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Differential diagnosis of Staphylococcus aureus from Staphylococcus epidermidis and Staphylococcus saprophyticus by alphazurine A dye.

Staphylococcal bacterial suspensions were streaked on Trypticase soy agar with 5% sheep blood culture plates. Paper discs containing alphazurine A, a triphenylmethane dye, were placed on the inoculated plates which were incubated at 37 degrees C for 24 hours. A wide zone of inhibition of growth of Staphylococcus aureus was present around the paper discs. Growth of Staphylococcus epidermidis and Staphylococcus saprophyticus was not inhibited.

Diagnosis, Differential↗

Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses.

Biomaterial-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of biomaterial-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. In this review, recent insights regarding factors functional in biofilm formation of S. epidermidis, their role in pathogenesis, and regulation of their expression are presented. Similarly, in S. aureus the biofilm mode of growth affects gene expression and the overall metabolic status. Experimental approaches for analysis of differential expression of genes involved in these adaptive responses and evolving patterns of gene expression are discussed.

Adaptation, Physiological↗

Staphylococcus epidermidis endocarditis and Staphylococcus epidermidis infection in an intensive care unit.

Staphylococcus epidermidis are the most common agents of prosthetic valve endocarditis (PVE). S. epidermidis isolated from the blood stream of patients with PVE are almost invariably multiple resistant to antibiotics. Antibiotic treatment alone gives unsatisfactory results and carries a mortality rate of 70-80%. That is why early surgical treatment is recommended. S. epidermidis is a less common cause of endocarditis in non-surgical patients, accounting for approximately 5% of the cases, which are mostly patients with pre-existent valvular heart disease. Generally, isolates from the latter patients are sensitive to most antibiotics, and the mortality rate is considerably lower. Recently coagulase-negative staphylococci have emerged as causative agents of septicaemia in patients hospitalized in intensive care units. Especially premature infants of very low-birth weight (less than 1500 g) receiving parenteral nutrition appear to carry a high risk of acquiring this kind of septicaemia. Although the staphylococci isolated from the blood of these patients are the same as in patients with PVE, generally multiple resistant to antibiotics, prognosis is far better than in cases of PVE. In our study carried out in a neonatal intensive care unit, two risk factors for coagulase-negative staphylococcal septicaemia were identified. First, nearly 20% of parenteral nutrition fluids used in the unit were found to be contaminated with coagulase-negative staphylococci, and a significant association was established between septicaemia and the infusion of contaminated fluids. Moreover, opsonization of staphylococci in infant serum proved to be severely deficient. Since host defence to staphylococci is dependent on optimal opsonization of these microorganisms, this defence may be severely compromised in the premature neonate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

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.

Adhesiveness↗

Effects of growth in the presence of subinhibitory concentrations of dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus biofilms.

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions of biofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.

Biofilms↗

Staphylococcus epidermidis and Staphylococcus xylosus in a secondary root canal infection with persistent symptoms: a case report.

The aim of this case report is to highlight the importance of maintaining the aseptic chain during endodontic treatment. This paper describes a clinical situation in which persistent symptoms had developed after the first appointment to treat a vital pulp case. Microbiological analysis of the case indicated that symptoms were probably due to a secondary root canal infection by two species of coagulase-negative staphylococci--Staphylococcus epidermidis and Staphylococcus xylosus. Symptoms had persisted in spite of using various intracanal antimicrobial agents and systemic antibiotic therapy.

Adult↗

Selective inhibition of hemolysin production by Staphylococcus epidermidis and Staphylococcus haemolyticus.

The effect of lincomycin on growth and hemolysin production by Staphylococcus epidermidis and Staphylococcus haemolyticus is presented. Lincomycin at subinhibitory concentration, clearly inhibited hemolysin yield without appreciably altering final growth density. The cells of these two microbes previously grown in the presence of lincomycin showed very low hemolytic activities upon disruption. The mode of lincomycin action on hemolysin production is discussed.

Hemolysin Proteins↗

Plasmids encoding for erythromycin ribosomal methylase of Staphylococcus epidermidis and Staphylococcus simulans.

Two 1.7 Md plasmids of Staphylococcus epidermidis and three ones of Staphylococcus simulans determining inducible macrolide-lincosamide resistance are identical as judged by restriction endonuclease fingerprinting. These plasmids designated pEI2101, pEI9105, pE1107, pEI1108 and pEI6104, respectively, belong to the incompatibility group 12. Dot-blot hybridization by photobiotin-labelled gene probe developed from S. aureus erythromycin ribosomal methylase gene showed cross hybridization between methylase-coding reference plasmids and the tested ones. The examined plasmids proved to be no transmissible in mating experiments into S. aureus recipients.

Drug Resistance, Microbial↗

Cloning and expression of methicillin resistance from Staphylococcus epidermidis in Staphylococcus carnosus.

A 6.2-kilobase chromosomal DNA fragment from a methicillin-resistant Staphylococcus epidermidis strain was cloned into Staphylococcus carnosus by using staphylococcal plasmid pCA44 as the vector. The recombinant plasmid obtained, pBBB21, conferred methicillin resistance on its host and was responsible for the synthesis of a low-affinity penicillin-binding protein (PBP), PBP 2'. PBP 2' determined by the S. epidermidis DNA and expressed as a membrane-bound PBP in S. carnosus reacted with monoclonal antibodies directed against PBP 2' of Staphylococcus aureus origin, and the cloned S. epidermidis DNA hybridized to the methicillin (mec)-specific DNA from S. aureus. These findings point to a common origin of the methicillin resistance determinant in staphylococci.

Bacterial Proteins↗

Transposase-dependent formation of circular IS256 derivatives in Staphylococcus epidermidis and Staphylococcus aureus.

IS256 is a highly active insertion sequence (IS) element of multiresistant staphylococci and enterococci. Here we show that, in a Staphylococcus epidermidis clinical isolate, as well as in recombinant Staphylococcus aureus and Escherichia coli carrying a single IS256 insertion on a plasmid, IS256 excises as an extrachromosomal circular DNA molecule. First, circles were identified that contained a complete copy of IS256. In this case, the sequence connecting the left and right ends of IS256 was derived from flanking DNA sequences of the parental genetic locus. Second, circle junctions were detected in which one end of IS256 was truncated. Nucleotide sequencing of circle junctions revealed that (i) either end of IS256 can attack the opposite terminus and (ii) the circle junctions vary significantly in size. Upon deletion of the IS256 open reading frame at the 3' end and site-directed mutageneses of the putative DDE motif, circular IS256 molecules were no longer detectable, which implicates the IS256-encoded transposase protein with the circularization of the element.

DNA Transposable Elements↗

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

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.

Adsorption↗

Use of trehalose-mannitol-phosphatase agar to differentiate Staphylococcus epidermidis and Staphylococcus saprophyticus from other coagulase-negative staphylococci.

Using a plate medium containing trehalose, mannitol, and phenolphthalein diphosphate (TMPA), we differentiated significant clinical isolates of Staphylococcus epidermidis by their lack of acid production in 18 h from other coagulase-negative staphylococci, with our results having a sensitivity (R. S. Galen and S. R. Gambino, Beyond Normality: The Predictive Value and Efficiency of Medical Diagnoses) of 100%, a specificity of 89.9%, and a positive predictive value of 94.8%. With a Taxo A bacitracin disk, which differentiates Staphylococcus species from Micrococcus species, no zone of inhibition was seen for 96% of all staphylococcal strains, with 5 of 26 strains of Staphylococcus saprophyticus exhibiting zone diameters up to 10 mm. By using resistance to a 5-microgram novobiocin disk, we differentiated S. saprophyticus, with our results having a sensitivity of 100%, a specificity of 97.1%, and a positive predictive value of 83.9% on TMPA. These two species represented 77.8% of coagulase-negative staphylococci isolated. Reference strains fo Staphylococcus and Micrococcus species were differentiated by TMPA. The cost of TMPA was compared with that of another method. TMPA was found to offer an inexpensive, sensitive method for rapidly differentiating coagulase-negative Staphylococcus isolates.

Agar↗

Induction of oxidative burst response in human neutrophils by adherent staphylococci. Comparison between Staphylococcus epidermidis and Staphylococcus aureus.

The ability of staphylococci adherent to silicone surfaces to induce superoxide anion (O2-) production by polymorphonuclear leukocytes (PMNs) was investigated and compared with the same activity induced by planktonic bacteria. The responses to Staphylococcus aureus strain E 2371 and Staphylococcus epidermidis strain ATCC 14990 were compared. The staphylococci were allowed to adhere to silicone catheters for 2 h at 37 degrees C. After opsonization of adherent bacteria in 30% human AB-positive serum, the induction of superoxide anion production by PMNs was measured in a cytochrome C reduction assay. Both bacterial strains, when adhered to the surfaces, were able to induce superoxide anion production by PMNs to about the same extent. Comparing adherent and planktonic bacteria with these two bacterial strains, it was found that planktonic S. epidermidis induced one to three times higher superoxide anion production than the adherent bacteria, whereas planktonic S. aureus induced four to seven times higher superoxide anion production than the adherent bacteria. Interstrain variation between the response to adherent and planktonic staphylococci was found. The lower phagocytic response to adherent staphylococci as compared to the response to planktonic organisms may interfere with the killing process and thereby contribute to poor clearance of these bacteria when adherent to foreign bodies such as catheters.

Humans↗

[Adhesion capacity and surface properties of Staphylococcus epidermidis and Staphylococcus saprophyticus].

We have comparatively studied the adherence of Staphylococcus epidermidis and S. saprophyticus to epithelial cells of a continuous cellular line of urinary origin (MDCK). The adherence of S. saprophyticus was significantly higher than that of S. epidermidis and this phenomenon was not related to the bacterial surface hydrophobicity or the ability of producing slime in vitro. The pre-incubation of the bacteria with sub-inhibitory concentrations of vancomycin, teicoplanin, amikacin, clindamycin, and roxitromycin did not modify the adherence ability of the microorganisms. Of the antimicrobial agents evaluated, only clindamycin significantly agents evaluated, only clindamycin, significantly inhibited the ability of producing slime of S. saprophyticus strains.

Animals↗

Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections.

Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules.

Adhesins, Bacterial↗

Staphylococcus epidermidis and Staphylococcus aureus in otitis media with effusion.

Bacteriologic investigation of middle ear effusion (MEE), external ear canal, and the nasopharynx was carried out on 458 patients with otitis media with effusion. Staphylococcus epidermidis was the most common bacteria in MEE, even after excluding the contaminants from the external ear canal, which had the same value of minimal inhibitory concentration as the paired MEE. The bacterial agreement of S epidermidis between MEE and the nasopharynx was extremely rare in contrast with Haemophilus influenzae, Streptococcus pneumoniae, and Branhamella catarrhalis, although the organism was also frequently isolated from the nasopharynx. Staphylococcus aureus, having the same minimal inhibitory concentration as that in the nasopharynx, was more frequently found in MEE than S epidermidis. The results suggest that S epidermidis found in MEE is not a pathogen, but rather a contaminant in many instances. Staphylococcus aureus seems to be a causative agent in otitis media with effusion.

Adolescent↗

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.

Animals↗

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.

Conjugation, Genetic↗