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Clinical studies have shown positive associations among sustained and intense inflammatory responses and the incidence of bacterial infections. Patients presenting with acute respiratory distress syndrome (ARDS) and high levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and IL-6, have increased risk for developing nosocomial infections attributable to organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter spp., compared to those patients with lower levels. Our previous in vitro studies have demonstrated that these bacterial strains exhibit enhanced growth extracellularly when supplemented with high concentrations of pure recombinant TNF-alpha, IL-1 beta, or IL-6. In addition, we have shown that the intracellular milieu of phagocytic cells that are exposed to supraoptimal concentrations of TNF-alpha, IL-1 beta, and IL-6 or lipopolysaccharide (LPS) favors survival and replication of ingested bacteria. Therefore, we hypothesized that under conditions of intense inflammation the host's micromilieu favors bacterial infections by exposing phagocytic cells to protracted high levels of inflammatory cytokines. Our clinical studies have shown that methylprednisolone is capable of reducing the levels of TNF-alpha, IL-1 beta, and IL-6 in ARDS patients. Hence, we designed a series of in vitro experiments to test whether human monocytic cells (U937 cells) that are activated with high concentrations of LPS, which upregulate the release of proinflammatory cytokines from these phagocytic cells, would effectively kill or restrict bacterial survival and replication after exposure to methylprednisolone. Fresh isolates of S. aureus, P. aeruginosa, and Acinetobacter were used in our studies. Our results indicate that, compared with the control, stimulation of U937 cells with 100-ng/ml, 1.0-microg/ml, 5.0-microg/ml, or 10.0-microg/ml concentrations of LPS enhanced the intracellular survival and replication of all three species of bacteria significantly (for all, P = 0.0001). Stimulation with < or =10.0 ng of LPS generally resulted in efficient killing of the ingested bacteria. Interestingly, when exposed to graded concentrations of methylprednisolone, U937 cells that had been stimulated with 10.0 microg of LPS were able to suppress bacterial replication efficiently in a concentration-dependent manner. Significant reduction in numbers of CFU was observed at > or =150 microg of methylprednisolone per ml (P values were 0.032, 0.008, and 0.009 for S. aureus, P. aeruginosa, and Acinetobacter, respectively). We have also shown that steady-state mRNA levels of TNF-alpha, IL-1 beta, and IL-6 in LPS-activated cells were reduced by treatment of such cells with methylprednisolone, in a concentration-dependent manner. The effective dose of methylprednisolone was 175 mg, a value that appeared to be independent of priming level of LPS and type of mRNA. We therefore postulate that a U-shaped relationship exists between the level of expression of TNF-alpha, IL-1 beta, and IL-6 within the phagocytic cells and their abilities to suppress active survival and replication of phagocytized bacteria.

Patients with unresolving acute respiratory distress syndrome (ARDS) have persistently elevated levels of proinflammatory cytokines in the lungs and circulation and increased rates of bacterial infections. Phagocytic cells hyperactivated with lipopolysaccharide (LPS), which induces high levels of proinflammatory cytokines in monocytic cells, are inefficient in killing ingested bacteria despite having intact phagocytic activity. On the other hand, phagocytic cells that are activated with an analogue of LPS that does not induce the expression of proinflammatory cytokines effectively ingest and kill bacteria. We hypothesized that in the presence of high concentrations of proinflammatory cytokines, bacteria may adapt and utilize cytokines to their growth advantage. To test our hypothesis, we primed a human monocytic cell line (U937) with escalating concentrations of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-6 and with LPS. These cells were then exposed to fresh isolates of three common nosocomial pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, and an Acinetobacter sp. In human monocytes primed with lower concentrations of proinflammatory cytokines (10 to 250 pg) or LPS (1 and 10 ng), intracellular bacterial growth decreased. However, when human monocytes were primed with higher concentrations of proinflammatory cytokines (1 to 10 ng) or LPS (1 to 10 micrograms), intracellular growth of the tested bacteria increased significantly (P <0.0001). These results were reproduced with peripheral blood monocytes obtained from normal healthy volunteers. The specificity of the cytokine activity was demonstrated by neutralizing the cytokines with specific antibodies. Our findings provide a possible mechanism to explain the frequent development of bacterial infections in patients with an intense and protracted inflammatory response.

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.

The pharmacokinetic disposition of vancomycin and ciprofloxacin was assessed in rabbits before the efficacy of these compounds in experimental staphylococcal endocarditis was compared. Ciprofloxacin was given in single intravenous bolus doses of 25 and 35 mg/kg and also in a multiple-dose regimen of 35 mg/kg every 6 h. Vancomycin was given in a similar manner in single doses of 17.5 and 25 mg/kg and in a multiple-dose regimen of 17.5 mg/kg every 6 h. Serum was sampled frequently after injections and analyzed by microbiologic assay for drug concentration. The pharmacokinetic parameters of clearance and steady-state volume of distribution were calculated by compartment-independent methods. These studies revealed that clearance of ciprofloxacin was reduced significantly after multiple doses (7.42 +/- 0.85 [standard deviation] versus 6.09 +/- 0.71 liters/h, P less than 0.01). Although the half-life and volume of distribution increased after multiple dosing, the differences were not statistically significant. The disposition of vancomycin following single doses was significantly altered after the 25-mg/kg dose compared with the 17.5-mg/kg dose. Half-life, clearance, and volume of distribution changed from 1.27 +/- 0.2 to 1.60 +/- 0.21 h (P less than 0.05), 0.54 +/- 0.05 to 0.39 +/- 0.04 liters/h (P less than 0.01), and 0.37 +/- 0.04 to 0.31 +/- 0.03 liters/kg (P less than 0.05), respectively. The disposition of ciprofloxacin was not altered with increases in dose size, and the disposition of vancomycin was not altered after multiple doses. If such alterations in the pharmacokinetic disposition of antimicrobial agents are unanticipated, the higher and more prolonged than expected serum concentrations may have an effect on the outcome of experimental infections.

An outbreak of antibiotic-associated colitis that occurred on a ward of a Michigan hospital during February-April, 1984, was studied by bacteriophage-bacteriocin typing. Stools from the seven involved patients yielded Clostridium difficile isolates of types B1537 or Cld7;B1537. C. difficile was recovered from 31.4% of environmental cultures obtained on the ward, and the majority of isolates were types B1537 or Cld7;B1537. When the ward was disinfected with unbuffered hypochlorite (500 parts per million (ppm) available chlorine), surface contamination decreased to 21% of initial levels and the outbreak subsequently ended. Phosphate buffered hypochlorite (1,600 ppm available chlorine, pH 7.6) was even more effective; its use resulted in a 98% reduction in surface contamination. These findings suggest that environmental contamination with C. difficile is important in the epidemiology of antibiotic-associated colitis, and that hypochlorite is effective in eliminating C. difficile from the hospital environment.

The study of the epidemiology of infection with Clostridium difficile would be aided by a way to type individual bacterial isolates. We therefore sought bacteriophages for use in typing. With mitomycin C exposure (3 micrograms/ml), filtrates from 10 strains of C. difficile had plaque-forming lytic activity on other C. difficile strains. Individual phage were passaged and made into high-titer stock preparations for typing. Electron microscopy revealed tailed phage particles from one such preparation. In addition to phage, inhibitory activity without distinct plaque formation consistent with bacteriocins was observed for 20 strains. C. difficile isolates from 16 patients taken 1 to 14 days apart were similar in their phage sensitivity pattern, whereas isolates from separate geographic locations showed a great variety of patterns. We conclude that bacteriophage should be useful for typing strains of C. difficile.

We compared the efficacy of ciprofloxacin with that of vancomycin by using the rabbit model of methicillin-resistant Staphylococcus aureus endocarditis. Endocarditis was treated with ciprofloxacin (25 mg/kg [body weight] intravenously every 8 h) or vancomycin (17.5 mg/kg intravenously every 6 h) for 3 days. Vancomycin and ciprofloxacin were equally efficacious in clearing bacteremia. Both reduced vegetation bacterial counts by 5 log10 CFU/g and renal and splenic bacterial counts by more than 3 log10 CFU/g as compared with untreated control rabbits after 26 h of infection (P less than 0.001). Both antimicrobial agents were able to eradicate the infectious process in an equivalent proportion of animals. No methicillin-resistant S. aureus that was recovered from ciprofloxacin-treated rabbits developed resistance to ciprofloxacin during therapy. Peak concentrations of ciprofloxacin in the sera of rabbits with endocarditis were significantly higher than those predicted by single-dose studies in uninfected rabbits. This finding was likely due to changes in the pharmacokinetics of the drug with multiple dosing and in infected versus uninfected rabbits. This study demonstrated that intravenously administered ciprofloxacin is as efficacious as vancomycin is in an in vivo model of a serious systemic methicillin-resistant S. aureus infection.

Between November 1981 and October 1984, 48 of 3,458 clinical isolates of Streptococcus faecalis at the University of Michigan Hospital showed high-level (greater than 2,000 micrograms/ml) resistance to gentamicin, as well as to all other clinically available aminoglycosides. Thirteen percent of clinical isolates in the University of Michigan Hospital currently demonstrate this level of resistance. Transfer of resistance to a plasmid-free streptococcal recipient was observed in filter matings for 44 of 48 such isolates. Analysis of transconjugants by agarose gel electrophoresis showed that gentamicin resistance was transferred alone in 8 isolates and was combined with other antimicrobial resistances on the same plasmid in the other 36 isolates. There were seven isolates which transferred the property of horse blood beta-hemolysis along with gentamicin resistance. Transfer of gentamicin resistance was either by conjugative plasmids or by a nonconjugative but mobilizable plasmid. Transfer frequencies on filters ranged from 4.7 X 10(-2) to 4.1 X 10(-8). Based on donor and transconjugant antibiotic resistance markers, agarose gel electrophoresis, transfer properties, and restriction enzyme analysis, the plasmid content of transconjugants was heterogeneous and could be classified into at least seven different patterns. These findings argue against clonal dissemination as the cause of the increased frequency of resistant strains and suggest that resistance to gentamicin appears on a variety of physically distinct conjugative and nonconjugative plasmids in S. faecalis.

R-plasmids originally isolated from Streptococcus pyogenes(pAC1,pAM15346), Streptococcus agalactiae(pIP501), and Streptococcus faecalis(pAM beta 1) were shown to be self-transferable on filter membranes from S. faecalis JH2-2 to Staphylococcus aureus recipients. The nonconjugative plasmid pAM alpha 1 was mobilized into S. aureus by pAM beta 1. Once in S. aureus, conjugative R-plasmids could be transferred to a second S. aureus recipient or back into S. faecalis. Determinants for chloramphenicol, clindamycin, erythromycin, and tetracycline resistances present on these streptococcal plasmids were expressed in S. aureus. Agarose gel electrophoresis, dye-buoyant centrifugation, and restriction endonuclease digestion showed that the plasmids were maintained intact as self-replicating elements in S. aureus recipients.

Agarose gel electrophoresis of the plasmid deoxyribonucleic acids from 60 gram-negative bacilli recovered during investigations of nosocomial epidemics was used to fingerprint the strains. This method was as specific at differentiating bacterial strains as more conventional phenotyping methods. In all cases, plasmid band fingerprints of epidermic strains isolates were identical whereas coisolate plasmid deoxyribonucleic acid patterns were different. Agarose gel electrophoresis of plasmid deoxyribonucleic acid is proposed as a method which can be used in conventional microbiology laboratories as an adjunct to or, possibly, replacement for other methods of identifying bacterial strains.

To measure the accuracy and consistency of a standardized method--retrospective chart review (RCR)--for estimating nosocomial infection rates (NIRs) in individual hospitals, the authors performed a series of pilot studies in four hospitals of different types. In comparison with a standard based on diagnoses made by physician-epidemiologists supervising intensive prospective data collection teams, the RCR method was found to have an average sensitivity of 0.74 (+/- 0.02 SE; range 0.69-0.78) and an average specificity of 0.964 (+/- 0.002; 0.945-0.991). These values were comparable to those of the physician-epidemiologists' diagnoses and varied less among the hospitals. Two independent teams of chart reviewers were found to have similar levels of sensitivity and specificity, and the reliability of diagnosis at the level of the individual chart reviewer averaged 0.94. In a restudy at one of the pilot hospitals at the midpoint of the actual Medical Records Survey (MRS), there was a substantial increase in sensitivityand a slight increase in specificity as a result of improvements made in the RCR method after the original pilot studies.