Slime-producing Staphylococcus epidermidis and S. aureus in acute bacterial conjunctivitis in soft contact lens wearers

In recent years, an increase in ocular pathologies related to soft contact lens has been observed. The most common infectious agents were Staphylococcus spp. Some strains produce an extracellular polysaccharidic slime that can cause severe infections. Polysaccharide synthesis is under genetic control and involves a specific intercellular adhesion (ica) locus, in particular, icaA and icaD genes. Conjunctival swabs from 97 patients with presumably bacterial bilateral conjunctivitis, wearers of soft contact lenses were examined. We determined the ability of staphylococci to produce slime, relating it to the presence of icaA and icaD genes. We also investigated the antibiotic susceptibility and Pulsed Field Gel Electrophoresis (PFGE) patterns of the clinical isolates. We found that 74.1% of the S. epidermidis strains and 61.1% of the S. aureus strains isolated were slime producers and showed icaA and icaD genes. Both S. epidermidis and S. aureus slime-producing strains exhibited more surface hydrophobicity than non-producing slime strains. The PFGE patterns overlapped in S. epidermidis strains with high hydrophobicity. The similar PFGE patterns were not related to biofilm production. We found scarce matching among the Staphylococcus spp. studied, slime production, surface hydrophobicity and antibiotic susceptibility.     

A novel application of Fourier-transformed infrared spectroscopy: classification of slime from staphylococci

It has been proposed that the virulence of nosocomial Staphylococcus infections associated with indwelling medical devices is related to the ability of the bacterium to colonise these materials by forming a biofilm composed of multilayered cell clusters embedded in a slime matrix. However, the pathogenic role of exopolysaccharide biofilms is not fully understood. A new method was sought for differentiating the structure of slime from two closely related bacterial strains, Staphylococcus aureus and Staphylococcus epidermidis. Using PCR it was confirmed that these strains were positive for the icaA and icaD genes and the complete ica operon (2.7 kb). Monosaccharide analysis by thin-layer chromatography revealed an identical profile for both strains, with xylose and glucose present among the four visible bands. Using Fourier-transformed infrared spectroscopy and hierarchical cluster analysis, three of four S. aureus samples (75%), and four of five S. epidermidis samples were grouped according to species. A novel FTIR approach in classifying slime produced by S. aureus and S. epidermidis is reported.     

Congo red agar plate method: improved accuracy and new extended application to Staphylococcus aureus.

In the last decade an increasing number of research studies have focused on the role of slime formation in Staphylococcus epidermidis and, more recently, also in S. aureus. In this context, much attention is being paid to evaluating the prevalence of slime production among bacteria strains isolated from clinical infections in an attempt to assess the role and the diagnostic value of this well recognised virulence marker. Such types of investigations require reliable techniques to identify slime producing strains. For years, even though based on a subjective chromatic evaluation, the Congo red agar plate (CRA) represented a reference phenotypic test for S. epidermidis. Only with the new introduction of PCR-based techniques, able to specifically identify the genes necessary for slime production, did the accurate genetic classification of slime producing bacteria become possible. In the present investigation, a comparison with new PCR methods confirmed the validity of the classic CRA test, implemented with minor refinements. Thanks to a few modifications it was also possible to adapt and extend the CRA test, making it also suitable to screen S. aureus strains.     

Comparison of three methods detection of slime production by Staphylococcus aureus and Staphylococcus epidermidis

In this article, slime production of Staphylococcus aureus and Staphylococcus epidermidis strains from infective skin lesions was evaluated by three different methods: Congo red agar method (CRA), Christensen tube method (CT) and spectrophotometric method (SC). All strains by CT method interpreted as negative (dark-claret or red colonies of the surface). 12 (37.5%) strains of S. aureus, 16 (50.0%) strains of S. epidermidis produced slime as shown by CT method, 6 (18.7%) strains of S. aureus, 8 (25,0%) strains of S. epidermidis by SC method. They also found a correlation of slime production by CT and SC method (p > 0.05).     

Biomaterial-associated staphylococcal peritoneal infections in a neutropaenic mouse model

Abstract Adhesion of staphylococcal cells to polyethylene with end point-attached heparin was quantified by bioluminescence. Staphylococcus epidermidis 3380 and the slime-producing S. epidermidis RP12 adhered to the highest extent, and S. lugdunensis 2342 to the least extent. Preincubation of the polymer with dialysis fluid reduced adhesion of S. epidermidis 3380 and RP12 but enhanced that of S. aureus , and preadsorption of the surface with fibronectin decreased subsequent adhesion of S. epidermidis and S. haemolyticus strains. When staphylococci were grown in the presence of a biomaterial their ability to activate peritoneal cells was decreased. The bactericidal activity was impaired, whereas ingestion of opsonized coagulase-negative staphylococci (CNS) strains was unaffected. With S. epidermidis RP12 the presence of biomaterial did not influence either phagocytosis or bactericidal effect of peritoneal cells. After intra-peritoneal challenge with staphylococcal strains, the organ uptake of S. aureus Cowan 1 was increased in normal mice whereas immunosuppressed mice died. CNS strains increased mainly in the peritoneal cavity of immunosuppressed mice. The uptake of bacteria in liver and kidneys was increased with S. epidermidis 3380, S. lugdunensis 2343 and S. schleiferi 667-88. Generally, CNS strains persisted in the peritoneal cavity of both normal and immunosuppressed mice. These data indicate that host defense mechanisms, mainly polymorphonuclear neutrophils, fail to eliminate CNS infections in the peritoneum, and that initial adhesion to an implanted biomaterial may be of lesser importance in the peritoneal cavity than in e.g. catheter-associated infections. There are strain-specific virulence factors of bacteria, and slime producing strains evade the host defense mechanisms more efficiently than non-slime producing strains.     

The effects of magnesium, calcium and EDTA on slime production byStaphylococcus epidermidis strains

Effect of magnesium, calcium and EDTA on slime production by 15 slime-positive and 13 slime-negative Staphylococcus epidermidis strains isolated from various clinical specimens was determined. The slime production on tryptic soy broth was significantly enhanced after addition of 128 mumol/L Mg2+. Similarly, the addition of Ca2+ caused a significant increase in slime production of all tested strains when concentration of Ca2+ exceeded 64 mumol/L. In contrast, in the presence of EDTA the slime production by all strains was significantly reduced. Hence Ca2+ and Mg2+ increase slime production of S. epidermidis. This finding is important in the context of the pathogenesis of biomedical implant infections caused by S. epidermidis.     

Septicemia due to coagulase-negative Staphylococcus in a community hospital.

The experience with septicemia due to coagulase-negative Staphylococcus at a 623-bed primary care hospital between 1980 and 1984 was reviewed. A total of 38 episodes in 37 patients were documented; data were available on 37 episodes in 36 patients. The organism accounted for 3.8% of all cases of septicemia and 6.7% of cases of nosocomial septicemia and was associated with 0.03% of all admissions. The incidence remained stable over the 5 years. The rate of survival 28 days after the episode was 78%. Most of the episodes (31) originated from infected vascular access sites. Of the 37 isolates 15 (41%), all S. epidermidis, were slime producing. S. epidermidis accounted for 33 of the isolates; of the 33, 5 were methicillin-resistant and slime producing. Various in-vitro susceptibility testing methods and testing for beta-lactamase production yielded conflicting results. Methicillin resistance, slime production and speciation as S. epidermidis were not confirmed as virulence markers. Five patients with methicillin-resistant organisms were treated with cephalosporins, and all recovered. These findings as well as examination of the literature do not support the recommendations that laboratories report such isolates as resistant to all beta-lactam agents and that vancomycin be given in all such infections. The different case mix in community hospitals as compared with university centres results in different patterns of nosocomial infection. Since the community hospital patient population is much larger, more information on the patterns of infections in these centres is needed.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Lack of biofilm contribution to bacterial colonisation in an experimental model of foreign body infection by Staphylococcus aureus and Staphylococcus epidermidis

The contribution of in vivo biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis was studied in an experimental model of foreign body infections. Increasing inocula (from 10(2) to 10(7) organisms) of ica-positive strains of S. aureus and S. epidermidis and their ica-negative isogenic mutants (the ica locus codes for a major polysaccharide component of biofilm) were injected into subcutaneously implanted tissue cages in guinea pigs. Surprisingly, bacterial counts and time-course of tissue cage infection by ica-positive strains of S. aureus or S. epidermidis were equivalent to those of their respective ica-negative mutants, in the locally infected fluids and on tissue-cage-inserted plastic coverslips.     

Staphylococci in orthopaedic surgical wounds.

From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.     

Adhesion dependence of coagulase-negative staphylococci to biomaterials and PIA polysaccharide synthesis on icaADBC operon presence

Both Staphylococcus epidermidis and Staphylococcus haemolyticus are important causes of infections associated with catheters and other medical devices. This infections result in significant morbidity, mortality and economic cost. It has recently been shown that not only S. epidermidis but also S. haemolyticus can produce slime and carries the ica operon responsible for and slime production. In the operon, coexpression of icaA and icaD is required for full slime synthesis. This study is focused on detecting icaA and icaD genes in S. haemolyticus and comparison of these two species. It turned out that strain representatives within the same species behave very differently and a single tested strain from each species is unlikely to be representative of the species as a whole. Contrary to S. epidermidis, S. haemolyticus strain appeared to carry no icaA-like and icaD-like genes, but was able to form biofilm in vitro.     

Vaccine potential of poly-1-6 beta-D-N-succinylglucosamine, an immunoprotective surface polysaccharide of Staphylococcus aureus and Staphylococcus epidermidis

Staphylococcus aureus and S. epidermidis are among the most common causes of nosocomial infection, and S. aureus is also of major concern to human health due to its occurrence in community-acquired infections. These staphylococcal species are also major pathogens for domesticated animals. We have previously identified poly-N-succinyl beta-1-6 glucosamine (PNSG) as the chemical form of the S. epidermidis capsular polysaccharide/adhesin (PS/A) which mediates adherence of coagulase-negative staphylococci (CoNS) to biomaterials, serves as the capsule for strains of CoNS that express PS/A, and is a target for protective antibodies. We have recently found that PNSG is made by S. aureus as well, where it is an environmentally regulated, in vivo-expressed surface polysaccharide and similarly serves as a target for protective immunity. Only a minority of fresh human clinical isolates of S. aureus elaborate PNSG in vitro but most could be induced to do so under specific in vitro growth conditions. However, by immunofluorescence microscopy, S. aureus cells in infected human sputa and lung elaborated PNSG. The ica genes, previously shown to encode proteins in CoNS that synthesize PNSG, were found by PCR in all S. aureus strains examined, and immunogenic and protective PNSG could be isolated from S. aureus. Active and passive immunization of mice with PNSG protected them against metastatic kidney infections after intravenous inoculation with eight phenotypically PNSG-negative S. aureus. Isolates recovered from kidneys expressed PNSG, but expression was lost with in vitro culture. Strong antibody responses to PNSG were elicited in S. aureus infected mice, and a PNSG-capsule was observed by electron microscopy on isolates directly plated from infected kidneys. PNSG represents a previously unidentified surface polysaccharide of S. aureus that is elaborated during human and animal infection and is a prominent target for protective antibodies.     

Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food

AIMS: To develop a multiplex PCR that allows the identification of bacteria belonging to the Staphylococcus genus and in particular to the species Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus isolated from food manufacturing plants. METHODS AND RESULTS: Five primer pairs were used in the multiplex PCR, one specific to the Staphylococcus genus and four specific to S. xylosus, S. saprophyticus, S. epidermidis and S. aureus species. All the 31 Staphylococcus reference strains yielded a specific PCR product with the genus-specific primers. Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus gave a specific PCR fragment with the corresponding species-specific primers. No amplification with the Kocuria, Macrococcus and Micrococcus strains was observed in our conditions. This multiplex PCR was performed on 30 strains of Gram-positive cocci isolated from different workshops and fermented sausages. Among them, 28 belonged to the Staphylococcus genus and 14 were identified to S. saprophyticus, four to S. xylosus, two to S. aureus and one to S. epidermidis. CONCLUSIONS: This multiplex PCR provided reliable and repeatable PCR results. It allowed the identification of a major part of the isolates, highlighting the predominance of the S. saprophyticus species in the workshops studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This tool is a useful way to screen the strains isolated from foodstuff and food environment and to monitor these species during the food processing.     

Pulsed-field gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative staphylococci

Thirteen Staphylococcus aureus and S. epidermidis strains obtained from nose and hand of two employees and one patient of a medical ward as well as two S. hemolyticus strains were analysed according to their restriction fragment length patterns (RFLP) by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI and SstII. Species identification of the isolates was performed by a system which includes 20 biochemical reactions. Furthermore, the antibiotic resistance patterns of the strains were determined. While several isolates exhibited identical antibiotic susceptibilities and biochemical profiles, differences in the RFLP were obtained. In three cases, S. epidermidis strains colonizing the skin showed an identical restriction profile as isolates from the mucous membranes of the same person. We concluded that the analysis of staphylococcal strains by PFGE is an important epidemiological tool with high discrimination power.     

Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS) strains by the API Staph System (Biomerieux). Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5%) were found to be slime producers by Christensen's test tube method whereas 58 strains (31%) were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05). Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1%) and erythromycin (47.1%) showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA) and oxacillin resistant coagulase-negative staphylococci (ORCNS), 97 (75.2%) and 36 (62.1%) respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4%) of 129 S. aureus strains were positive for beta-lactamase enzyme. However, 78 (81.25%) of 96 beta-lactamase positive S. aureus strains were beta-lactamase positive ORSA isolates, but none of them had vancomycin resistance.     

The superoxide dismutase gene sodM is unique to Staphylococcus aureus: absence of sodM in coagulase-negative staphylococci

Superoxide dismutase (SOD) profiles of clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were determined by using whole-cell lysates and activity gels. All S. aureus clinical isolates exhibited three closely migrating bands of activity as previously determined for laboratory strains of S. aureus: SodM, SodA, and a hybrid composed of SodM and SodA (M. W. Valderas and M. E. Hart, J. Bacteriol. 183:3399-3407, 2001). In contrast, the CoNS produced only one SOD activity, which migrated similarly to SodA of S. aureus. Southern analysis of eight CoNS species identified only a single sod gene in each case. A full-length sod gene was cloned from Staphylococcus epidermidis and determined to be more similar to sodA than to sodM of S. aureus. Therefore, this gene was designated sodA. The deduced amino acid sequence of the S. epidermidis sodA was 92 and 76% identical to that of the SodA and SodM proteins of S. aureus, respectively. The S. epidermidis sodA gene expressed from a plasmid complemented a sodA mutation in S. aureus, and the protein formed a hybrid with SodM of S. aureus. Both hybrid SOD forms as well as the SodM and SodA proteins of S. aureus and the S. epidermidis SodA protein exist as dimers. These data indicate that sodM is found only in S. aureus and not in the CoNS, suggesting an important divergence in the evolution of this genus and a unique role for SodM in S. aureus.     

Transfer of resistance plasmids from Staphylococcus epidermidis to Staphylococcus aureus: evidence for conjugative exchange of resistance.

The ability of Staphylococcus epidermidis to transfer antimicrobial resistance to Staphylococcus aureus was tested by mixed culture on filter membranes. Two of six clinical isolates examined were able to transfer resistance to S. aureus strains 879R4RF, RN450RF, and UM1385RF. Subsequent S.aureus transconjugants resulting from matings with S. epidermidis donors were able to serve as donors to other S. aureus strains at similar frequencies. Cell-free and mitomycin C-induced filtrates of donors and transconjugants showed no plaque-forming ability. Addition of DNase I, citrate, EDTA, calcium chloride, and human sera to mating mixes and agar showed no effect on transfer. Nonviable donor cells were unable to transfer resistance and transfer did not occur at 4 degrees C. Cell-to-cell contact was required since transfer did not occur in broth or when filters of donor and recipient, respectively, were placed back-to-back so cells were not in direct contact. Analysis of DNA from S. epidermidis isolate UM899, its subsequent S. aureus transconjugants, and cured derivatives demonstrated that all resistance markers which transferred resided on plasmids. Mating experiments suggested a central role for the gentamicin plasmid pAM899-1 in the transfer process. It is concluded that our results are consistent with a conjugative transfer of resistance from S. epidermidis to S. aureus analogous to plasmid transfer demonstrated in streptococcal species for plasmids such as pAM beta 1. This represents a novel mechanism for gene exchange among staphylococci.     

Antibiotic sensitivity of the main opportunistic pathogenic aerobic bacteria

Antibiotic sensitivity of 1421 strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa and Klebsiella spp. was studied. Gentamicin, levomycetin (chloramphenicol) and ristomycin proved to be the antibiotics of choice in treatment of purulent inflammatory diseases caused by S. epidermidis and S. aureus. For antibiotic therapy of infections caused by gram-negative organisms gentamicin and polymixin might be recommended.