Anemia of chronic renal failure: characterization in the mouse and correction with human recombinant erythropoietin.

Anemia is a cardinal feature of chronic renal failure (CRF) which contributes significantly to the clinical syndrome of chronic uremia. We have conducted a detailed examination of the hematological changes in CRF in the inbred mouse strain C57BL/6J. As in the human situation, CRF mice presented major hematological changes affecting primarily the erythroid cell series. Despite the presence of abundant iron stores in the bone marrow, the CRF mice developed a hypoproliferative anemia of a severity commensurate with the degree of renal impairment. The levels of circulating erythropoietin (EPO) in CRF mice were not significantly different from those in normal control littermates and were therefore inappropriately low for the degree of anemia. In contrast acutely bled control mice with normal renal function showed a significant inverse correlation between the serum EPO level and hemoglobin concentration, indicating an appropriate response to anemia. The chronic administration of recombinant human EPO raised the hemoglobin concentration of CRF mice, a therapeutic effect which was independent of the initial degree of anemia. These observations suggest that this animal model has wide applicability for the study of anemia secondary to CRF.

An assay to measure antibiotic efficacy against Staphylococcus epidermidis biofilms on implant surfaces.

Infection is a major limitation of implantable devices. Optimal antibiotic therapeutic regimes have not yet been defined. Implant-associated infections have a number of differentiating characteristics, which include the predominance of Staphylococcus epidermidis and other skin bacteria of normally low pathogenicity as the causative agents, together with a relative resistance to host defenses and to antibiotic therapy. These properties have been ascribed to the ability of the bacteria to exist on implant surfaces in the biofilm phase, which is protective. An assay of antibiotic activity using a standardized bacterial biofilm preparation of S. epidermidis is described. The assay is used to evaluate the relative efficacy of antibiotics to sterilize the biofilm, when they are used singly, or in double or triple combinations. The modulating effects of changing antibiotic concentrations and modifying the environment with CAPD variables (fresh and spent dialysis fluid, common PD solution additives) are also measured and the data summarized. It is hoped that, by using this and similar assays, individualized optimal therapeutic regimes of implant-associated infections may be logically planned.

Preoperative assessment of skin colonization and antibiotic effectiveness in total knee arthroplasty.

A bacteriologic screening procedure was performed to preoperatively assess the skin flora of 152 total knee arthroplasty patients and to determine the appropriate prophylactic antibiotic and skin-cleansing technique. Staphylococcus epidermidis resistant to standard prophylactic antibiotics was present in 4.6% of patients, whereas 44% of the patients had Staphylococcus aureus, which is poorly eradicated by standard cleansing techniques. Preoperative assessment of skin colonization has demonstrated that standard preoperative antibiotics and skin-cleansing techniques will not completely eradicate all pathogenic skin bacteria in every case. Preoperative screening effectively identifies bacterial skin flora and allows for the modification of antibiotic selection and preoperative cleansing to eradicate resistant bacterial organisms and thereby decrease the risk of postoperative prosthetic infections.

The differential activity of aminoglycoside antibiotics with rifampin explored in a kinetic in vitro model of implant-associated infection (Staphylococcus epidermidis).

Implant-associated infections offer resistance to antibiotic treatment and possibly do so because the causative bacteria which reside on the artificial surface are enclosed in a protective matrix (biofilm) shielding the bacteria against the action of host defenses and antibiotic action. We have investigated in vitro the effect of various antimicrobial agents on biofilms of Staphylococcus epidermidis, which is the main organism responsible for implant-associated infections. Rifampin was found to exert superior activity, albeit incomplete, against S. epidermidis biofilms using an assay system which enabled the determination of the kinetics of antibiotic action over five days. In a large screening study looking for agents capable of completing the action of rifampin, gentamicin was unexpectedly found to be antagonistic to rifampin. The present study was undertaken to investigate further the activity of gentamicin and five other aminoglycoside antibiotics using a wider range of concentrations (2.5-20 micrograms/mL). The main findings were a marked synergy with rifampin demonstrated by streptomycin, producing a bactericidal outcome, which contrasted sharply with the indifference or antagonism shown by the other aminoglycosides. We then studied in further detail the effect of separate combinations of streptomycin and gentamicin with rifampin over a wider concentration range of each agent (1.25-40 micrograms/mL). Streptomycin showed strong rapid synergy with rifampin even at the lowest concentration of each antibiotic. Gentamicin demonstrated a concentration-related antagonism towards rifampin which was independent of rifampin concentration. The data support the conclusion that streptomycin, like cell-wall active antibiotics, exerts a potent synergy with rifampin against S. epidermidis biofilms, and that the other aminoglycosides, predominantly gentamicin, strongly antagonize rifampin action.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-kill efficacy of antibiotics in combination with rifampin against Staphylococcus epidermidis biofilms.

Infections associated with implants are frequently resistant to conventional antibiotic therapy. This resistance has been ascribed to the presence of bacteria on the artificial surface within a protective glue-like matrix forming a bacterial biofilm. We have demonstrated that experimental biofilms of Staphylococcus epidermidis, the main pathogen associated with implantation, are exquisitely sensitive to the action of rifampin. This effect of rifampin is incomplete, however, with the emergence of rifampin-resistant survivors that readily repopulate the biofilm. Studies were therefore performed to determine the effect of combinations of 13 different antibiotics with rifampin against standardized S. epidermidis biofilm preparations in an in vitro assay enabling the kinetic measurement of antibiotic action over a five-day period. The antibiotic combinations with rifampin demonstrated unsuspected divergent patterns of antimicrobial activity against the biofilms: 1. rapid synergy with rifampin was observed with cell-wall active antibiotics (cloxacillin, cephalothin, cefazolin, and cefamandole), whereas slower synergy occurred with vancomycin, ciprofloxacin, tetracycline, and amikacin; 2. some antibiotics (tobramycin, erythromycin, clindamycin, fusidic acid) did not influence the outcome; 3. gentamicin unexpectedly showed marked antagonism to rifampin. These results are relevant to the design of optimal therapeutic regimens for the management of resistant implant-associated infections.

An assay of Staphylococcus epidermidis biofilm responses to therapeutic agents.

Implant-associated infections tend to become persistent, resisting host defences and antibiotic therapy. Routine clinical laboratory testing of bacterial isolates in the fluid phase for antibiotic susceptibility may not be predictive of therapeutic outcomes and therefore a number of antibiotic regimes have been formulated empirically. The resistance of implant-associated infections has been ascribed to the production by bacteria, when adherent to the implant surface, of a shielding matrix of polymerized carbohydrates protecting the enclosed bacteria from immune defences and antibiotics. This complex of surface, bacteria and matrix is termed a biofilm. We describe a technique to evaluate the efficacy of antimicrobial agents directed against biofilm-forming strains of Staphylococcus epidermidis (a major cause of implant infections) utilizing standardized biofilm preparations formed on glass. The impact of the antimicrobial agents was assessed quantally using the end-point of permanent cessation of metabolic activity (cell death) of the entire biofilm determined by the loss of ability to reduce 2,3,5-triphenyltetrazolium chloride to a visible red intracellular precipitate of formazan. The relative rate of action of differing antimicrobial agents could be determined by the minimum period of exposure of the biofilm to the agents that is required to bring about sterilization, the clinically relevant marker. A wide range of antimicrobial substances could be evaluated, including chemical disinfectants, immunoreactive substances, antibiotics; singly and in combination; and the modifying effects of interaction with non-antibacterial therapeutic agents and other environmental factors. The technique is simple, inexpensive, reproducible and readily adaptable to the clinical situation where evaluation of therapeutic regimes for individual cases of prosthetic device-associated infection is required routinely with despatch and ease of interpretation.

A mouse model of implant-associated infection.

Infections of implanted devices are of increasing frequency and importance, representing a significant limitation of many therapeutic modalities. There are puzzling features of implant-associated infection including the changes in microbial flora, the tendency to chronicity and impaired responses to conservative modes of treatment. The concept of the bacterial biofilm as a shielding mechanism generated by bacteria adherent to artificial surfaces has recently been proposed as an explanation for these features. The biofilm is a term applied to a complex comprising the implant surface, adherent bacteria and a specialized matrix enclosing the bacteria. The matrix of the biofilm is an electrostatically charged glue-like extracellular polymer derived by bacterial enzymes acting on tissue carbohydrates, formed by bacteria when adherent to surfaces. This matrix binds the bacteria to the surface providing a sequestration affording selective protection against harmful elements of the environment, especially mechanisms of host defenses and antimicrobial agents. These biological systems are complex to study because of the dynamic interaction of the microbial variables, host defenses, properties of synthetic materials and the biofilm matrix itself. There is a need for a laboratory model in which the variables can be controlled permitting the researcher to examine the outcomes of modifying one variable at a time in a planned and orderly manner. The practical way to attain this end is the conduct of studies in a stable reproducible animal model of localized biofilm-implant infection. Staphylococcus epidermidis is a representative of the class of microorganisms predominant in implant-associated infection. This paper describes the development of a model utilizing an implant-S. epidermidis-biofilm infection localized to the peritoneal cavity of the mouse. The natural history of the infection has been well documented and is stable in all respects for periods exceeding 3 months. This chronicity is especially advantageous in analyzing the impact of long-term therapeutic modalities and necessary periods of recovery and assessment. A representative example of an experimental use of this model to determine the relative efficacy of antibiotic therapeutic regimes is described, demonstrating its scope and efficacy.

The effect of the fiber components cellulose and lignin on experimental colon neoplasia.

Sixty Sprague-Dawley rats were pair-fed one of three nutritionally identical diets. One diet contained "low-fiber" (3.8% crude fiber); the others contained "high fiber" (28.7% crude fiber) composed of either cellulose or lignin. Although both "high fiber" diets had similar stool bulking effects, only the cellulose diet was associated with a reduction in 1,2-dimethylhydrazine (DMH)-induced colon neoplasms. The cellulose diet was also associated with distinct changes in the gut bacterial profile and with a lowered serum cholesterol.

Impact of dialysis fluid on the susceptibility of Staphylococcus epidermidis biofilms to rifampin.

Routine laboratory testing of the susceptibility of infecting strains of S. epidermidis to antibiotics is based on the assumptions that the infection is in contact with the stable environment of blood or tissue fluid and that the bacteria are in a susceptible phase of growth. In infections of the continuous ambulatory peritoneal dialysis (CAPD) intraperitoneal catheter, the bacteria are commonly in the resistant biofilm phase of growth, are applied to the surface of the catheter, and are exposed to the nonphysiological impact of peritoneal dialysis solutions. The possible impact of these two variables is not examined in routine testing of antibiotic susceptibility. We describe an assay of antibacterial action on a strain of S. epidermidis in the biofilm phase, exposed to a neutral control environment and to dialysis solutions, both fresh and spent. Rifampin, which has an incomplete action in a neutral control environment, was used as a sensitive indicator of possible synergistic or antagonistic interaction of the biofilm bacteria with the dialysis environments. We demonstrated that peritoneal dialysis solutions (1.5% dextrose), both fresh and spent, were synergistic with rifampin action against S. epidermidis biofilms, converting the incomplete killing action to a total bactericidal outcome, clinically essential for cure. Fresh dialysis solution was more active than spent, but in both instances the synergy was complete, indicating that the crucial complementation of the bactericidal action of rifampin was independent of pH. These findings have implications in the management of catheter-associated infections due to rifampin-susceptible strains of S. epidermidis.

Vancomycin therapy of experimental peritoneal catheter-associated infection (Staphylococcus epidermidis) in a mouse model.

Catheter-related sepsis, principally with S. epidermidis, remains a main complication of continuous ambulatory peritoneal dialysis (CAPD). A possible reason for the antibiotic resistance often displayed by these infections is the presence of bacteria growing in a protective biofilm on the catheter surface. We developed a reproducible stable model of experimental peritoneal catheter-associated infection with S. epidermidis in the mouse and used this model to examine the therapeutic efficacy of vancomycin. The response to vancomycin treatment given daily (15 mg/kg body weight) for periods from 1-14 days, relating the proportion of successful outcome (sterilization of implant infection) over time, was typical of an S-shaped biological response curve. These results extend our previous observations in vitro of the activity of vancomycin against S. epidermidis biofilm preparations and serve as a rational basis for the experimental evaluation of synergy and antagonism in the treatment of implant-associated infection.

The effect of peritoneal dialysis solutions on rifampin action against Staphylococcus epidermidis in the fluid and biofilm phases of growth.

Continuous ambulatory peritoneal dialysis (CAPD) catheter-associated infections can be resistant to conservative therapeutic measures despite the apparent susceptibility of the infecting bacterial strains to antibiotics. This chronicity of infection has been ascribed to the development of bacteria on the surfaces of catheter material within a protective polysaccharide shield or matrix. CAPD catheters are unique in being exposed to nonphysiological environments of dialysis fluids of varying physical properties and constitution. We determined that the rate of growth of S. epidermidis in the fluid phase in laboratory media or fresh dialysis solution was significantly influenced by pH. An acid milieu (pH 5) impaired growth compared to a neutral milieu (pH 7). Spent dialysis fluid, however, which had an approximately neutral pH, also impaired bacterial growth, the assumption being that this results from properties of spent fluid unrelated to pH. Using rifampin as the test antibiotic, we noted that its action on S. epidermidis was unaffected by pH or by the differing milieu of fresh and spent fluid. Bacterial growth was arrested regardless of the milieu. We further examined the potential of dialysis fluids to modify the interaction between rifampin and the matrix-enclosed bacteria of standardized S. epidermidis biofilms. We determined that the pH of the dialysis solution was a critical factor influencing the outcome of exposure of the biofilm to rifampin. An acid pH enhanced antibiotic activity, whereas a neutral pH was associated with antibiotic antagonism. These findings have implications in the management of S. epidermidis catheter-associated infections using rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)

The modulation of rifampin action against Staphylococcus epidermidis biofilms by drug additives to peritoneal dialysis solutions.

Many drugs are added to peritoneal dialysis solutions in continuous ambulatory peritoneal dialysis (CAPD) patients, principally during episodes of peritonitis when antibiotics and nonantibiotic agents are often administered together. To evaluate the influence of nonantibiotic drug additives on antibiotic action, we determined the effect of ten drug additives on the antimicrobial activity of rifampin against S. epidermidis biofilms. None of the additives had any direct effect on biofilm bacteria. While five additives did not affect rifampin action, the other five strongly antagonized rifampin. Insulin and heparin were among these five antagonists. This antagonism suggests that a significant degree of inhibition of antibiotic action may occur by the coadministration of agents in the treatment of peritonitis, particularly in diabetic patients.

Experimental Staphylococcus epidermidis implant infection in the mouse. Kinetics of rifampin and vancomycin action.

Staphylococcus epidermidis implant infections remain a therapeutic challenge; they frequently result in failure of conservative management and require removal of the implant. This resistance to antibiotic therapy has been ascribed to the presence of a protective bacterial biofilm at the surface of the implant. An animal model of implant associated infection has been developed in which preformed bacterial biofilm catheter segments are implanted intraperitoneally, resulting in a chronic standardized localized infection. The authors have determined the superior rapid action of rifampin (Cieba-Geigy) compared to vancomycin hydrochloride (Eli Lilly) and determined that the combination is superior to either used alone. No rifampin resistant surviving infection was noted, which indicates the significant contribution of mammalian host defenses. This animal model is an excellent vehicle for the study of Staphylococcus epidermidis implant associated infection and the evaluation of the relative efficacy of antibiotic regimens, singly and in combination.

Assessment of gelatin-supplemented BACTEC blood culture medium in a pediatric hospital.

Sodium polyanetheolesulfonate (SPS), an anticoagulant used in blood culture media, adversely affects the isolation of Neisseria meningitidis. The addition of gelatin appears to counteract this effect. Studies using the radiometric BACTEC system, however, have noted a lower isolation rate of other bacteria from gelatin-supplemented media. We wished to evaluate the effect of the addition of gelatin (1.2%) to a nonradiometric BACTEC aerobic medium (NR6A) on the recovery of N. meningitidis and other pathogens. The NR6A medium with gelatin (NR6A analogue) also contained a lower concentration of SPS (0.025% vs 0.035%). We did 6045 paired comparisons of blood cultured in routine NR6A medium and the NR6A analogue. Eight isolates of N. meningitidis were recovered, five only from the gelatin-supplemented medium and three from both bottles. There was no statistically significant difference in total recovery of aerobic and facultative bacteria or Candida species from both bottles. Haemophilus influenzae was detected earlier in the nonsupplemented NR6A medium. We conclude that the use of the NR6A analogue medium appeared to increase the yield of N. meningitidis without adversely affecting the recovery of other common pathogens, although the recovery of H. influenzae was slightly delayed.

The modulation by fresh and spent peritoneal dialysis fluids of antibiotic kinetics directed against Staphylococcus epidermidis biofilms.

The presence of CAPD peritoneal catheters has an association with S. epidermidis infections which are relatively resistant to antibiotic therapy. This resistance has been ascribed to the formation by the bacteria of a protective matrix on the catheter surface enclosing the bacteria to form a bacterial biofilm. The rate of action of 14 antibiotics on standardized S. epidermidis bacterial biofilms over a 5 day exposure period and the modifying effects of fresh and spent peritoneal dialysis fluids were assayed. There were significant differences in the rates of antibiotic action. Fresh dialysis solution markedly augmented rifampin activity, and to a lesser extent, vancomycin, cloxacillin and teicoplanin but antagonized ciprofloxacin. Spent dialysis fluid generally augmented the activity of antibiotics, notably cloxacillin and cephalothin. Rifampin was bactericidal in both fresh and spent dialysis fluids. Fusidic acid was a poor performer in all tests.

Natural history of chronic Staphylococcus epidermidis foreign body infection in a mouse model.

The development and characterization of a mouse model of chronic Staphylococcus epidermidis foreign body infection was done with two clinical isolates that differed in degree of extracellular slime production. Segments of Silastic catheters bearing preformed S. epidermidis biofilms were implanted intraperitoneally, and mice were assessed after 3 and 6 months. Both test strains of S. epidermidis persisted at the implant site through the 6-month follow-up in 80% of the mice, regardless of the degree of slime production. There was no evidence of overt animal morbidity, and microbiologic assessment of other peritoneal sites did not reveal dissemination of bacteria from the infected focus. In comparison with control mice, animals harboring chronic foreign body infection presented marked peripheral neutrophilia and mild anemia.

Staphylococcus epidermidis biofilms: unexpected outcome of double and triple antibiotic combinations with rifampin.

Staphylococcus epidermidis, in contact with artificial surfaces, may produce a protective glue-like matrix or biofilm. The authors demonstrated that rifampin alone among 35 antibiotics penetrated the biofilm within a 24 hr exposure producing a major but incomplete killing. Antibiotics of the cell-wall active class (including vancomycin) were synergistic with rifampin, completing the bactericidal action. The addition of these antibiotics to a rifampin-vancomycin combination did not alter the synergy. Other antibiotics (including aminoglycosides) antagonized rifampin activity. This antagonism of rifampin was maintained when the antibiotics were added to the rifampin-vancomycin synergistic combination. These results may have implications for the choice of optimal therapeutic regimens in the management of implant-associated infection.

Comparative rates of antibiotic action against Staphylococcus epidermidis biofilms.

The relative resistance of S. epidermidis implant-associated infections to antibiotic therapy has been ascribed to a protective function of the gluelike biofilm matrix produced by strains of S. epidermidis in contact with artificial surfaces. Using a standardized S. epidermidis biofilm assay we determined the periods of exposure required by various antibiotics to produce cessation of biofilm metabolic activity. Rifampin has the superior rate of action, producing substantial disruption of biofilm activity by 7 hr of exposure, but leading to replacement of the susceptible bacterial cells by rifampin-resistant mutant survivors. Other antibiotics required longer periods of exposure, in excess of 48 hr, but produced a bactericidal outcome. Combinations of antibiotics with rifampin produced strikingly divergent results. Cefazolin and vancomycin (cell wall active antibiotics) produced a bactericidal outcome at 16 hr of exposure, whereas gentamicin (aminoglycoside) neutralized the rapid action of rifampin with metabolic activity maintained at 48 hr. We confirmed the selectively protective function of the S. epidermidis biofilm with regard to antibiotic action. In vitro biofilm assays may be of value in guiding antibiotic therapy in S. epidermidis implant-associated infection.

Antistaphylococcal activity of pentamidine.

Pentamidine isethionate was bacteriostatic against Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus sanguis, Micrococcus sp., and Candida albicans. S. aureus was inhibited by concentrations of 16 to 64 micrograms/ml and killed by 64 to greater than or equal to 128 micrograms/ml. Staphylococcal killing was consistently greater in the presence of cations and was unaffected by methicillin resistance.