Comparison of arbitrarily primed PCR with restriction endonuclease and immunoblot analyses for typing Clostridium difficile isolates.

Arbitrarily primed PCR (AP-PCR) was used to genotype 26 clinical isolates of Clostridium difficile previously analyzed by immunoblotting (IB) and 20 isolates typed by restriction endonuclease analysis (REA) with HindIII. Two levels of differentiation were achieved with the AP-PCR approach by use of two different arbitrary primers. With the 19-mer arbitrary primer T-7 (first level of differentiation), a good correlation was found between IB and AP-PCR typing. Twenty isolates grouped into six IB types were separated into seven major AP-PCR types. These seven AP-PCR groups were further discriminated into 12 subtypes after genotyping with the arbitrary primer PG-05 (second level of differentiation). The remaining six isolates, all of different IB types, showed a unique and distinct DNA banding pattern with both of the arbitrary primers, T-7 and PG-05. Twenty isolates representing 20 REA types from 15 REA groups were resolved into 13 AP-PCR DNA profiles with the arbitrary primer T-7. A good correlation was found at this level of differentiation between the major REA groups, Y and M, and AP-PCR typing. While AP-PCR with this primer failed to differentiate isolates in REA groups J, G, R, and B, AP-PCR with PG-05 resolved these four isolates into four distinct AP-PCR types. In addition, one of three M strains and one of four Y strains displayed a slightly different DNA banding pattern by AP-PCR (with PG-05) from that of the other strains in the group. We conclude that AP-PCR is a rapid and sensitive method which not only complements other typing schemes but also may be a substitute and prove to be especially suited for immediate epidemiological tracking of nosocomial infections due to C. difficile.

Safety of parenteral third-generation cephalosporins.

Knowledge of side effects associated with different cephalosporins may be of help to prescribers. There are several side effects that are common to all cephalosporins, but overall, cefotaxime and ceftizoxime cause the fewest adverse reactions. Bleeding is probably the most common serious side effect of cephalosporins. Moxalactam causes coagulopathy and bleeding more often than do other cephalosporins, probably because it is carboxylated and has a methylthiotetrazole side chain. Cefoperazone also has a methylthiotetrazole side chain and may cause bleeding, particularly when used in doses greater than 4 g per day. Ceftriaxone has a similar side chain and there is some evidence that it can induce a coagulopathy. Coagulation tests should be monitored when any of the third-generation cephalosporins are given to patients with a high risk of bleeding. Disulfiram-like reactions are also related to the side chains associated with coagulation defects and have been reported when patients receiving cefoperazone, moxalactam, or ceftriaxone have ingested alcohol. Seizures have been reported with ceftazidime, but are uncommon. Hematologic reactions are rare with all third-generation cephalosporins. Benign diarrhea and Clostridium difficile colitis probably occur most often with moxalactam, cefoperazone, ceftazidime, and ceftriaxone, but there are few good data on this issue. Ceftriaxone has the unique ability to cause sludge (also referred to as pseudolithiasis) to form in the gallbladder, particularly in children. This may be associated with nausea, anorexia, epigastric distress, and colic, and is usually detected using ultrasonography. The sludge dissolves and symptoms subside after therapy is discontinued. None of the third-generation cephalosporins is clearly significantly nephrotoxic, even when combined with aminoglycosides. Most of the third-generation cephalosporins have surprisingly few serious side effects, which make them attractive for use in the treatment of a wide variety of serious infections.

Retrospective evaluation of piperacillin use in a university hospital.

A retrospective review was conducted to evaluate the appropriateness of empiric and definitive piperacillin use and to determine if less expensive, more appropriate antibiotics could have been used. The criteria were approved by the chief of infectious diseases and the drug utilization review committee at this institution. One hundred courses of piperacillin use in adult patients were reviewed. Therapy was categorized as appropriate in 78 of the 100 courses: 21 were appropriate empiric, 32 were appropriate empiric changed to definitive due to culture and sensitivity reports, and 25 were appropriate definitive. Reasons for this high percentage of appropriate use include: 1) 40% of the infections involved Pseudomonas aeruginosa; 2) 18% of the organisms that were sensitive to piperacillin were resistant to ticarcillin and mezlocillin; and 3) 64% of the courses of therapy involved critically ill patients with diagnoses such as neutropenia secondary to cancer chemotherapy, burns, sepsis, and hospital-acquired pneumonia. Although only 19.7% of the 11,845 grams of piperacillin used were categorized as inappropriate, the cost is relatively high (annualized to $19,648) and cost savings could be realized if piperacillin use were monitored more closely.

Epidemiological aspects of Clostridium difficile in a pediatric hospital and its role in diarrheal disease.

The influence of antibiotics on the frequency of colonization by Clostridium difficile and the presence of its cytotoxin in infants and older children was examined to determine its role in diarrheal disease. Cytotoxin was more closely associated with cases of diarrhea, both in infants and in children than the microorganism, although not significantly. The isolates were typed by means of sensitivity to bacteriophages and bacteriocins and their cytotoxigenic potential was also determined. Less than 30% of the colonized patients had toxigenic strains. A study of strain variability over a four-year period in the same hospital showed that two bacteriophage-bacteriocin types and non-toxigenic strains predominated. The common presence of non-toxigenic strains could explain in part the lack of correlation between isolation of Clostridium difficile and diarrhea. Most of the non-toxigenic strains showed moderate resistance to tetracycline, a property which might explain their ability to persist for long periods in the hospital.

Nosocomial Clostridium difficile reservoir in a neonatal intensive care unit.

A new bacteriophage/bacteriocin typing system was used to study Clostridium difficile colonization in a neonatal intensive care unit. C. difficile was isolated from 21 of 62 (34%) stools from 15 of 37 (41%) infants. Colonization was reduced during antimicrobial therapy and for about 1 week thereafter. One of five nurses and one of two parents studied were carriers. Eight isolates were cultured from environmental surfaces. Thirty of 31 C. difficile isolates were found to be a single type, Cld 6,9,10,13; bacteriocin 1320,1537,2304. No C. difficile was found in 29 specimens obtained in the delivery room from mothers and infants, and there was no association of early colonization with vaginal delivery. The data provide strong evidence for nosocomial acquisition of C. difficile by infants in the neonatal intensive care unit. No obvious pathologic role for C. difficile could be identified among colonized infants. Among 22 C. difficile isolates from 7 adult inpatients with diarrhea and 13 healthy infants attending the center's well baby clinic, 4 were the same type as the strain found in the intensive care nursery. Only one of these patients had had direct contact with the neonatal intensive care unit, indicating that the nursery strain may also be found elsewhere in the community.

Bacteriophage and bacteriocin typing scheme for Clostridium difficile.

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.

Fluorescent-antibody test for detection of Clostridium difficile in stool specimens.

We evaluated a direct fluorescent-antibody test to detect Clostridium difficile, the most frequent cause of antibiotic-associated colitis. C. difficile organisms were injected into the ear veins of New Zealand White rabbits to induce antibodies, and the globulin fractions of their sera were conjugated to fluorescein isothiocyanate. The resulting conjugate strongly stained all 40 isolates of C. difficile tested. It also stained isolates of C. sordellii, C. bifermentans, C. chauvoei, and C. sporogenes, but not 20 other clostridial isolates or 10 isolates from other species. Results of testing fecal smears with the direct fluorescent-antibody method were compared with results of testing stools for C. difficile toxin and of culturing for C. difficile on a selective medium. A total of 158 fecal specimens from patients with antibiotic-associated diarrhea were tested. In these patients, the fluorescent-antibody test agreed with culture and toxin testing in 93% of the specimens. However, in normal adults, 62% of the fecal specimens from which C. difficile could not be cultured were positive by the fluorescent-antibody test. Absorption of the conjugate with C. sordellii led to a loss of reactivity to other clostridia as well as to 18 of 20 isolates of C. difficile.

Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile.

Isolation of Clostridium difficile from fecal specimens has been facilitated by the development of a selective and differential medium, cefoxitin-cycloserinefructose agar (CCFA). We substituted 0.1% sodium taurocholate for the 2.5% egg yolk in CCFA and compared the growth of 15 isolates of C. difficile on the resulting medium with growth on conventional CCFA. The taurocholate-containing medium (TCCFA) quantitatively recovered vegetative forms of C. difficile in the same numbers as CCFA medium. Recovery of spores was a mean 1.7 log(10) higher on TCCFA than on CCFA. Thirty-six of 60 patient stool specimens growing C. difficile gave a heavier growth on TCCFA than on CCFA, and 9 failed to yield C. difficile on CCFA. TCCFA detected spores of 75 colony-forming units per ml from artificially inoculated fecal specimens when conventional stool culturing techniques were used. Fluorescence of colonies of C. difficile was more intense on TCCFA than on CCFA. TCCFA was simpler to prepare and, overall, was more sensitive than CCFA.

Suppression of Clostridium difficile by normal hamster cecal flora and prevention of antibiotic-associated cecitis.

Administration of normal cecal homogenates decreased numbers of viable Clostridium difficile and prevented cecitis in antibiotic-challenged hamsters. Cecal anaerobes appeared to suppress C. difficile.

Nosocomial outbreak of Candida parapsilosis fungemia related to intravenous infusions.

Candida parapsilosis is rarely isolated from blood cultures. Our hospital surveillance detected an increased rate of isolation of C parapsilosis during a four month period. Fourteen postoperative patients receiving intravenous (IV) hyperalimentation and eight burn patients receiving IV albumin were involved. Hectic fever, the major clinical manifestation, was seen in 61% of cases. Therapy in the postoperative patients consisted merely of discontinuing IV catheters and hyperalimentation, while amphotericin B was needed in five of eight burn patients to control persistent fungemia. Epidemiologic analysis identified a source of the organism in the IV-additive preparation room where C parapsilosis was found contaminating a vacuum system. Organisms apparently refluxed into IV bottles when aliquots were removed to accommodate additives. Of 103 patients who received fluids prepared with the contaminated system, 21% became infected with C parapsilosis. Infection surveillance was instrumental in detection and control of the outbreak. Routine guideline should be established to insure the sterility of IV fluids containing additives.

Antibiotic-induced colitis implication of a toxin neutralised by Clostridium sordellii antitoxin.

A toxin(s) has been demonstrated in the stools of two patients with antibiotic-associated colitis. This toxin(s) was heat-labile, was rapidly lethal for hamsters, increased vascular permeability in rabbit skin, and was cytotoxic for cells in tissue-culture. It was neutralised by Clostridium sordellii antitoxin but not by antitoxins prepared against other clostridia; Escherichia coli, and Vibrio cholerae toxins. These characteristics were identical to those of a toxin implicated in the aetiology of antibiotic-induced colitis in the hamster. One patient improved rapidly after treatment with oral vancomycin, and at the same time the toxin disappeared from the stool.

Experimental endocarditis due to Pseudomonas aeruginosa. II. Therapy with carbenicillin and gentamicin.

Rabbits with left-sided endocarditis due to Pseudomonas aeruginosa were treated with a high dose (7.5 mg/kg) of gentamicin for six days, a low dose (5 mg/kg) of gentamicin for six days or two weeks, carbenicillin (400 mg/kg) for six days or two weeks, or a combination of the lower dose of gentamicin and carbenicillin (400 mg/kg) for six days or two weeks. Sterilization of cardiac vegetations was accomplished more effectively in rabbits given the higher dose of gentamicin for six days (44% with sterilized vegetations) or combination therapy for 14 days (64%) than in rabbits given the lower dose of gentamicin or carbenicillin alone for two weeks (23.5% and none, respectively). Combination therapy for two weeks prevented relapse in all of six treated rabbits that were followed after treatment; in contrast, all four animals treated with carbenicillin and four of six animals treated with the lower dose of gentamicin relapsed after two weeks of treatment. Levels of creatinine in serum became elevated in four of nine rabbits that received gentamicin along for two weeks. Therapeutic success was associated with a synergistic antibiotic combination, a peak bactericidal titer in serum of greater than or equal to 1:16, and a high level of gentamicin in serum.

Gastrointestinal side effects of clindamycin and ampicillin therapy.

Hospitalized patients who received clindamycin or ampicillin were evaluated for gastrointestinal side effects for a period of up to six weeks after therapy was discontinued. Of 104 patients receiving clindamycin therapy, 31 (29.8%) developed diarrhea, and two (1.9%) developed pseudomembranous colitis (PMC). Of 138 patients receiving ampicillin, 24 (17.3%) developed diarrhea, and one (0.7%) developed PMC. Diarrhea persisting for three days or more was noted in 13 (12.5%) of the patients receiving clindamycin and in seven (5.1%) of those receiving ampicillin. The tendency to develop diarrhea was positively correlated with serious illness, abdominal or pelvic sepsis, and total dosage of clindamycin. Examination of stools from a patient with PMC that was associated with clindamycin therapy showed a decrease in the number of anaerobic bacteria from the numbers found in stool cultures of normal controls. Those patients who did not develop diarrhea also had fewer anaerobic bacteria and coliform organisms. Lymphocytes from the patient with PMC were hyporeactive to phytohemagglutinin and hyperreactive to clindamycin.

Experimental endocarditis due to Pseudomonas aeruginosa. I. Description of a model.

Rabbits with sterile, right ventricular cardiac vegetationss were challenged with Staphylococcus aureus and serum-susceptible or -resistant isolates of Pseudomonas aeruginosa. None of the rabbits challenged with serum-susceptible P. aeruginosa died or had greater than 10(2) colony-forming units (cfu)/g cultured from vegetations two weeks later. In contrast, 78% of animals challenged with serum-resistant P. aeruginosa died within three weeks, and 74% sacrificed at three days had organisms cultured from vegetations. All of the animals challenged with S. aureus died, and all had greater than 10(8) cfu of P. aeruginosa/g in vegetation tissue at three days. There was a significantly greater number of organisms (P less than 0.001) in the vegetations of animals dying of S. aureus infection than in those of animals with P. aeruginosa endocarditis (mean, 10(9.6) vs. 10(7.5) cfu/g, respectively). Left-sided endocarditis was produced in 100% of rabbits injected with serum-resistant P. aeruginosa. These models could be used for studies of the pathogenesis and therapy of P. aeruginosa endocarditis.

Factors affecting the frequency infection in renal transplant recipients.

A retrospective review of 149 patients receiving 162 renal transplants showed that 83% of these patients developed one or more infections during a follow-up period averaging one year. In 32 (73%) of 44 deaths, infection was an important contributing cause. In only four (9%) of the deaths were the patients free of infection at the time of death. The Klebsiella-Enterobacter group was the most common agent causing pneumonitis and sepsis. Cryptococcus neoformans caused seven of 11 cases of meningitis. Pseudomonas was the most frequent agent associated with infections documented during postmortem examinations. In a short-term controlled study comparing daily and alternate daily therapy with prednisone, the alternate daily group had significantly (P less than .05) more infections per patient, especially in patients who had no evidence of rejection (P less than .025).

Therapy of Pseudomonas aeruginosa infections with tobramycin.

The efficacy of tobramycin in doses of 2.7 to 5.6 mg/kg per day in 29 courses of therapy in 25 hospitalized patients with serious Pseudomonas aeruginosa infections was studied. Eighty-three percent of the P. aeruginosa strains showed zones of inhibition of 16 mm or more around a 10-mug tobramycin disk in the Bauer-Kirby disk method. Tobramycin minimal inhibitory concentration ranged from <0.05 to 1.5 mug/ml (microtiter twofold dilution method); for gentamicin they ranged from 0.05 to 6.2 mug/ml; corresponding geometric means were 0.19 and 0.49 mug/ml. Therapy was given for a median of 10 days (mean 19, range 1 to 83). The clinically satisfactory response rate for the 29 courses of therapy was 52%: critically ill, 44%; seriously ill, 50%; moderately ill, 80%. The response rates for various sites of infection were bone and cartilage, 100%; urinary tract infection, 56%; wound, 50%; respiratory tract, 67%; septicemia, 40%; abscess, 0%; burns, 44%. No adverse reactions were seen. Serum concentration (mug/ml +/- standard deviation) of tobramycin determined by an agar-well plate method, were 4.81 +/- 2.17 (1 h); 3.24 +/- 1.43 (2 h); 2.35 +/- 1.30 (4 h); and 1.40 +/- 1.09 (8 h). Tobramycin appears to be as effacacious as gentamicin in the treatment of serious P. aeruginosa infections and has a theoretical advantage of lower minimal inhibitory concentration for P. aeruginosa. The data suggest that, for life-threatening infections, dosages of tobramycin may need to be increased over those used in this study.