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.     

Neutrophil-stimulating activity of staphylococci based on reactive chemiluminescence data

The neutrophil-stimulating properties of 38 S. aureus strains and 32 S. epidermidis strains were studied in the reaction of luminol-mediated chemiluminescence. All S. aureus strains and 29 S. epidermidis strains were found to possess neutrophil-stimulating activity, the mean activity index for S. aureus being significantly higher. The stimulating activity of the strains varied within a wide range (the variation coefficient was 120.0 +/- 21.9%) and did not correlate with the content of protein A in bacterial cells and the degree of their hydrophoby. The opsonization of staphylococci with normal human serum enhanced the neutrophil reaction 1.5- to 100-fold and simultaneously leveled out the chemiluminescence indices in experiments with different strains (the variation coefficient was 8.0 +/- 1.5%). The nature of the neutrophil-stimulating effect of staphylococci and its relationship to the exploratory reactions of phagocytes are discussed.     

Different staphylococcal strains elicit different levels of production of T-helper 1-inducing cytokines

Cytokine production has been implicated in the pathogenic mechanisms of infections caused by the staphylococci, since these bacteria may act as strong cytokine inducers. To gain deeper insight into the Th1 immune response activated by these bacteria, we have analyzed the interferon (IFN), interleukin-12 (IL-12) and IL-18-inducing activities of different Staphylococcus aureus (S. aureus), S. epidermidis and S. saprophyticus strains in human monocytes and murine bone marrow macrophages. A large majority of the S. aureus strains elicited the simultaneous production of IL-12 p70 and IFN-alpha in the human monocytes, while the S. epidermidis and S. saprophyticus strains induced only a low level of production, if any, of these cytokines. Furthermore, a majority of the S. aureus strains induced significantly higher IL-12 p70 and IL-18 titers in the murine bone marrow macrophages than did the S. epidermidis and S. saprophyticus strains. As IL-12, IL-18 and IFN-alpha stimulate Th1 differentiation synergistically, we suggest that S. aureus strains bias the immune response toward a Th1 phenotype, whereas S. epidermidis and S. saprophyticus strains provide a weaker stimulus for the production of Th1-inducing cytokines, and accordingly possibly elicit a less extensive Th1-associated adaptive immunity.     

Slime production by Staphylococci isolated from prosthesis-associated infections.

Clinical isolates of Staphylococcus epidermidis are frequently referred to produce a biofilm, known as slime, involved in adherence to medical devices and in resistance to host defences. A high frequency of slime producing Staphylococcus aureus strains was never reported, at least in the case of human isolates. In the present study the production of slime by clinical isolates of S. aureus and S. epidermidis from catheter associated infections and from post-surgical infections was studied by a sensitive method based on culturing the isolates on Congo red agar. The study demonstrates that in nosocomial surgical infections, considered separately from catheter-associated infections, S. aureus emerges as a more prevalent etiologic agent than S. epidermidis, with a proportion of slime producing strains markedly high.     

Pathogenicity of Staphylococcus lugdunensis, Staphylococcus schleiferi, and three other coagulase-negative staphylococci in a mouse model and possible virulence factors

Staphylococcus lugdunesis and Staphylococcus schleiferi, two newly described species, have been isolated from numerous types of human infections. We compared the pathogenicity of 30 strains of S. lugdunensis, S. schleiferi, Staphylococcus epidermidis, Staphylococcus warneri, and Staphylococcus hominis, using a mouse model in which a foreign body preadhered with the test strain was implanted subcutaneously, followed by injection of the test strain. All five species of staphylococci produced abscesses. Staphylococcus epidermidis, S. schleiferi, and S. lugdunensis yielded species means of 76-91% abscess formation; 80-100% of the infected foreign bodies and tissues were culture positive. These three species were more virulent than S. warneri or S. hominis, which produced abscesses in 54 and 65% of mice, respectively; only 10-48% of the infected samples were culture positive. Transmission electron microscopy of pure cultures of selected strains showed that all species possessed glycocalyx. All species produced a variety of possible virulence factors, such as alpha and delta hemolysins, as well as the aggressins lipase and esterase. The production of exoenzymes did not always correlate with virulence as demonstrated by abscess formation in mice.     

Protein A research in 1200 strains of Staphylococcus (using a rapid detection technic by passive hemagglutination with glutaraldehyde treated erythrocytes]

The technique used was that of passive hemaglutination of red cells of sheep prepared with glutaraldehyde and sensitized by anti-erythrocyte antibodies. A protein-A was not found in S. epidermidis strains, but was present in 96.3 per cent of 689 strains of S. aureus from human, animal and food origins.     

Production of penicillinase by staphylococcal strains of different origins

A total of 319 strains of S. aureus and 729 strains of S. epidermidis belonging to different biovars isolated from the skin and nasal mucosa of 349 persons representing 8 independent groups were tested. On the whole production of penicillinase was more often observed in the strains of S. aureus than in the strains of S. epidermidis. Within the first species this property was more often detected in the strains of biovar I as compared to the other biovars. However, the frequency of the penicillinase-producing strains within S. aureus and the biovars of S. epidermidis markedly varied.     

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).     

Immunological detection of penicillin-binding protein 2'' in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.     

武汉地区医院感染葡萄球菌的耐药性监测

目的了解武汉地区医院感染葡萄球菌的耐药现状。方法采用回顾性分析方法,对2003年1月到2007年12月我院分离的1373株金黄色葡萄球菌和259株表皮葡萄球菌的耐药性进行分析。药敏试验采用K—B纸片法,判断标准根据美国临床实验室标准化委员会（NCCLS）的标准。结果2003年1月到2007年12月我院分离到金黄色葡萄球菌1373株,其中耐甲氧西林的金黄色葡萄球菌（MRSA）有697株,对甲氧西林敏感株（MSSA）有587株,表皮葡萄球菌有259株,其中耐甲氧西林的表皮葡萄球菌（MRSE）有92株,对甲氧西林敏感株（MSSE）有142株。MRSA、MRSE对临床常用的抗生素几乎均耐药,只有对万古霉素和替考拉宁100％敏感;MSSA、MSSE对临床常用抗生素较敏感,但是对青霉素和红霉素耐药率均大于70％。结论武汉地区医院感染MRSA和MRSE对大部分临床常用抗生素均已高度耐药,对万古霉素和替考拉宁依然高度敏感。了解医院感染葡萄球菌的耐药状况,对临床合理选用抗生素十分重要。     

Comparative study of the binding of human blood plasma fibronectin with clinical strains of staphylococci

The interaction of 62 S. aureus clinical strains and, respectively, 20 and 17 isolated S. epidermidis and S. saprophyticus strains with human blood plasma fibronectin (FN) has been studied. The specific interaction of FN with bacteria has been evaluated simultaneously by the binding of 125I with FN (method 1), the FN-mediated agglutination of staphylococci (method 2) and the character of colonies formed in 0.15% agar medium containing FN (method 3). The data obtained in this investigation indicated that all S. aureus strains under study react with FN to a different extent. When evaluating the binding of FN with bacteria, the most pronounced correlation was observed between methods 1 and 3. None of the methods used in this investigation has revealed interaction between FN and S. epidermidis and S. saprophyticus strains under study. The authors suggest that a preliminary inference on the capacity of the isolated clinical strains of staphylococci for reaction with FN may be made from the character of colonies formed in 0.15% agar medium containing FN.     

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.     

Sensitivity to vancomycin and teicoplanin of coagulase-negative staphylococci isolated from clinical specimens

The frequency of resistance and elevated resistance to teicoplanin and vancomycin among 689 strains of coagulase-negative staphylococci isolated in one year from clinical specimens was determined. Using ATB.STAPH test, a resistance was shown mainly among strains of S. epidermidis and S. haemolyticus. The elevated resistance to teicoplanin was much more frequently observed than to vancomycin. About 27% of isolated strains of S. haemolyticus and 6.8% of S. epidermidis were classified as resistant. Among other species only single strains were recognised as resistant: one strain of S. xylosus, one of S. cohni and one of S. intermedius. 94.7% of S. epidermidis and 100% of S. haemolyticus strains classified as resistant to teicoplanin in ATB showed MIC values 14 mg/l. Moreover it was shown that 26.3% of these strains of S. epidermidis and 33.3% of S. haemolyticus had MBC of teicoplanin values equal to or higher than 32 mg/l.     

The combined effects of plasma and hydrogel coating on adhesion of Staphylococcus epidermidis and Staphylococcus aureus to polyurethane catheters

Abstract The adhesion of three Staphylococcus epidermidis and three S. aureus clinical isolates, to uncoated and hydrogel-coated polyurethane catheters was tested, following pretreatment of catheters with human plasma. Plasma significantly decreased the adhesion of S. epidermidis strains to uncoated polyurethane catheters, but had no significant effect on the adhesion to hydrogel-coated catheters. The influence of plasma on adhesion of S. aureus strains to catheters was strain dependent. Plasma significantly increased the adhesion of one strain (SA6) to uncoated catheters. For two other strains (SA3 and SA14) plasma produced no clear effect on their adhesion to uncoated catheters; adhesion values for each strain showed either a small but significant increase or a replicate-dependent increase or decrease. However, plasma significantly increased the adhesion of all S. aureus strains to hydrogel-coated polyurethane catheters. Overall, with the exception of one batch culture of S. epidermidis strain SE3 tested, attachment to plasma-treated hydrogel-coated catheters was statistically significantly lower, by up to 85%, than attachment to plasma-treated uncoated catheters for both S. epidermidis and S. aureus .     

The effect of staphylococci on the manifestation of opsonic cooperation in the complement system

The influence of the cultures of 14 Staphylococcus aureus and Staphylococcus epidermidis strains on the capacity of factor C3b of the complement for mediating the adhesive reaction of human neutrophils was studied. In experiments with 5 out of 11 S. aureus strains the essential weakening of reactions was registered, while one of the strains considerably enhanced reactions. Out of 3 S. epidermidis strains, the weakening of C3b-dependent reaction was noted in one case. The activity of the cultures did not correlate with the gelatinase properties of staphylococci and was absent in all gelatinase-positive Pseudomonas aeruginosa strains. The model worked out by the authors may be used for studying the influence of bacterial metabolites and biologically active substances on the effector properties of factor C3b of the complement.     

Cross-reactions between Staphylococcus epidermidis and 23 other bacterial species

By quantitative immunoelectrophoretic methods, 43 antigens were found in a mixture of sonicated preparations of four Staphylococcus epidermidis strains, using corresponding rabbit antiserum. Two of the antigens were identified as cell wall teichoic acid and a peptidoglycan antigen, respectively. Using this antigen/antibody reference system, cross-reactions between S. epidermidis antigens and antigens from other bacterial species were investigated. Fourteen of the S. epidermidis antigens cross-reacted with antigens from all S. aureus strains investigated. Only few cross-reactions were found between S. epidermidis and bacteria not belonging to the Micrococcaceae. The antigenic relatedness, expressed as a matching coefficient, seems promising for taxonomic work.     

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.