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PubMed2006

Coordinated and differential control of aureolysin (aur) and serine protease (sspA) transcription in Staphylococcus aureus by sarA, rot and agr (RNAIII).

Previous studies have shown that production of extracellular proteases in Staphylococcus aureus is stimulated by agr (RNAIII) and mgrA, and repressed by sarA. Protease expression is also repressed by rot, however this effect is generally observed only in agr mutants. Several other regulators (sarR, sarV, sarS, sae) that may impact protease expression have been described. As the interactions between all regulators that control protease gene expression are not fully understood, the present study was undertaken to elucidate the regulatory network governing aureolysin (aur) and staphylococcal serine protease (sspA) transcription. The regulation of both genes was studied as activation of the serine protease (SspA) zymogen requires aureolysin. For this purpose we have analyzed the effect of different combinations of regulatory mutations. The present study clearly shows that the positive effect of agr (RNAIII) on aur and sspA transcription requires rot, which is in accordance with the hypothesis that RNAIII acts by neutralizing Rot activity through binding [McNamara, P.J., Milligan-Monroe, K.C., Khalili, S., Proctor, R.A., 2000. Identification, cloning, and initial characterization of rot, a locus encoding a regulator of virulence factor expression in Staphylococcus aureus. J. Bacteriol. 182, 3197-3203]. Concomitantly, overexpression of rot in agr(+) strains or inactivation of rot in strains with low levels of RNAIII clearly affected aur and sspA transcription, indicating that the inhibiting effect of RNAIII on Rot could be titrated. Furthermore, our present data support that the only role of RNAIII in aur and sspA regulation is to counteract the repressive activity of Rot. Apart from an apparent direct positive effect of mgrA on sspA and aur transcription, these genes were mainly controlled through repression by sarA and rot, which seemed to occur via binding of SarA and Rot to the aur and sspA promoters, respectively. Maximum transcription of aur and sspA was obtained when both repressors were absent, either in a sarA mutant where Rot is neutralized by RNAIII during post-exponential phase, or in an agr sarA rot triple mutant. Interestingly, aur was much more sensitive to repression by sarA than by rot, whereas sspA was equally suppressed by sarA and rot. On the other hand, sspA was more sensitive to repression by rot than aur. Thus, SarA and Rot seemed to act independently in an additive way. Inactivation of sarR and sarS had no apparent effect on aur and sspA transcription, although overexpression of these regulators suppressed aur and sspA transcription, respectively, likely in a direct way as indicated by DNA binding experiments. In conclusion, our results indicate that aur and sspA transcription are coordinately regulated but can also be individually modulated by agr, sarA, rot, sarS, sarR, and mgrA. A provisional model for the regulation of aur and sspA transcription is presented.

Bacterial Proteins↗

PubMed2004

Inhibitory effects of Caesalpinia sappan on growth and invasion of methicillin-resistant Staphylococcus aureus.

In the present study, we investigated antimicrobial activity of Caesalpinia sappan against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and effect of Caesalpinia sappan extract on the invasion of MRSA to human mucosal fibroblasts (HMFs). Chloroform, n-butanol, methanol, and aqueous extracts of the Caesalpinia sappan showed antimicrobial activity against standard methicillin-sensitive Staphylococcus aureus (MSSA) as well as MRSA. Methanol extract of Caesalpinia sappan demonstrated a higher inhibitory activity than n-butanol, chloroform, and aqueous extracts. In the checkerboard dilution method, methanol extract of Caesalpinia sappan markedly lowered the minimal inhibitory concentrations (MICs) of ampicillin and oxacillin against MRSA. To determine whether methanol extract of Caesalpinia sappan inhibits the MRSA invasion to HMFs, the cells were treated with various sub-MIC concentrations of methanol extract and bacterial invasion was assayed. MRSA invasion was notably decreased in the presence of 20-80 microg/ml of Caesalpinia sappan extract compared to the control group. The effect of Caesalpinia sappan extract on MRSA invasion appeared dose-dependent. These results suggest that methanol extract of Caesalpinia sappan may have antimicrobial activity and the potential to restore the effectiveness of beta-lactam antibiotics against MRSA, and inhibit the MRSA invasion to HMFs.

Caesalpinia↗

PubMed2005

Detection of icaA and icaD loci by polymerase chain reaction and biofilm formation by Staphylococcus epidermidis isolated from dialysate and needles in a dialysis unit.

Staphylococcus epidermidis, a coagulase-negative staphylococcus, is a major cause of infections associated with indwelling medical devices. Certain strains produce slime and form biofilm on polymer surfaces, where their pathogenicity is associated with biofilm formation. In this report, we investigated the presence or absence of the intercellular adhesion icaA and icaD genes by polymerase chain reaction, and phenotypic biofilm production was examined by qualitative Congo red agar (CRA) assay. A total of 32 strains of S. epidermidis were identified from dialysates and needles 4h after the initiation of dialysis. Qualitative biofilm production revealed that 16 (50%) strains produced slime on CRA plates. Among the 23 strains positive for the ica operon, 15 were biofilm positive on CRA, eight were biofilm negative, and one was icaA and icaD negative but produced slime. These results show that the ability of S. epidermidis to produce slime is not associated with the presence of icaA and icaD genes.

Bacterial Adhesion↗

PubMed2006

Epidemiology, clinical and laboratory characteristics of Staphylococcus aureus bacteraemia in a university hospital in UK.

There has been a persistent increase in the number of meticillin-resistant Staphylococcus aureus (MRSA) and meticillin-susceptible Staphylococcus aureus (MSSA) bacteraemia in the UK. This prospective study included 147 episodes of S. aureus bacteraemia in 139 patients over a 14 month period, from 1 November 2001 to 31 December 2002. Eighty-seven (59%) episodes in 84 patients and 60 (41%) in 56 patients were due to MRSA and MSSA, respectively. An intra-vascular device (29, 33%) and a soft-tissue (15, 25%) source were the commonest identifiable foci for bacteraemia in the MRSA and MSSA groups, respectively. Attributable mortality in the MRSA group was higher than the MSSA group (33% vs 16%; P=0.03) but there was no statistical difference for either attributable (P=0.35) or crude (P=0.39) mortality between the two groups, when adjusted for age, respiratory focus and inappropriate antibiotic therapy. A respiratory source (P=0.02) and inappropriate antibiotic therapy (P=0.02) were associated with attributable mortality in the MRSA group whereas advanced age was the only risk factor (P=0.02) in the MSSA group. The present study shows that S. aureus bacteraemia continues to be a serious infection mostly affecting the elderly and emphasizes the need for improved strategy in the control and management of this condition.

Adolescent↗

PubMed2005

Severe non-pneumonic necrotising infections in children caused by Panton-Valentine leukocidin producing Staphylococcus aureus strains.

Two cases of infection with Panton-Valentine Leukocidin (PVL) producing strains of Staphylococcus aureus are reported. A 15-year-old insulin dependent diabetic developed toxic shock syndrome and an abscess in the deep tissue around his left hip. A 34-day-old infant presented with a right orbital cellulitis with an intra-orbital collection and septicaemia. In both cases PVL-producing strains of Staphylococcus aureus were isolated. Both surgery and prolonged antibiotic combination regimens were required to eradicate the infection. The cases reported here demonstrate the wide range of clinical presentations seen with PVL producing strains, which have so far been mainly associated with furuncles and necrotising pneumonia.

Abscess↗

PubMed2006

Osteomyelitis of the ear canal caused by Staphylococcus lugdunensis.

Osteomyelitis of the temporal bone caused by Staphylococcus lugdunensis has never been reported before. This infection was diagnosed in a 51-year-old diabetic man. S. lugdunensis is an unusually virulent coagulase negative staphylococcus and has been implicated in a variety of severe infections. Considering the serious morbidity and mortality associated with osteomyelitis and in particular with S. lugdunensis, special awareness and alertness are required when otitis externa, which predisposes to osteomyelitis, is refractory to treatment.

Humans↗

PubMed2006

DNA microarray for the detection of therapeutically relevant antibiotic resistance determinants in clinical isolates of Staphylococcus aureus.

An oligonucleotide microarray was constructed for the rapid and sensitive molecular detection of antibiotic resistance determinants in Staphylococcus aureus. The array is equipped with oligonucleotide capture probes for the detection of 10 clinically and therapeutically relevant antibiotic resistance genes and -mutations (mecA, aacA-aphD, tetK, tetM, vat(A), vat(B), vat(C), erm(A), erm(C), grlA-mutation) as well as several control probes. A microarray concept was established including multiplexed PCR amplification, DNA labeling, hybridization and data processing. This concept was applied to clinical Staphylococcus aureus isolates and results were concordant with those from standard genotypic and phenotypic resistance testing. Our microarray concept offers rapid and accurate identification of antibiotic resistance profiles. It is easily expandable and thus can be adapted to changing clinical and epidemiological requirements in clinical diagnosis as well as in epidemiological studies.

DNA Probes↗

PubMed2004

The application of peptide nucleic acid probes for rapid detection and enumeration of eubacteria, Staphylococcus aureus and Pseudomonas aeruginosa in recreational beaches of S. Florida.

A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, Staphylococcus aureus and Pseudomonas aeruginosa, was achieved within 6-8 h of processing. Following 5 h of incubation on TSA, soybean peroxidase-labeled peptide nucleic acid probes (Boston Probes, Boston, MA) targeting species-specific 16S rRNA sequences of P. aeruginosa and S. aureus were used to hybridize microcolonies of the target species in-situ. In addition, a universal probe for 16S rRNA sequences was used to target the eubacteria. Probes were detected after a light generating reaction with a chemiluminescent substrate and their presence recorded on Polaroid film. The probes showed limited cross-reactivity with mixed indigenous bacteria extracted from seawater and sand by shaking with phosphate-buffered saline (PBS). Specificity and cross-reactivity was tested on the reference bacterial genera Pseudomonas, Staphylococcus, Vibrio, Shigella, Salmonella, Acinetobacter, Enterobacter, Escherichia and Citrobacter. These tests confirmed that the probes were specific for the microorganisms of interest and were unaffected by high salt levels. The results of the PNA chemiluminescent in situ hybridization were compared with traditional plate count methods (PCM) for total 'freshwater' eubacteria, S. aureus and P. aeruginosa. Counts of eubacteria and S. aureus were comparable with numbers obtained from traditional plate counts but levels of P. aeruginosa were higher with PNA than with PCM. It is possible that PNA is more sensitive than PCM because it can detect microcolonies on the agar surface that never fully develop with the plate count method. We conclude that the in situ hybridization technique used here represents an important potential tool for the rapid monitoring of novel indicator organisms in beaches and recreational waters.

Acinetobacter↗

PubMed2006

Proinflammatory effects of Staphylococcus aureus exotoxin B on nasal epithelial cells.

OBJECTIVE: The aim of this study was to evaluate the effects of Staphylococcus aureus exotoxin B (SE-B) on proinflammatory cytokine/chemokine releases in primary nasal epithelial cell cultures (NECC) of subjects with and without chronic rhinosinusitis (CRS). STUDY DESIGN AND SETTING: NECC (CRS: n = 14; CONTROLS: n = 11) were stimulated with SE-B. Protein concentrations of interleukin-(IL)-1beta, IL-6, and IL-8 levels were measured in NECC supernatants by ELISA before (T0) and after 24 hr stimulation with SE-B (T1). RESULTS: T0: supernatants of the NECC of CRS patients contained significant lower levels of IL-8 (2.1 ng/ml) compared to CONTROLS (IL-8: 6.2 ng/ml; P < 0.01). T1: SE-B induced a significant increase of IL-6 in NECC (P < 0.001). IL-1beta was not detectable. CONCLUSIONS: This is the first study evaluating the effects of exotoxins on NECC. SE-B showed proinflammatory effects on NECC. SIGNIFICANCE: Our data suggest that resident NECC are involved in immunological responses to Staphylococcus aureus toxins, supplementing the so-called "superantigen hypothesis" in CRS.

Cells, Cultured↗

PubMed2004

[Methicillin-resistant Staphylococcus aureus nosocomial infections in ICU: risk factors, morbidity and cost].

OBJECTIVE: Methicillin resistance and infections caused by methicillin-resistant Staphylococcus aureus represent a growing problem and a challenge for health-care institutions. We evaluated risk factors, morbidity and cost of infections caused by methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus. DESIGN: We performed an un-matched case-control study in an 20-bed medical intensive care unit from 1994-2001 at Nantes teaching hospital, France. All patients with pneumonia, bacteraemia and urinary MRSA (cases) or MSSA (controls) nosocomial infections were included in the study. RESULTS: Twenty four patients with MRSA infection were compared to 64 patients with MSSA infections. Patients with MRSA infection were older (56 vs. 45 years, P < 0.01), had longer length of stay (47 vs. 35 days, P < 0.05) and were infected later (22 vs. 10 days, P < 0.00001) than patients with MSSA infection. No difference was observed between the two groups according to the Omega index, acute simplify index and mortality. MRSA infections involved extra cost due to antimicrobial treatment (184 vs. 72 Euros, P < 0.005) and length of stay (37,278 vs. 27,755 Euros, P < 0.05). CONCLUSION: Patient infected by MRSA seems to be different from patient infected by MSSA but without consequence on Omega index and mortality. But methicillin-resistance involves extra cost due to antimicrobial treatment and length of stay.

Costs and Cost Analysis↗

PubMed2006

[Detection of nasal colonization methicillin-resistant Staphylococcus aureus: a prospective study comparing real-time genic amplification assay vs selective chromogenic media].

UNLABELLED: In contrast to "classical" genic amplification, real-time genic amplification can be performed in every laboratory without the need of sophisticated isolation procedures. Moreover, real-time genic amplification allows an early detection of meticillin resistant Staphylococcus aureus colonization, 2 hours compared to 1 or 2 days for culture. OBJECTIVE: In order to assess the feasibility on Smartcycler of the IDI-MRSA real-time genic amplification assay in comparison with chromogenic media. METHODS: A prospective study has been initiated in July 2004: nasal swabs were taken from patients entering the ICU, vascular surgery, diabetology and geriatry wards. During a 4 months period, 682 specimens have been obtained from 508 patients. RESULTS: Sixty-four (9.3%) patients were positive by genic amplification and selective agar culture (CHROMagar MRSA, MRSASelect and/or ORSAB), 19 (2.9%) were positive by genic amplification only (3 of these patients were under antibiotic treatment); 572 specimens remained negative by both methods. The sensitivity and specificity of this assay were 100% and 96% respectively with a positive predictive value of 70% and negative predictive value of 100%. Initially 82 nasal specimens were unresolved (12%). 38 were resolved following a freeze-thaw cycle. Thus, 44 (6.4%) were unresolved specimens. Comparison between CHROMagar MRSA and MRSASelect showed a good correlation for the detection at 24 hours (5.5% and 5.6% respectively). These two chromogenic media allowed a much better detection of MRSA than ORSAB medium within 24H. CONCLUSION: The results obtained by the early real-time genic amplification for the detection of meticillin resistant Staphylococcus aureus are promising. Despite 6.4% amplification failure, we consider that IDI-MRSA real-time genic amplification assay represents a significant breakthrough in the detection of colonization.

Culture Media↗

PubMed2006

Clonality of clinical methicillin-resistant Staphylococcus epidermidis isolates in a neonatal intensive care unit.

OBJECTIVE: Study of the clonality of methicillin-resistant Staphylococcus epidermidis responsible of epidemic infections in a neonatal intensive care unit. PATIENTS AND METHODS: All S. epidermidis isolates (mecA+) were collected during the epidemic period (December 2003-September 2004) from different pathological products of newborns. Isolates were characterized by genotyping in pulsed-field gel electrophoresis and by electrophoretic profiles obtained by PCR-based analysis of inter-IS256 spacer polymorphisms. RESULTS: Twenty methicillin-resistant S. epidermidis isolates were collected from newborns during the epidemic period and represented 41.6% of the total isolates of S. epidermidis, which is the first Staphylococcus species isolated from the unit. These isolates were collected from blood cultures (80%), vascular catheters (5%), pus (10%), and intra-tracheal tube (5%). Six genotypic profiles were individualized: type A, type B, type C, type D, type E, and type F, with clear dominance of type A. Five different PCR patterns were found with poor correlation to genotypes defined by PFGE. CONCLUSION: Neonatal nosocomial outbreak of methicillin-resistant S. epidermidis was caused by multiple clones of this species with predominance of one epidemic and multiresistant clone. This clone may be transmitted between babies and was able to persist in the unit. PCR IS 256 proved to be less discriminative than PFGE for typing MRSE.

Equipment Contamination↗

PubMed2005

Analysis of the beta-lactamase plasmid of borderline methicillin-susceptible Staphylococcus aureus: focus on bla complex genes and cadmium resistance determinants cadD and cadX.

Borderline methicillin-susceptible Staphylococcus aureus strains are a rather homogeneous group, characterized by MICs of penicillinase-resistant penicillins (PRPs) at or just below the susceptibility breakpoint. Other features unique to this group include the presence of a pBW15-like beta-lactamase plasmid, the association with phage complex 94/96, and the production of a PRP-hydrolyzing beta-lactamase activity in addition to the classical penicillinase activity. The four HindIII fragments of pBORa53, a pBW15-like plasmid from the well-studied borderline S. aureus strain a53, were cloned in Escherichia coli, sequenced and analyzed. The plasmid (17,334 bp in size) contains 14 open reading frames (ORFs) and a complete copy of transposon Tn552, which harbors the three genes of the bla complex (blaZ, blaR1, and blaI) necessary for penicillinase production. Among the other 11 ORFs identified, two were homologous to cadmium resistance determinants of Staphylococcus lugdunensis and to the cadD and cadX genes recently detected in S. aureus. Consistent with this, strain a53 was found to be cadmium resistant. From a collection of 30 S. aureus isolates with borderline PRP MIC levels, 27 matched strain a53 in the positive amplification reactions with all of the four primer pairs targeting the cadD-cadX region, the presence of the 17.3-kb plasmid, and the level of cadmium resistance. The well-established S. aureus laboratory strain ATCC 29213 was also found to express cadD-cadX-mediated cadmium resistance. pBORa53 could be re-isolated from transformants obtained by transferring it into a PRP-susceptible recipient. However, while the transformants demonstrated levels of cadmium and penicillin resistance similar to those of strain a53, they remained fully susceptible to PRPs.

Bacterial Proteins↗

PubMed2004

In vitro antimicrobial activity of orbifloxacin against Staphylococcus intermedius isolates from canine skin and ear infections.

The objective of the study was to evaluate the in vitro activity of orbifloxacin against Staphylococcus intermedius strains isolated in France from canine skin and ear infections. The minimum inhibitory concentrations (MICs) of orbifloxacin against 240 field S. intermedius isolates (69 skin and 171 ear isolates) ranged from 0.016 to 8 mg l(-1), with MIC50 and MIC90 equal to 0.5 and 1 mg l(-1), respectively. Only one strain, a pyoderma isolate was resistant (MIC=8 mg l(-1)). Orbifloxacin was tested at different concentrations for killing rate against five isolates obtained from pyoderma cases and against a reference strain (Staphylococcus aureus ATCC 29213). Orbifloxacin expressed a concentration-dependent bactericidal activity against the S. aureus reference strain, but a time-dependent bactericidal activity against S. intermedius. Orbifloxacin induced bactericidal effect against the S. intermedius strains tested with concentrations equal to or two times MIC.

Animals↗

PubMed2004

In vitro growth inhibition of major mastitis pathogens by Staphylococcus chromogenes originating from teat apices of dairy heifers.

Earlier field observations suggest that teat apex colonization by Staphylococcus chromogenes pre-partum in dairy heifers protects udder quarters against elevated somatic cell counts early post-partum. To explain these findings, the in vitro inhibitory capability of S. chromogenes from teat apices of heifers towards some major mastitis pathogens was tested using a modified cross-streaking method. Two out of 10 S. chromogenes isolates, both originating from two different teats from the same heifer, consistently inhibited growth of all Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis strains, but none of the Escherichia coli strains. The present study, therefore, supports the protective effect of teat apex colonization by S. chromogenes by in vitro production of inhibitory substances.

Animals↗

PubMed2005

Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes.

Staphylococcus chromogenes is closely related to Staphylococcus hyicus, which is recognised as the causative agent of exudative epidermitis (EE) in pigs. S. chromogenes is part of the normal skin flora of pigs, cattle and poultry and has so far been considered non-pathogenic to pigs. A strain of S. chromogenes producing exfoliative toxin type B, ExhB, was identified by the use of a multiplex PCR specific for the exfoliative toxins from S. hyicus. The exfoliative toxin from S. chromogenes reacted in immunoblot analysis with polyclonal and monoclonal antibodies specific to ExhB from S. hyicus and had an apparent molecular weight of 30 kDa. Sequencing the gene encoding the exfoliative toxin from S. chromogenes revealed that the molecular weight of the toxin with the signal peptide and the mature toxin was 30,553 and 26,694 Da, respectively. Comparison of the exhB genes from S. chromogenes strain VA654 and S. hyicus strain 1289D-88 showed differences in seven base pairs of the DNA sequences and in two amino acid residues in the deduced amino acid sequences. Pigs were experimentally inoculated with S. chromogenes strain VA654. By clinical observations and histopathological evaluation of the skin alterations, all pigs revealed development of generalized exudative epidermitis. No toxin producing S. hyicus was isolated from the pigs and all ExhB-positive bacterial isolates were identified as S. chromogenes. This confirmed that the disease-causing agent was the inoculated S. chromogenes strain VA654. The results of this study show that S. chromogenes may cause exudative epidermitis in pigs.

Amino Acid Sequence↗

PubMed1996

Coagulase-negative Staphylococcus endophthalmitis after cataract surgery with intraocular vancomycin.

PURPOSE: We studied a case of postoperative coagulase-negative Staphylococcus endophthalmitis in a 79-year-old man who had undergone cataract extraction in which vancomycin had been used intraoperatively in the infusion fluid. METHODS: The medical records were reviewed for the clinical history, ocular findings, and the vitreous and anterior chamber culture results. RESULTS: Acute, postoperative coagulase-negative Staphylococcus endophthalmitis developed in the patient in whom vancomycin had been used intraoperatively in the infusion fluid. CONCLUSIONS: Restraint is urged in the prophylactic use of vancomycin in the infusion fluids.

Acute Disease↗

PubMed1988

Neutrophil stimulating activity released by Staphylococcus-stimulated mononuclear leukocyte conditioned medium. Further characterization and partial purification.

Culture medium conditioned by human mononuclear leukocytes (MNL) stimulated with formalin fixed heat-killed Staphylococcus aureus induces a small respiratory burst in human neutrophils, and dramatically increases the response of neutrophils to stimuli such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The data presented show that the activity is not unique to Staphylococcus aureus. Similar neutrophil modulating activities were produced by medium conditioned by MNL cultured in the presence of Streptococcus pneumonia, and Group B streptococcus. The activity was relatively resistant to heating; significant reduction of activity was observed only when 80 degrees C was reached. Neutrophil stimulating activity production by stimulated MNL was dependent on protein and RNA synthesis and the activity appeared to be released by the non-adherent fraction of the MNL, suggesting that it is not of macrophage origin. The activity was not sensitive to soya bean trypsin inhibitor, but was sensitive to trypsin and was not removed when stimulated conditioned medium was depleted of immunoglobulin and albumin by affinity chromatography. Purification by gel filtration on Sephadex G-100 and high-performance liquid chromatography with Bio-Sil TSK250 columns showed that the major activity had an apparent molecular weight of 35,000-43,000 under conditions in which ionic interactions and association with albumin were reduced; by using polyethylene glycol or high salt (0.46 M Na+) in the elution buffer.

Cell Adhesion↗