococcus aureus in clinical samples: a sensitive screening approach.

A rapid and sensitive method for excluding the presence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples was developed and evaluated. The method utilised an MRSA-selective enrichment broth for 16 h, followed by PCR quantification of the nuc gene. Samples below a quantitative PCR threshold were reported as MRSA-negative. Broths from PCR-positive samples were subcultured for MRSA isolation. Clinical samples (n = 334) in a constructed high prevalence population were analysed in parallel with a selective plating method. The new broth-PCR assay increased the number of positive samples by 35% (49 vs. 66), and 94% of negative samples were reported within 24 h. To reduce costs and workload, 665 clinical samples were grown separately in enrichment broth and then pooled in the PCR step. The broth-PCR assay increased the number of MRSA positive samples from 11 to 15 compared with selective plating. Most (89%) of the culture-negative samples were also PCR-negative and could be reported within 24 h. The growth of 25 European EMRSA strains was tested in the selective enrichment broth. On average, the MRSA strains showed a 300 000-fold increase in CFU, compared with 30-fold for the eight methicillin-sensitive Staphylococcus aureus strains tested.

Bacterial Proteins↗

Staphylococcus lugdunensis: clinical spectrum, antibiotic susceptibility, and phenotypic and genotypic patterns of 39 isolates.

Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci with the potential to cause clinically significant infections. The spectrum of infections was investigated in 39 isolates of S. lugdunensis from 38 patients. Most (73%) infections were located below the waist, while those above the waist were mainly (5/7) breast abscesses. Most isolates were susceptible to the antibiotics tested, although 15.4% were beta-lactamase-positive and could be identified by the disk-diffusion method for penicillin G. There was very good concordance between the disk-diffusion method and the Etest method for oxacillin resistance. Pulsed-field gel electrophoresis (PFGE) showed that 56% of the isolates belonged to one SmaI pulsotype, while phenotypic analysis by the Phene Plate system identified three main phenotypic groups. Although the S. lugdunensis isolates analysed were obtained from different patients, treated in different wards and hospitals during a 4-year period, there was a low degree of diversity, both genotypically and phenotypically. For this reason, PFGE is not suitable for the analysis of an outbreak situation, and the homogeneity observed may indicate that S. lugdunensis is a genetically conserved species of coagulase-negative Staphylococcus.

Adolescent↗

Structure and putative origin of a plasmid from Staphylococcus hyicus that mediates chloramphenicol and streptomycin resistance.

The structure and functional organization of Staphylococcus hyicus plasmid pSCGp3EB that mediates chloramphenicol and streptomycin resistance (CmrSmr) is described and compared with another CmrSmr plasmid, pSCS12, from Staphylococcus sciuri. Both plasmids appeared to be formed by co-integrate formation between plasmids that very closely resemble the chloramphenicol resistance (Cmr) plasmid pC221 and the streptomycin resistance (Smr) plasmid pS194. In addition to the established recombination site B (RSB) in pC221 and pS194, another area suitable for recombination immediately downstream of the cat gene in pC221 and upstream of the str gene in pS194 has been identified. Co-integration at these sites would lead to the structures we have observed in the wild-type CmrSmr plasmids pSCGp3EB and pSCS12.

Amino Acid Sequence↗

Morphological and physiological changes of Staphylococcus aureus exposed to hypochlorous acid.

AIMS: To characterize hypochlorous acid (HOCl) stress resistance of Staphylococcus aureus and to assess physiological state and changes in cell morphology. METHODS AND RESULTS: Clinical wild-type strain of S. aureus was used in the stress with HOCl at concentrations ranging from 0 to 4 mg l(-1). Concentrations below 1.5 mg l(-1) caused no significant drop in viability. During 2 h of HOCl stress at 2 mg l(-1), there was appearance of minicells capable of passing through the 0.45 microm pores of filtration membranes. Intracellular proteins increased gradually to reach a level of 51% of dry weight and an enhanced synthesis of at least two proteins of 23 and 220 kDa was concluded. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus can undergo morphological and physiological changes during 2 h of exposure to 2 mg l(-1) of HOCl, which represents an adaptative response towards the hypochlorous acid stress. This evolution limits the use of 0.45 microm pores size membrane filters for research on S. aureus in waters and the clinical environment.

Bacterial Proteins↗