In vitro study of bacterial growth in continuous ambulatory peritoneal dialysis fluids.

We examined the in vitro survival of bacteria in continuous ambulatory peritoneal dialysis effluents of patients with clinical peritonitis and those without peritonitis. Standard strains of coagulase-negative staphylococci (CNS), Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were inoculated into the fluids, and portions were plated for bacterial counts at 0.5, 4, 24, 48, 72, and 96 h. Unused dialysate fluid was also inoculated simultaneously. Our results show that CNS increased minimally up to 48 h in the noninfected continuous ambulatory peritoneal dialysis effluents and decreased by 96 h, whereas survival was only minimal in the infected effluent. S. aureus showed trends similar to those of CNS, but differences in survival in infected and noninfected effluents were less marked. By contrast, E. coli and P. aeruginosa increased by greater than 1,000-fold in all solutions tested. Based on the above findings, it is likely that a proportionate number of culture-negative cases of peritonitis are due to gram-positive cocci, especially CNS, which are not retrievable by standard culture techniques because of poor survival rate.     

Rapid identification of fibronectin, vitronectin, laminin, and collagen cell surface binding proteins on coagulase-negative staphylococci by particle agglutination assays.

Seventeen strains of ten different species of coagulase-negative staphylococci were shown to interact with collagen, laminin, fibronectin, and vitronectin immobilized on latex beads. Different species of coagulase-negative staphylococci have different capacities to agglutinate proteins. Cells of 18 strains of Staphylococcus haemolyticus reacted more strongly than did cells of 18 Staphylococcus epidermidis strains with proteincoated latex beads, although no significant difference in cell surface hydrophobicity or charge could be shown. The cell surface receptors of S. haemolyticus were more heat and protease resistant than were Staphylococcus aureus receptors. Strains of Staphylococcus saprophyticus isolated from urinary tract infections showed a high capacity to adhere to laminin. The ability to agglutinate fibronectin and collagen was common among coagulase-negative staphylococci isolated from other infections; 55% (31 of 56) and 63% (35 of 56) agglutinated fibronectin and/or collagen. S. haemolyticus and S. epidermidis bound to both N-terminal (29-kDa) and C-terminal (120-kDa) fragments of fibronectin.     

Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein.

Staphylococcus aureus and the coagulase-negative staphylococci are commonly responsible for peritonitis in renal patients undergoing continuous ambulatory peritoneal dialysis. To simulate growth conditions in vivo, staphylococci isolated from peritoneal infections were cultured in used human peritoneal dialysate (HPD). Immunoblotting experiments using cell wall preparations from these staphylococci revealed the presence of the host iron-binding glycoprotein transferrin bound to S. aureus, S. epidermidis, S. capitis, S. haemolyticus, and S. hominis but not to S. warneri or S. saprophyticus. Similar results were obtained by incubating broth-grown staphylococci with human transferrin, although, in contrast to S. aureus, the coagulase-negative staphylococci bound more transferrin after growth in iron-restricted broth. To determine whether the staphylococci express a saturable specific receptor for human transferrin, the interaction of human 125I-transferrin with the staphylococci was examined. Both S. aureus and S. epidermidis bound the radiolabelled iron-saturated ligand in a time- and concentration-dependent manner. From competition binding assays, the affinity (Kd) and number of receptors were estimated for S. epidermidis (Kd, 0.27 microM; 4,200 receptors per cell) and S. aureus (Kd, 0.28 microM; 4,200 receptors per cell). S. epidermidis but not S. aureus receptor activity was partially iron regulated. Human apotransferrin and iron-saturated transferrin and rabbit and rat transferrins competed equally well for the staphylococcal receptor. Bovine and porcine transferrins and ovotransferrin as well as human and bovine lactoferrins were much less effective at competing with human transferrin. Treatment of whole staphylococci with protease abolished transferrin binding, indicating the involvement of cell surface protein. Western blots (immunoblots) of cell wall preparations probed with human transferrin revealed the presence of a 42-kDa transferrin-binding protein common to both S. aureus and S. epidermidis. On Western strip blots, the binding of human transferrin to this protein was blocked by labelled human transferrin but not by albumin, immunoglobulin G, or bovine transferrin or ovotransferrin. To assess the conservation of the 42-kDa transferrin-binding protein, cell wall proteins of S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S. warneri, and S. saprophyticus were Western blotted and probed with human transferrin. Only S. warneri and S. saprophyticus lacked the 42-kDa wall protein, consistent with their inability to bind transferrin. These data show that the staphylococci express a specific receptor for human transferrin based at least in part on a common 42-kDa cell wall protein.     

Phenotypic and genotypic mupirocin resistance among Staphylococci causing prosthetic joint infection

Mupirocin MICs and mupA presence were determined in 108 staphylococci causing prosthetic joint infection. Zero of 35 isolates (0%) of methicillin-susceptible Staphylococcus aureus, 4/15 (27%) methicillin-resistant S. aureus isolates, 3/16 (19%) methicillin-susceptible coagulase-negative staphylococci, and 11/42 (26%) methicillin-resistant coagulase-negative staphylococci were mupirocin resistant. mupA was detected in all five high-level mupirocin-resistant staphylococci and one mupirocin-susceptible staphylococcus.     

Association of slime with pathogenicity of coagulase-negative staphylococci causing nosocomial septicemia.

To assess the role of slime in the pathogenesis of nosocomial bloodstream infections caused by coagulase-negative staphylococci, we compared the characteristics of 27 nosocomial bloodstream isolates with those of 27 skin isolates from non-hospital personnel. Of 27 bloodstream isolates, 14 were judged to be significant by a clinical index, and 13 were contaminants. Slime production was observed in 13 of 14 significant isolates but in only 3 of 13 contaminants (P = 0.0003) and 4 of 27 skin isolates (P = 0.0001). The 14 pathogens were identified as Staphylococcus epidermidis. Only 7 of 13 contaminants and 9 of 27 skin isolates belonged to the same species (P less than 0.006). Slime-producing strains of S. epidermidis represented 13 of 14 pathogens but only 2 of 13 contaminants (P less than 0.0003). Neither adherence to Teflon catheters nor phagocytosis and killing of coagulase-negative staphylococci by polymorphonuclear leukocytes was significantly influenced by slime production. Nevertheless, the identity of the organism and the slime production test predicted the clinical significance of blood isolates of coagulase-negative staphylococci with an overall accuracy of 89%.     

Treatment of peritonitis in continuous ambulatory peritoneal dialysis with intraperitoneal ceftriaxone

In 16 patients under CAPD, 29 cases of bacterial peritonitis were observed, with clinical manifestations in 23. The mean cell count in peritoneal dialysis fluid was 5608/mm3 with 4991/mm3 polymorphonuclear, Leukocytes Causative pathogens were Staphylococcus in 14 cases, Streptococcus in 6, Bacillus in one, Enterobacteria in 5, Pseudomonas aeruginosa in 1 and Moraxella in 1. Three cultures were negative. First choice treatment was a daily intraperitoneal injection of 1 g of ceftriaxone. 79.3% of patients recovered within 5 days. Failure were due to a Methicillin-resistant Staphylococcus epidermidis in one case, a Streptococcus faecalis in two cases, and a Staphylococcus aureus in three observations, which two were responsible of abscess round catheter peritoneal. Mean ceftriaxone concentrations 24 hours after the intraperitoneal injection were 50.6 mg/l (range: 3.3-141 mg/l) in serum and 58.1 mg/l (range: 4.3-180 mg/l) in dialysate. These concentrations are greater than most of ceftriaxone's MICs for susceptible bacteria, a finding that confirm the value of treatment with a single daily intraperitoneal injection of ceftriaxone.     

Incidence of constitutive and inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci in a community and a tertiary care hospital

The incidences of inducible clindamycin resistance at two hospitals (an inner-city hospital and a suburban community hospital) were 7 and 12% for methicillin-resistant Staphylococcus aureus, 20 and 19% for methicillin-susceptible S. aureus, and 14 and 35% for coagulase-negative staphylococci, respectively. Given the variability of inducible resistance to clindamycin found in our two hospitals, we conclude that susceptibility testing of staphylococci should include the disk diffusion induction test (D-test).     

Infection peritonitis in patients undergoing continuous ambulatory peritoneal dialysis: microbiological review during an four-year period

The purpose of this study was to analyse the microbiological characteristics of infectious peritonitis in patients undergoing continuous ambulatory peritoneal dialysis. This study was conducted at the CHU Nancy from 1999 to 2002. The diagnosis of peritonitis was based on cloudy peritoneal effluent (>100 cells per mm(3)) with an elevated leukocyte count (>50%), on isolation of bacteria or fungi and on symptoms such as abdominal discomfort or pain. The majority of infections associated with continuous ambulatory peritoneal dialysis were caused by Gram-positive bacteria (68%), Gram-negative bacteria (31%), and Candida (1%). The coagulase-negative staphylococci were the most common cause of peritonitis. The antibiotic sensitivity of species corresponded to community-acquired isolation.     

Prevalence of inducible clindamycin resistance in macrolide-resistant Staphylococcus spp.

Between January 2002 and December 2003, macrolide-resistant isolates of Staphylococcus aureus (n = 45) and coagulase-negative staphylococci (CoNS; n = 75) from a Greek hospital were examined phenotypically for inducible clindamycin resistance. The constitutive macrolide resistance phenotype predominated (60%) in S. aureus, followed by the inducible (35%) and the clindamycin-susceptible (5%) phenotypes. In CoNS, the inducible phenotype was more common than the constitutive phenotype (50% vs. 41%). There was a significant incidence of inducible clindamycin resistance, and screening of all staphylococci is necessary in order to differentiate inducibly resistant isolates from those that are truly sensitive.     

Influence of carbon dioxide on growth and antibiotic susceptibility of coagulase-negative staphylococci cultured in human peritoneal dialysate.

Used peritoneal dialysis fluid was collected from patients undergoing continuous ambulatory peritoneal dialysis, and its pH and composition were assessed after incubation in either air or air with 5% CO2. Precipitation of calcium, magnesium, phosphate, and proteins occurred in the dialysis fluid incubated in air at 37 degrees C and was associated with a mean pH increase of 1.23 U. Incubation of dialysis fluid in air with 5% CO2 prevented precipitation and maintained pCO2 and pH levels at those found physiologically. Coagulase-negative staphylococcal strains isolated from patients with peritonitis tended to grow less well in dialysis fluid incubated in air than in dialysis fluid incubated in the carbon dioxide-enriched atmosphere. MICs of cefuroxime, ciprofloxacin, and vancomycin for seven strains of coagulase-negative staphylococci in dialysis fluid were markedly affected by atmosphere type (16 of 21 MICs). Of these 16 atmosphere-dependent MICs, 14 were at least fourfold higher in air than in air with 5% CO2.     

Staphylococcus aureus colonization and infection in patients on continuous ambulatory peritoneal dialysis.

Staphylococcus aureus is the most common cause of peritonitis in patients undergoing peritoneal dialysis in Brazil. Using restriction endonuclease analysis of plasmid DNA, we investigated the importance of chronic carriage of S. aureus in the development of peritonitis in patients on continuous ambulatory peritoneal dialysis at the Division of Nephrology, Escola Paulista de Medicina, Sao Paulo, Brazil. A total of 117 isolates (30 patients) of S. aureus were available for typing, including 51 isolates (22 patients) from the nares, 58 isolates (27 patients) from pericatheter skin, and 8 isolates (6 patients) from peritoneal fluid, from patients with peritonitis. Restriction endonuclease subtyping showed that although most patients harbored more than one subtype of S. aureus, in the majority of patients nasal and/or pericatheter skin isolates with identical restriction endonuclease digest patterns were recovered on more than one occasion. Furthermore, 95% of patients with both nasal and pericatheter colonization were colonized with the same subtypes at both sites. All of the patients with peritonitis were infected with a subtype which colonized the nares, pericatheter skin, or both. These results demonstrate the importance of an endogenous source of S. aureus in the development of continuous ambulatory peritoneal dialysis-associated peritonitis.     

Change in bacterial aetiology of peritoneal dialysis-related peritonitis over 10 years: experience from a centre in south-east Asia

This study reviewed 1787 episodes of peritoneal dialysis (PD)-related peritonitis in 544 patients between 1994 and 2003. The overall rate of peritonitis was 0.68 episodes/year of PD, but decreased from 1.10 to 0.46 episodes/year between 1994 and 2003. The incidence of peritonitis caused by coagulase-negative staphylococci declined between 1994 and 1998 from 0.21 to 0.06 episodes/year of PD, coinciding with a reduction in the use of spike PD sets. There was a 60.1% response rate to antibiotics throughout the period, but the percentage of cases that required modification of the initial empirical antibiotic regimen rose from 13.6% to 58.7%, indicating that treatment should be individualised.     

Effect of milk on fibronectin and collagen type I binding to Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis.

Tryptic soy broth (TSB)-grown cells of Staphylococcus aureus isolated from acute and chronic bovine mastitis bound mainly 125I-fibronectin (Fn) [corrected], whereas strains of nine species of coagulase-negative staphylococci showed a predominant interaction with 125I-collagen (Cn) [corrected] type I. A particle agglutination assay (PAA) was used to examine the interaction of coagulase-negative staphylococci with 125I-Fn and 125I-Cn immobilized on latex. All 368 coagulase-negative staphylococci demonstrated high 125I-Cn and moderate to low 125I-Fn interactions in the PAA. Cn-PAA reactivity was high among strains of Staphylococcus xylosus (84.2%), Staphylococcus simulans (77.8%), Staphylococcus epidermidis (76.7%), and Staphylococcus hyicus (74.3%), whereas all six Staphylococcus capitis strains clumped Cn-PAA reagent. Incubating TSB-grown cells in 10% skim milk for 1 h decreased the 125I-Fn- and 125I-Cn-binding affinity in most of the S. aureus and coagulase-negative staphylococci, while growth in 10% skim milk for 18 h resulted in more than 90% decrease or complete loss of interaction with these proteins. Decreased 125I-Fn binding in the presence of milk was correlated with protease production but not with 125I-Cn binding.     

Morphology of bacterial attachment to cardiac pacemaker leads and power packs.

The mode of growth of the bacteria adherent to the surfaces of various components of cardiac pacemakers infected with coagulase-negative staphylococci was examined by scanning and transmission electron microscopy. Within developing adherent microcolonies, coccoid cells could be clearly seen to be enveloped by an amorphous material that condensed radically upon dehydration for electron microscopy, producing an amorphous residue in scanning electron micrographs and a fibrous anionic ruthenium red-staining residue in transmission electron micrographs. The differences in morphologies of the condensed residues may reflect differences in exopolysaccharides elaborated by the microorganisms and the degree to which materials of host origin are incorporated. The coccoid cells of coagulase-negative staphylococci were less clearly seen in thick, mature, adherent biofilms on heavily colonized surfaces because the condensed residue of their exopolysaccharide glycocalyces covered, and sometimes occluded, the bacteria cells. These coagulase-negative staphylococcal strains appeared to produce more exopolysaccharide material than did the strains infecting various intravascular catheters.     

Production of siderophore by coagulase-negative staphylococci and its relation to virulence

The ability to produce siderophore is considered to be a virulence factor for many pathogenic bacteria. To determine if siderophore production by coagulase-negative staphylococci (CNS) was related to virulence, 40 clinical isolates of CNS cultured from peritoneal dialysis fluid were compared with 38 commensal skin isolates. Siderophore activity was detected using the chrome azurol S liquid assay. Using precursor studies,Staphylococcus epidermidis isolates were shown to be more likely to produce the siderophore staphyloferrin A. Production of staphyloferrin B amongst non-Staphylococcus epidermidis species was associated with clinical isolates rather than commensal isolates, and therefore may play a role in pathogenicity.     

Dialysis catheter infection related peritonitis: incidence and time dependent risk

The aim of this study was to conduct an in-depth analysis of the relationship of exit site and tunnel infection (ES/TI) to peritonitis and catheter loss in peritoneal dialysis patients, with emphasis on the incidence and risk of infection over time. Bacterial epidemiologies of 63 consecutively implanted catheters were studied for a combined total of 1,248 dialysis months. Analyses of bacterial profiles, infection rates, probabilities of time to first infection, and catheter survival were performed. The probability of first ES/TI and peritonitis was greatest during the first postimplant year. The earlier in dialysis history that patients developed an infection, the more infection prone they continued to be during the course of their dialysis experience. Staphylococcus aureus was the predominant organism for both ES/TI and peritonitis. The incidence of S. aureus infection was greatest during the first year and decreased over time on dialysis. S. aureus ES/TI caused significant risk for subsequent development of peritonitis, and 93% of ES/TI related peritonitis episodes were caused by this organism. Half of all ES/TIs that led to related peritonitis occurred by 3.5 months, and 100% by 12.8 months postimplant. S. aureus ES/TI related peritonitis led to catheter loss in 85% of cases. Our study identified a high risk period for infection for as long as 12 months postimplant. The inherent characteristics of ES/TI related peritonitis suggest that prevention should focus on both the organism and time period at risk. These findings are important in considering issues regarding S. aureus prophylaxis regimens versus nasal carrier treatment protocols.     

Comparative performances of six agglutination kits assessed by using typical and atypical strains of Staphylococcus aureus.

Six commercial agglutination tests designed for the identification of Staphylococcus aureus were compared by using a strain collection which included 512 staphylococci representing 33 species (318 isolates of Staphylococcus aureus [including 144 oxacillin resistant], 46 S. epidermidis isolates, 15 S. haemolyticus isolates, 12 S. saprophyticus isolates, 29 S. schleiferi isolates, 30 S. lugdunensis isolates, and 62 other coagulase-negative staphylococci). This group also included a proportion of strains with unusual phenotypes (e.g., 19 coagulase-negative S. aureus isolates, 26 clumping factor-negative S. aureus isolates, and 4 S. aureus isolates each with a double deficiency). The overall sensitivity for identification of typical and atypical S. aureus was high with the Staphaurex Plus test (Murex Biotech) (99.7%), the Pastorex Staph Plus test (Sanofi Diagnostics Pasteur) (99.7%), and the Slidex Staph Plus test (bioMérieux) (100%). The overall rate of specificity was affected by the unusual inclusion in this study of a high proportion of non-S. aureus species, such as S. lugdunensis and S. schleiferi, which express a clumping factor and therefore produce a positive result with the agglutination tests.     

Evaluation of the LightCycler Staphylococcus M GRADE kits on positive blood cultures that contained gram-positive cocci in clusters

We evaluated the Roche LightCycler Staphylococcus M(GRADE) kits to differentiate between Staphylococcus aureus and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing S. aureus), the assay was 100% sensitive and 98.44% specific for S. aureus and 100% sensitive and specific for coagulase-negative staphylococci.     

Trimethoprim resistance determinants encoding a dihydrofolate reductase in clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci

The molecular and biochemical basis of resistance to high concentrations (MIC greater than or equal to 1000 mg/L) of trimethoprim (Tpr(H] was examined in Australian isolates of Staphylococcus aureus and coagulase-negative staphylococci. The Tpr(H) determinant (dfr A) was located within a 2.75-Kb Bg/II fragment on the S. aureus aminoglycoside-resistance plasmids pSK1 and pSK16 as judged by comparative restriction mapping with two naturally-occurring variants, pSK9 and pSK14, which did not encode trimethoprim resistance. This was confirmed in DNA-DNA hybridisation experiments in which a 0.9-Kb sequence of pSK1 DNA was used as a specific probe for the Tpr(H) gene. Plasmid DNA from three strains of coagulase-negative staphylococci, and the chromosomal material of one other isolate, were found to share homology with the probe DNA. Dihydrofolate reductases produced by these strains were virtually identical to the type S1 enzyme encoded by the S. aureus plasmid pSK1. Interspecies transfer may have been responsible for the conservation of Tpr(H) gene sequences among staphylococci.     

Antiserum agar method for identification of Smith type exopolysaccharides in clinical isolates of Staphylococcus aureus.

We used an antiserum agar method to identify clinical Staphylococcus aureus strains producing an exopolysaccharide antigenically identical to the S. aureus Smith diffuse strain. S. aureus blood isolates were obtained from 137 patients, and three additional isolates were obtained from bone debridement. The 140 patients were clinically divided into the following groups: endocarditis (7 patients); pneumonia, empyema, or both (33 patients); intravascular device (34 patients); superficial or wound infection or both (35 patients); deep tissue infections (18 patients); and 6, unknown bacteremias (13 patients). Ninety (64.3%) of the total 140 S. aureus isolates were found to produce precipitin halos on the antiserum agar. The percentage was greatest in the isolates from the endocarditis group (100%) and least in deep tissue infections (55.5%). The presence of clinical S. aureus strains producing exopolysaccharides antigenically identical to the Smith diffuse strain exopolysaccharide appears to be a common phenomenon.