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Toxin A has historically been regarded as the primary virulence determinant in Clostridium difficile infection, but naturally occurring toxin A-negative, toxin B-positive (A-/B+) C. difficile strains are known to be virulent. To determine the role of toxin B in these strains, we immunized hamsters with a toxoid prepared from purified toxin B to determine whether they would be protected from lethal challenge with an A-/B1 strain of C. difficile. [ABSTRACT FROM AUTHOR]

Background. North American and European hospitals have reported outbreaks of Clostridium difficile-associated disease with unexpectedly high mortality caused by a newly recognized group of C. difficile strains, group BI. Our objective was to compare, in hamsters, the virulence of a historical nonepidemic BI type, BI1, with that of 2 recent epidemic BI types, BI6 and BI17, and with that of 2 standard toxigenic strains, K14 and 630. Methods. For each strain, 10 hamsters were given 1 dose of clindamycin, followed 5 days later with 100 C. difficile spores administered by gastric inoculation. Outcomes were recorded. Results. The hamster model demonstrated variations in mean times from inoculation to death (for BI6, 40 h; for BI1, 48 h; for K14, 49 h; for BI17, 69 h; for 630, 102 h; for BI6, BI1, and K14 vs. 630, P < .01; for BI17 vs. 630, P < .05) and from colonization to death (for BI1, 7 h; for BI17, 13 h; for BI6, 16 h; for K14, 17 h; for 630, 52 h; for BI1, BI17, BI6, and K14 vs.630, P < .01). Conclusion. Group BI strains were not more rapidly fatal than the standard toxinotype 0 strain K14 but were more rapidly fatal than the standard toxinotype 0 strain 630. BI6, the most common BI type in our collection, was particularly virulent in hamsters, consistently causing death within 48 h of inoculation. [ABSTRACT FROM AUTHOR]

Binary toxin CDT or its genes have been identified in some strains of Clostridium difficile that also produce the large clostridial toxins, toxins A and B (A+B+CDT+), including a newly recognized epidemic strain in the United States and Canada. To study the effects of binary toxin alone, we characterized 4 binary toxin CDT-positive only (A-B-CDT+) C. difficile strains. Unlike other clostridial binary toxins, binary toxin CDT required exogenous trypsin for activation. Supernatants from all A-B-CDT+ strains caused marked fluid accumulation in the rabbit ileal loop assay after concentration and trypsinization. In addition, the ileal loop response was neutralized by antisera raised against other binary toxin-producing clostridia. Challenge of clindamycin-treated hamsters with these strains resulted in colonization but not diarrhea or death. Binary toxin CDT may play an adjunctive role to toxins A and B in the pathogenesis of C. difficile-associated disease but by itself may not be sufficient to cause disease. [ABSTRACT FROM AUTHOR]

Studies clostridium difficile-associated disease (CDAD) due to toxigenic strains. Prevention in hamsters by colonization by nontoxigenic C. difficile after administration of clindamycin. Prevention of CDAD during treatment with antibiotics through the administration of Cm-resistant nontoxigenic C. difficile strain, M13; Need to balance the benefits of use of Cm-resistant nontoxigenic C. difficile to prevent CDAD against the risk that resistance might be transferred to other enteric bacteria.

Studies suggest that asymptomatic colonization with Clostridium difficile (CD) decreases the risk of CD-associated disease (CDAD) in humans. A hamster model was used to test the efficacy of colonization with 3 nontoxigenic CD strains for preventing CDAD after exposure to toxigenic CD. Groups of 10 hamsters were given 10[sup 6] nontoxigenic CD spores 2 days after receiving a single dose of clindamycin. Five days later, the hamsters were given 100 spores of 1 of 3 toxigenic CD strains previously shown to cause mortality within 48 h. Each nontoxigenic strain prevented disease in 87%-97% of hamsters that were challenged with toxigenic strains. Failure to prevent CDAD was associated with failure of colonization with nontoxigenic CD. Colonization with nontoxigenic CD strains is highly effective in preventing CDAD in hamsters challenged with toxigenic CD strains, which suggests that use of a probiotic strategy for CDAD prevention in humans receiving antibiotics might be beneficial. [ABSTRACT FROM AUTHOR]

Presents a study which correlated the inferred human virulence potential of Clostridium (C.) difficile strains to their pathogenicity in the hamster model. Materials and methods; Clostridium difficile isolates used in a hamster model of C. difficile infection; Primers used in the amplification of toxin genes in C. difficile; Antibiotic susceptibilities of toxigenic strains of C. difficile.

Nosocomial Clostridium difficile infection was investigated at a hospital with 15 cases of C. difficile diarrhea per 1000 discharges. From January 1991 to May 1991, patients admitted or transferred to five wards or units had weekly rectal swabs taken for culture; in addition, all cytotoxin-positive stools were cultured. Restriction enzyme analysis (REA) was used for molecular typing. Among 205 isolates from 39 patients with C. difficile diarrhea and 67 asymptomatically colonized, 55 distinct REA banding patterns were identified. Evidence for patient-to-patient transmission was limited, in that numerous strains were found even among clustered cases of diarrhea. Patients who acquired C. difficile in the community or other hospitals constituted 32% of culture-positive patients and contributed 44% of the REA types. Diversity of C. difficile strains was in part the result of patients acquiring C. difficile in the community or other hospitals. High incidences of nosocomial C. difficile diarrhea do not necessarily indicate clonal epidemics. [ABSTRACT FROM PUBLISHER]

The systemic and mucosal humoral response to toxin A, the primary virulencefactor of Clostridium difficile, was measured in sera and intestinal secretions from 21 patients with C. difficile diarrhea, 9 asymptomatic C. difficile fecal excretors, and 10 noncolonized control subjects. Toxin A-specific IgG was higher in convalescent sera of the patients with diarrhea (mean ± SE, 990 ± 260 ng/rnl.) than in acute sera (620 ± 150 ng/ml.), in sera from asymptomatic excretors (410 ± 140 ng/ml.), or control subjects (320 ± 50 ng/ml.; P < .05 convalescentvs. control). The pattern of toxin A-specific serum IgA and intestinal secretory IgA levelswas similar to that of serum IgG in these groups. Neutralization of toxin A was demonstrated in 5 of 14 convalescent sera but only 1 of 13 acute sera (P = .04). However, the presence of neutralizing activity was independent of the subsequent clinical response. Therefore, most patients convalescent from C. difficile diarrhea demonstrate systemic and mucosal antibodies to toxin A, but these antibodies following natural infection do not appear to alter the clinical course of C. difficile infection. [ABSTRACT FROM PUBLISHER]

The frequency of introduction and spread of specific Clostridium difficile strains among hospitalized patients were assessed by serial cultures of patients admitted to a medical-surgical ward with endemic C. difficile-associated diarrhea. Stool cultures were obtained from 634 (94%) of 678 consecutive admissions to the ward (ward admissions), and all C. difficile isolates were typed by restriction endonuclease analysis. Sixty-five ward admissions introduced C. difficile to the ward, and 54 initially culture-negative admissions acquired C. difficile on the ward. Ward admissions hospitalized within the prior 30 days in the medical center were more likely to be culture-positive for C. difficile at admission to the study ward than those not previously hospitalized at the institution (16% vs. 7%, P < .001). Nosocomial acquisition of a C. difficile strain was preceded by a documented introduction of that strain to the ward by another asymptomatic ward admission in 16 (84%) of 19 instances, suggesting that C. difficile-colonized new admissions are a major source of nosocomial C. difficile infections. [ABSTRACT FROM PUBLISHER]

Mutational changes occurring at amino acid codons 84 and 85 located in the gyrA gene of methicillin-resistant Staphylococcus aureus (MRSA) were studied using radiolabeled oligonucleotide probes to examine the incidence of these ciprofloxacin resistance determinants in 30 MRSA isolates from Indiana, Minnesota, and Tennessee. Four separate oligonucleotide probes, one each corresponding to the wild-type sequence, a mutation at codon 84 (nucleotide 251), a mutation at codon 85 (nucleotide 253), and mutations at both, were used to examine the total genomic DNA from each of the 30 isolates, which had been restricted, electrophoresed, and Southern blotted. The probes indicated that 15 of the 28 ciprofloxacin-resistant isolates gave results consistant with a single mutation at codon 84. Four of the 28 ciprofloxacin-resistant strains had results consistent with a mutation at codon 84 and possibly at codon 85. The two ciprofloxacinsensitive isolates from Tennessee showed homology with the wild-type probe sequence. Five isolates (4, Minnesota; I, Tennessee) had no homology with any probe. By oligonucleotide probes, ciprofloxacin-resistant MRSA from diverse geographic regions contained similar gyrA mutations at codons 84 or 85 in 19 of 28 ciprofloxacin-resistant isolates. [ABSTRACT FROM PUBLISHER]

Bacterial agglutination and polyacrylamide gel electrophoresis (PAGE) were methods evaluated for typing strains of Clostridium difficile. A panel of four antisera, obtained by immunizing rabbits with washed whole cells of different strains of C. difficile, produced distinctive patterns of agglutination. Ethylenediaminetetraacetate (EDTA) extracts subjected to PAGE also produced distinctive protein profiles. Excellent correlation between the two methods was observed when geographically distant isolates were typed without knowledge of their clinical origin. Both typing methods should receive further evaluation for their value as tools for epidemiological studies. [ABSTRACT FROM PUBLISHER]

Rabbit peripheral blood and glycogen-stimulated peritoneal neutrophils were labeled with [111In]indium oxine and transfused intravenously into recipient rabbits with experimental abdominal abscesses due to Staphylococcus aureus. Peritoneal neutrophils harvested 4 hr after glycogen infusion localized within the abscesses to a greater extent than did peripheral blood neutrophils (P < .002). In an in vitro chemotaxis under-agarose assay, peripheral blood neutrophils had greater random migration (P < .002) and directed migration (P < .01) than did peritoneal cells. In an in vitro glass slide adherence assay, peritoneal neutrophils were more adherent than were blood neutrophils (P < .05). The discrepancy between in vivo and in vitro findings may be due to the increased adherence of peritoneal neutrophils. Glycogen-stimulated peritoneal neutrophils have been exposed in vivo to C5a, which is known to decrease migration and increase adherence in vitro of polymorphonuclear neutrophils; consequently, in vivo exposure of neutrophils to C5a may mean in vitro migration data may be misleading in predicting results in vivo. [ABSTRACT FROM PUBLISHER]

The penetration of cephapirin into extravascular spaces of various volumes and ratios of surface area to volume (SA/V) was studied in an in vitro kinetic model. The SA/V ratio was found to be an important determinant of the kinetics of these spaces. The greater the surface area in relation to the volume of the extravascular space, the more closely its kinetics mimicked those of the intravascular space - that is, the higher the absolute peak concentration of drug achieved in the extravascular space, the greater the peak-to-trough fluctuation and the more quickly the peak concentration of drug was reached. Extravascular spaces with similar SA/V ratios demonstrated similar kinetics. Data from various models of extravascular fluid in humans and animals were reviewed, and the kinetics of these extravascular spaces were also found to be determined, at least in part, by the SA/V ratios of the spaces. [ABSTRACT FROM PUBLISHER]

Ascites was induced in dogs by partial ligation of the inferior vena cava. Concentrations of ampicillin, penicillin G, oxacillin, cloxacillin, dicloxacillin, nafcillin, and methicillin in ascitic fluid and serum were each determined in three animals. All antibiotics were administered intramuscularly in a dose of 15 mg/kg (dry weight) for both single-dose and multiple-dose (eight doses at 4-hr intervals) studies. Binding of antibiotics to serum and ascitic fluid proteins was measured by ultracentrifugation. After single doses the highly protein-bound drugs (oxacillin, cloxacillin, dicloxacillin, and nafcillin) had lower percentages of penetration (ratio of peak in ascitic fluid to that in serum, multiplied by 100) than did methicillin, ampicillin, and penicillin G, which have a lower degree of protein binding. The effect was partially overcome by repetitive doses, but five to six doses were usually required to reach equilibrium. In addition to protein binding, serum kinetics (particularly the log mean total concentration of drug in serum after multiple doses) were important determinants of antibiotic concentrations in ascitic fluid. The total antibiotic concentration in ascitic fluid at equilibrium can be accurately calculated from the log mean serum concentration and the percentages of protein binding in serum and ascitic fluid. [ABSTRACT FROM PUBLISHER]

The kinetic interaction of cephalothin and cefamandole with human serum protein and of cephalothin with canine serum protein was studied with use of a rate-of-dialysis method. Binding of antibiotic to serum proteins was complete in <1 min and was immediately reversible. Human serum bound 76.9% of cephalothin and 73.7% of cefamandole at 10 µg/ml, and canine serum bound 41.3% of cephalothin at 10 µg/ml when this technique was used. Quantitative binding determined by ultra-centrifugation was 78.5% for cephalothin and 80.1% for cefamandole in human serum and 45.6% for cephalothin and 32.5% for cefazolin in canine serum. There was no delayed binding of these antibiotics to either human or canine serum. In addition, a large number of antibiotic-binding sites appeared to be present on both human and canine serum protein when the antibiotic concentration was increased to 105 µg/ml. [ABSTRACT FROM PUBLISHER]

A preparative ultracentrifuge method was standardized for determination of quantitative binding of cephalothin, cefamandole, cefazolin, cefaclor, erythromycin, gentamicin, and chloramphenicol to human serum proteins. At achievable in vivo concentrations, serum binding was 78.5% for cephalothin, 79.9% for cefamandole, 88.5% for cefazolin, 23.5% for cefac1or, 41.9% for erythromycin, 22.7% for gentamicin, and 59.5% for chloramphenicol. Techniques that use semipermeable cellophane or diaflow membranes, cross-linked dextran, inhibition of bacterial growth, protein precipitation, or liquid partitioning all have inherent problems with either the ligand or the antibiotic adversely interacting with the experimental apparatus. Ultracentrifugation provides a rapid, reproducible technique for protein-binding determinations of the classes of antibiotics described. [ABSTRACT FROM PUBLISHER]

The aminoglycoside antibiotic tobramycin was given intramuscularly to 15 patients with infected body fluids (empyema in five patients, peritonitis in five, peritonitis and empyema in one, tracheobronchitis in three, and infection of the pacemaker pocket in one). The infecting bacteria included Pseudomonas aeruginosa, Klebsiella, Enterobacter, Serratia, Escherichia coli, Proteus species, and Staphylococcus aureus. The mean dose of tobramycin was 1.7 mg/kg given intramuscularly every 8 hr for nine to 10 days. Levels of tobramycin in specimens of serum and infected body fluid obtained simultaneously were measured at various intervals after a dose of the antibiotic. Comparison was made between levels of tobramycin and minimal inhibitory concentrations (MICs) of the infecting bacteria. Bacteriological and clinical failures were common if the level in body fluid did not exceed the MIG In patients with levels of tobramycin in body fluid that were higher than the MIC, cures were frequent. Drainage of infected body fluids is a necessary part of successful therapy of these infections. [ABSTRACT FROM PUBLISHER]