In vitro measurement of the adherence of Staphylococcus epidermidis to plastic by using cellular urease as a marker.

A rapid and sensitive in vitro assay was developed to quantitatively assess the adherence of Staphylococcus epidermidis to a hydrophobic plastic surface. The assay is based upon the detection of cell-associated urease activity as a marker of bacteria remaining adherent to the polystyrene microwells of flat-bottomed, 96-well tissue culture plates. Using ATCC 35984, a slime-producing strain of S. epidermidis, the assay could detect as few as 3 x 10(3) bacteria and was linear to 3.5 x 10(7) bacteria. The adherence of both slime-positive and slime-negative coagulase-negative staphylococci could be evaluated by using this method. This assay could be used to examine factors which influence the adherence of individual S. epidermidis strains to hydrophobic surfaces and to develop agents or coating materials which suppress the adherence of coagulase-negative staphylococci to biomedical implants.     

Identification of Staphylococcus epidermidis using a 16S rRNA-directed oligonucleotide probe

Staphylococcus epidermidis is the most medically significant of the coagulase-negative staphylococci. An oligonucleotide probe (pSe) for identification of S. epidermidis was defined by comparing the sequences of the 16S rRNA variable region V6 from numerous coagulase-negative staphylococci. In order to increase the sensitivity of the detection, polymerase chain reaction amplification of the variable region with primers based on the conserved flanking sequences was applied. The detection limit of the polymerase chain reaction assay combined with pSe probe was shown to be 1 fg which corresponds to about one single bacterium. Additionally, a sensitive, non-radioisotopic system with chemiluminescence detection was tested.     

PCR-based identification of Staphylococcus epidermidis targeting gseA encoding the glutamic-acid-specific protease

The frequency of the gseA gene encoding a glutamic acid-specific serine protease, GluSE, of Staphylococcus epidermidis was investigated. DNA hybridization analysis demonstrated that gseA existed exclusively in S. epidermidis but not in other bacteria examined. A single step PCR assay with a set of designed primers yielded amplification of gseA from all 69 clinical isolates of S. epidermidis taken from patients and healthy adults, whereas production of GluSE was observed in 74% (51/69) of the isolates. Furthermore, none of the 46 clinical isolates of other species of coagulase-negative staphylococci and 45 clinical isolates of Staphylococcus aureus showed amplification, except a Staphylococcus capitis strain. However, this strain was positive for a S. epidermidis-specific DNA region and the DNA sequence of the 16S rRNA gene showed 99% identity with that of S. epidermidis. Therefore, these results indicated that the present PCR assay for gseA was ubiquitous and highly specific for detection of S. epidermidis.     

Physico-chemical properties of staphylococci of different species in resistance to human thrombodefensins

Testing 54 strains of staphylococci (Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. capitis) revealed that S. aureus in contrast to coagulase-negative staphylococci (CNS) is more resistant to bactoriocidal action of human thrombodefensins (resistance index: 60.3 vs 25.6%), less hydrophilicolipophilic balance-HLB: -0.42 vs -0.64) and less charged (x-potential: -32.4 vs -35.6 mV). In groups of staphylococci (S. aureus and CNS) correlation links of bacterial resistance to human thrombodefensins with their HLB and x-potential (r=-0.32...-0.36). By In vitro experiments, it was shown that 5 passages of staphylococci in meat-peptonic broth with human thrombodefensins (50 mkg protein/ml) lead to adaptation of bacteria followed by the formation of resistance to cationic peptides from thrombocytes, a decrease of hydrophobicity and x-potencial. The role of physico-chemical properties in providing thrombodefensin-resistance of staphylococci as a developmental factor of infectious-and-inflammatory process and persistence of bacteria was confirmed with Salmonella infection.     

Antimicrobial sensivity and ability to slime production of koagulaze-negative staphylococci

The aim of this study was to assess the ability of slime production ofcoagulase-negative staphylococci (CONS) and evaluate the susceptibility of bacteria to antibiotics. Strains were isolated from clinical specimens obtained from hospitalized patients. The most frequently isolated species were S. epidermidis (51%), S. hominis (18%), S. haemolyticus (13%). The result of this study shows that 61% of S.epidermidis produce slime on CRA (Congo red agar), whereas none of the tested S. haemolyticus strains has this ability. All examined strains were susceptible to vancomycin, linezolid and quinupristin/ dalfopristin. The majority of strains were susceptible to minocycline, fusid acid, nitrofurantoin and rifampicin. Sixty six percent of isolates were determined as methicillin-resistant coagulase-negative staphylococci.     

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

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

Adhesion of Staphylococcus epidermidis and Staphylococcus saprophyticus to a hydrophobic biomaterial

The relative surface charge and hydrophobicity of 16 strains of Staphylococcus epidermidis showed large variations. For this species no relationship between the two surface parameters was found. A highly negative surface charge was observed in all seven encapsulated strains (one S. epidermidis and six Staphylococcus saprophyticus strains). The adhesion of the staphylococci to fluorinated polyethylene-propylene films was not related to the relative surface charge and the hydrophobicity of the bacteria. On films pre-exposed to human plasma, the bacterial adhesion was substantially reduced. Mechanisms involved in the adhesion of coagulase-negative staphylococci to this biomaterial are discussed.     

Lysozyme Production as an Aid for Identification of Potentially Pathogenic Strains of Staphylococci

Lysozyme production is a frequent property of potentially pathogenic staphylococci. In the present study, 1,186 strains of human origin, 85 strains of animal origin, and 156 strains of Staphylococcus albus (epidermidis) were tested. Of 1,114 coagulase-positive strains of human and animal origin, 1,098 were lysozyme-positive (98.5%). On the other hand, of 157 coagulase-negative strains which, based on further investigations, belong to the potentially pathogenic staphylococci, all were lysozyme-positive. All of the 156 strains (100%) belonging to the species S. albus (epidermidis) were lysozyme-negative. We conclude that lysozyme production is a better index of potentially pathogenic staphylococci than the measurement of free coagulase, especially in cases of strains of animal origin. It is possible that lysozyme production allows a differentiation between pathogenic and nonpathogenic coagulase-negative staphylococci.     

Composition of AME encoding gene in Gram positive Cocci--study on spread of aminoglycoside resistance

Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Composition of six genes encoding AMEs (including lately described aph 2"-genes) was investigated by PCR for 16 clinical isolates of Enterococcus faecalis, 16 clinical isolates of coagulase-positive (S. aureus) and 13 clinical isolates of coagulase negative staphylococci (S. haemolyticus, S. epidermidis) collected in Gdańsk region (Northern Poland) in the years 1998-2001. Diversity of AME encoding gene profiles (composition) was used to analyze spread of AME encoding gene among and within studied group of cocci. According to presence of particular genes we distinguish eleven different AME encoding gene profiles: seven profiles were unique for particular species while the most common was shared among S. aureus, coagulase negative staphylococci and enterococci. Regarding profile frequency statistical analysis (Fstat, AMOVA, cluster analysis UPGMA) shows: the difference between S. aureus and enterococci and coagulase-negative staphylococci, lack of difference between enterococci and coagulase-negative staphylococci, higher variability within than between studied species and presence of multispecies cluster. On the basis of the reports about ability of staphylococci to synthesis enterococcal pheromones, this finding lets assume that spread of aminoglycoside resistance gene among gram (+) cocci is limited only by the ability of stains to synthesis or induction of synthesis conjugation protein.     

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.     

Identification of Micrococcaceae in Clinical Bacteriology

The cellular morphology, identifying physiological characteristics, and a key to the human genera of Micrococcaceae are presented with flow charts for identification of aerobic and anaerobic isolates. These flow charts can be amended as desired, depending upon the degree of accuracy desired. Micrococcaceae isolates in a 350-bed private general hospital during a 15-week period are tabulated to show relative numbers of the different genera and species, with their probable relationship to infection or contamination. Only 11 of the 220 Micrococcaceae isolates were not Staphylococcus; no Sarcina or Peptococcus were isolated. Of the Staphylococcus isolates, 61% were S. epidermidis. Almost 18% of the S. aureus isolates were coagulase-negative. Of the S. aureus isolates, 80% of the coagulase-positive isolates were infecting agents, as were 67% of the coagulase-negative S. aureus isolates, compared to only 48% of S. epidermidis isolates. Two of four Gaffkya isolates but only one of seven Micrococcus isolates were infecting agents. If coagulase production is used as the sole criterion for speciation of staphylococci, and Micrococcus is not differentiated from Staphylococcus, the term "coagulase-negative staphylococci" does not differentiate three distinct levels of pathogenicity. Coagulase-negative S. aureus is more virulent than S. epidermidis or Gaffkya, which are more virulent than Micrococcus or Sarcina.     

Coagulase-negative Staphylococci isolated from patients. III. Phage typing

Coagulase-negative staphylococci of clinical origin were subjected to phage typing by means of phages from the experimental Dutch (Verhoef) and American (Paris) sets. These sets of phages were used to study 153 and 378 strains, respectively. The Dutch phages could lyse 30.1%, and the American ones 19.6% of the cultures. The strains belonging to the species S. epidermidis were lysed in 34.3% and 32.4% of cases, respectively, which is indicative of the fact that the American phages possess a more pronounced specificity in respect to S. epidermidis. The unsufficient effectiveness of typing phages does not yet allow one to evaluate the outlook for the method of phage typing in the study of coagulase-negative staphylococci.     

Expression of glycocalyx by coagulase-negative Staphylococcus species isolated from bovine milk

Two hundred and six strains of coagulase-negative Staphylococcus species were assessed for expression of glycocalyx on serum soft agar, india ink and adherence techniques. The organisms were maintained on trypticase soy agar plates at 4 degrees C for 30 d (120 strains) or stored at -80 degrees C in skim milk for 90 d (60 strains). Additionally, 26 milk samples from cows known to have excreted coagulase-negative staphylococci were used to inoculate serum soft agar directly. Nine of 26 direct culture samples and 43 of 180 strains maintained for an extended period had diffuse-type growth on serum soft agar. The proportion that exhibited an unstained halo by india ink was similar regardless of storage time. Slime production determined by in vitro adherence revealed a higher proportion of positive strains than had been predicted by serum soft agar or india ink techniques. More strains of Staphylococcus chromogenes, Staph. epidermidis, Staph. hominis, Staph. simulans and Staph. warneri expressed glycocalyx than other coagulase-negative Staphylococcus species. These results suggest that most coagulase-negative staphylococci produce slime rather than a capsule. However, evidence for classical encapsulation was demonstrated in several strains by india ink. The finding that Staphylococcus species other than Staph. aureus isolated from bovine milk are capable of glycocalyx production may be of importance in investigations on the relationship between staphylococci and host defence mechanisms.     

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.     

Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture

The notification of "Gram-positive cocci, possibly staphylococcus" in a blood culture drawn from a seriously ill patient is responsible for a large amount of vancomycin prescribing in institutions where methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of bacteraemia. A duplex real-time TaqMan polymerase chain reaction targeting the species-specific nuc gene, and the mecA gene encoding methicillin-resistance, was developed as a tool for rapid identification and detection of S. aureus and methicillin-resistance, and optimised for immediate as-needs testing. Three different DNA extraction methods achieved varying DNA quality, with PCR inhibition the main problem. Serial blood cultures (n=120) identified as possible staphylococci on Gram stain from our clinical laboratory were examined. There was one false negative result for a methicillin-resistant Staphylococcus epidermidis, which was positive on repeat testing, and one false negative result due to DNA extraction failure for MRSA from peritoneal dialysate inoculated into blood culture medium. Sensitivity and specificity of 97% and 100%, respectively, were obtained for mecA; and sensitivity and specificity of 98% and 100%, respectively, for nuc. Detection of slow-growing coagulase-negative staphylococci as co-infecting strains may be reduced. The assay quickly and reliably identified S. aureus in mixed infection, and identified methicillin resistance in both S. epidermidis and S. aureus strains.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 x 10(9) cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A x 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20 degrees C reduced cell-surface hydrophobicity of all species, while storage at 4 degrees C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4 degrees C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

Effects of a topical essential oil-containing formulation on biofilm-forming coagulase-negative staphylococci

AIMS: To evaluate the antimicrobial effects of Polytoxinol (PT), a topical essential oil-based formulation, against biofilm positive strains of coagulase-negative staphylococci. METHODS AND RESULTS: Using a microtitre plate assay we measured inhibitory effects for PT against a selection of biofilm-forming clinical isolates of coagulase-negative staphylococci. Susceptibility varied considerably (MIC = 0.6-20 000 ppm). For the most tolerant clinical isolate (Staphylococcus warneri) biofilm growth was inhibited by a 32-fold lower PT concentration than planktonic growth. This inhibition of biofilm development, which was not observed with the other test isolates, was related to an inhibition of the initial phase of S. warneri cell adherence to the polystyrene surface. CONCLUSION: The antimicrobial efficacy of PT was verified against clinical isolates of coagulase-negative staphylococci in vitro. PT was able to inhibit biofilm formation in the most tolerant isolate at sub-inhibitory concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: These observations indicate that an ability to prevent biofilm formation, independently of effects on cell viability may contribute to the in vivo topical efficacy of essential oils.     

Coagulase-Negative,Novobiocin-Resistant Staphylococci on the Skin of Animals and Man,on Meat and in Milk

The occurrence of coagulase-negative, novobiocin-resistant staphylococci, i.e. Staphylococcus cohnii, Staphylococcus saprophyticus, Staphylococcus sciuri and Staphylococcus xylosus, on the skin of animals and man has been studied. On cultures from cats, cows, dogs, guinea pigs, mice, rabbits and sheep studied, such organisms were predominant among the coagulase-negative staphylococci. From the skin of the hands of 21 of 38 persons whose professions brought them into contact with animals, e.g. inséminât ors, slaughterhouse workers and veterinarians, coagulase-negative, novobiocin-resistant staphylococci were isolated. This finding contrasted with that regarding 50 persons lacking such contacts, of whom only 1 harboured such bacteria. S. saprophyticus was isolated only from those slaughterers presenting with wounds on their hands. Coagulase-negative, novobiocin-resistant staphylococci were also isolated from every second specimen collected from the surface of meat at a slaughterhouse. No difference in the culture results could be demonstrated from specimens collected before and after cutting-up of the carcasses. Of 26 strains of coagulase-negative, DNase-negative staphylococci isolated from milk with pathological CMT, all but 5 were novobiocin-resistant. Fifteen were classified as S. xylosus, 4 as S. sciuri and 1 as S. cohnii. Of another 15 DNase-positive strains, 3 were resistant to novobiocin. Finally, clinical infections with coagulase-negative, novobiocin-resistant staphylococci in man, e.g. urinary tract infections caused by S. saprophyticus, are considered in relation to possible contagious reservoirs and modes of spread.     

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.     

Expression of glycocalyx by coagulase-negative Staphylococcus species isolated from bovine milk

Two hundred and six strains of coagulase-negative Staphylococcus species were assessed for expression of glycocalyx on serum soft agar, india ink and adherence techniques. The organisms were maintained on trypticase soy agar plates at 4°C for 30 d (120 strains) or stored at -80°C in skim milk for 90 d (60 strains). Additionally, 26 milk samples from cows known to have excreted coagulase-negative staphylococci were used to inoculate serum soft agar directly. Nine of 26 direct culture samples and 43 of 180 strains maintained for an extended period had diffuse-type growth on serum soft agar. The proportion that exhibited an unstained halo by india ink was similar regardless of storage time. Slime production determined by in vitro adherence revealed a higher proportion of positive strains than had been predicted by serum soft agar or india ink techniques. More strains of Staphylococcus chromogenes, Staph. epidermidis, Staph. hominis, Staph. simulans and Staph. warneri expressed glycocalyx than other coagulase-negative Staphylococcus species. These results suggest that most coagulase-negative staphylococci produce slime rather than a capsule. However, evidence for classical encapsulation was demonstrated in several strains by india ink. The finding that Staphylococcus species other than Staph. aureus isolated from bovine milk are capable of glycocalyx production may be of importance in investigations on the relationship between staphylococci and host defence mechanisms.