Interaction between fluconazole and amphotericin B in mice with systemic infection due to fluconazole-susceptible or -resistant strains of Candida albicans.

The interaction between fluconazole (Flu) and amphotericin B (AmB) was evaluated in a murine model of systemic candidiasis for one Flu-susceptible strain (MIC, 0.5 microg/ml), two strains with intermediate Flu resistance (Flu mid-resistant strains) (MIC, 64 and 128 microg/ml), and one highly Flu-resistant strain (MIC, 512 microg/ml) of Candida albicans. Differences in fungal densities in kidneys of infected mice after 24 h of therapy and in survival rates at 62 days of mice treated with an antifungal drug or a combination of antifungal drugs for 4 days were compared. For the Flu-susceptible and Flu mid-resistant strains, the combination of Flu and AmB was antagonistic, as shown by both quantitative culture results and survival. The interaction was additive for the highly Flu-resistant strain. These results suggest that the combination of Flu and AmB should be used with caution in infections due to fungi that are usually susceptible to both antifungal agents and as empirical antifungal drug therapy.

The dnaK/dnaJ operon of Haemophilus ducreyi contains a unique combination of regulatory elements.

Haemophilus ducreyi, which causes the genital ulcer disease chancroid, requires high basal levels of the 60-kDa heat-shock (hs) protein GroEL in order to survive and adhere to host cells in the presence of common environmental stresses. In contrast, the 70-kDa hs protein, DnaK, a negative modulator of the hs response in prokaryotes, is not produced at as high a level as GroEL. Because of these differences, we were interested in identifying regulatory elements affecting the expression of the H. ducreyi dnaK/dnaJ operon. First, the genes encoding H. ducreyi DnaK (Hsp70) and DnaJ (Hsp40) were sequenced. The deduced amino acid sequences shared 82.8 and 63. 9% identity with the Escherichia coli DnaK and DnaJ homologs, respectively. Despite the presence of highly similar (but not identical) hs promoter sequences preceding both the H. ducreyi groES/groEL and dnaK/dnaJ operons, transcription levels for groEL were found to exceed that of dnaK. Subsequently, other genetic elements that could contribute to a lower basal expression of dnaK in H. ducreyi were identified. These elements include: (1) a complex promoter for dnaK consisting of four transcriptional start points (two for sigma32 and two for sigma70) identified by primer extension; (2) a putative binding site for Fur (a transcriptional repressor of iron-regulated genes) that overlaps the initiating AUG of dnaK; and (3) the potential for extensive secondary structure of the long leader sequences of the dnaK transcripts, which could interfere with efficient translation of DnaK. This unique combination of regulatory elements may be responsible for the relatively low-level expression of dnaK in this fastidious genital pathogen.

Comparison of restriction enzyme analysis, arbitrarily primed PCR, and protein profile analysis typing for epidemiologic investigation of an ongoing Clostridium difficile outbreak.

During an outbreak of diarrhea in a general hospital in 1992, 166 Clostridium difficile isolates from 102 patients were typed by restriction enzyme analysis (REA), arbitrarily primed PCR (AP-PCR), and protein profile analysis (PP) techniques. A total of 18 types and 5 subtypes were identified by REA, 32 types were identified by AP-PCR, and 9 types were identified by PP. Analysis of the data indicated the presence of a predominant strain among 76, 75, and 84% of the isolates by REA, AP-PCR, and PP, respectively. Subsequently, 45 C. difficile isolates which had been collected in 1990 from 33 patients in the same hospital following a significant increase in the number of cases of diarrhea caused by C. difficile were studied by REA, AP-PCR, and PP typing techniques. Thirteen types and one subtype were identified by REA, 12 types were identified by AP-PCR, and 5 types were identified by PP. As with the isolates from 1992, a dominant strain was identified. This strain was represented by 53, 64, and 70% of the total number of isolates when the strains were typed by REA, AP-PCR, and PP, respectively. Every isolate (210 of 211) from both 1990 and 1992 that was available for typing was typeable by all three methods. Furthermore, the same dominant strain was identified in both 1990 and 1992 by each method. This study demonstrates that each of the three typing methods can be useful in epidemiologic investigations of C. difficile outbreaks and that one strain can be dominant in an institution over a number of years.

Pharmacodynamics of fluconazole in a murine model of systemic candidiasis.

In this study we defined the pharmacodynamic parameter that optimizes outcome in deep-seated Candida albicans infections treated with fluconazole. Using a murine model of systemic candidiasis, we conducted single-dose dose-ranging studies with fluconazole to determine the dosage of this drug that resulted in a 50% reduction in fungal densities (50% effective dose [ED50]) in kidneys versus the fungal densities in the kidneys of untreated controls. We found that the ED50 of fluconazole given intraperitoneally was 4.56 mg/kg of body weight/day (95% confidence interval, 3.60 to 5.53 mg/kg/day), and the dose-response relationship was best described by an inhibitory sigmoid maximal effect (Emax) curve. To define the pharmacodynamics of fluconazole, we gave dosages lower than, approximating, and higher than the ED50 of fluconazole (range, 3.5 to 5.5 mg/kg/day, equivalent to the ED16 to the ED75) to various groups of infected animals using three dose-fractionation schedules. For each total dose of fluconazole examined, the dose-fractionation schedules optimized the ratio of the area under the concentration-time curve (AUC) to the MIC (the AUC/MIC ratio), the ratio of the maximum concentration of drug in serum (Cmax) to the MIC, and the time that the drug remained above the MIC for the infecting C. albicans isolate. Similar reductions in fungal densities in kidneys were seen between groups that received the same total dose of fluconazole in one, two, or four equally divided doses. Thus, dose-fractionation studies demonstrated that the pharmacodynamic parameter of fluconazole that best predicted outcome was the AUC/MIC ratio.

BOX-polymerase chain reaction-based DNA analysis of nonserotypeable Streptococcus pneumoniae implicated in outbreaks of conjunctivitis.

Nonserotypeable isolates predominate in epidemic conjunctivitis caused by Streptococcus pneumoniae. Previous evaluations of outbreaks of pneumococcal conjunctivitis have relied on epidemiologic factors and the nontypeability of the isolates to infer that a single clone was involved. In the present study, BOX-polymerase chain reaction DNA analysis was used to characterize nonserotypeable S. pneumoniae isolated by conjunctival culture during a recent conjunctivitis outbreak and to compare these isolates with those from outbreaks described earlier. The recent outbreak was caused by a single pneumococcal clone. Outbreaks in separate parts of the United States in 1980-1981 were all caused by the same clone. Cluster analysis revealed a high degree of genetic relatedness among isolates causing conjunctivitis compared with that among other nonserotypeable S. pneumoniae, with the closest relatedness being found among the 1996 and 1980-1981 conjunctival isolates.

Alterations in levels of DnaK and GroEL result in diminished survival and adherence of stressed Haemophilus ducreyi.

Haemophilus ducreyi is a hemin-requiring bacterium causing the genital ulcer disease chancroid. Previously we demonstrated that the heat shock protein GroEL was immunogenic and possibly highly expressed in a mammalian host. The present study was initiated to (i) determine the relative amounts of GroEL expressed by H. ducreyi during in vitro exposure to stresses and (ii) evaluate whether a high level of GroEL is directly or indirectly required for survival and adherence of stressed H. ducreyi. Using scanning densitometry of sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles, we found that H. ducreyi expressed high basal levels of GroEL, averaging fivefold greater than in Escherichia coli. These high GroEL levels increased up to twofold upon exposure of the organism to heat shock or high levels of hydrogen peroxide and during adherence to two human genital cell lines. Furthermore, when the gene for DnaK was present on a multicopy plasmid in H. ducreyi, a 1.8-fold increase in DnaK and a 2.3-fold reduction in GroEL were seen. These results suggest that DnaK serves as a negative modulator of H. ducreyi GroEL. Subsequently we found that H. ducreyi with lower GroEL had diminished ability to survive when challenged by heat and oxidative stresses. In addition, the long, parallel chains characteristic of virulent strains of H. ducreyi were absent when GroEL was lowered, so that fewer bacterial cells adhered to the human cells. These results suggest that the unusually high basal levels of GroEL are involved, either directly or indirectly, in the survival, chaining, and adherence of H. ducreyi in the presence of the combined stresses of the host environment.

Laboratory diagnosis of chancroid using species-specific primers from Haemophilus ducreyi groEL and the polymerase chain reaction.

To enhance laboratory identification of Haemophilus ducreyi, the causative agent of the genital ulcer disease chancroid, a polymerase chain reaction (PCR) assay was developed using target DNA sequences from the essential H. ducreyi gene, groEL. Positive reactions were obtained in this PCR assay with 139 isolates of H. ducreyi from patients in worldwide locations from the 1940s to the 1990s. In contrast, 24 other bacterial species were negative. When genital ulcer specimens from 162 African patients with clinically diagnosed chancroid were evaluated, 66 were culture positive. The sensitivity of PCR as compared with culture was 89% (59 of 66), and specificity was 79% (76 of 96). However, representative samples of the 20 culture-negative, PCR-positive specimens were confirmed as positive by a second PCR assay using different H. ducreyi-specific primers. Thus, combined results of culture and PCR detected H. ducreyi in 86 specimens, with resolved sensitivities of 92% (79 of 86) for PCR, and 77% (66 of 86) for culture. These results suggest that PCR assays for H. ducreyi have great potential for augmenting or replacing problematic cultural techniques.

Typhoid fever at a resort hotel in New York: a large outbreak with an unusual vehicle.

The largest outbreak of typhoid fever in the United States since 1981 occurred in 1989 among guests and staff at a New York hotel. There were 43 culture-confirmed and 24 probable cases among guests, 1 culture-confirmed case and 1 asymptomatic culture-positive case among hotel employees, and 1 culture-confirmed secondary case. Twenty-one persons were hospitalized and 2 had bowel perforation. Breakfast on 13 June was the only meal consumed by all ill persons (relative risk, infinite; P = .004). In a case-control study, case-patients were more likely than controls to have consumed orange juice (odds ratio, 5.6; 95% confidence interval, 1.1-54.7), which had been prepared in a 208-L container with ample opportunity for hand contact. No other food was associated with illness. S. typhi was isolated from the stool of an asymptomatic food worker who handled orange juice but who was not known to be a typhoid carrier. S. typhi is a foodborne pathogen with continuing potential to cause large outbreaks in the United States.

Molecular analysis of the Haemophilus ducreyi groE heat shock operon.

Chancroid is a sexually transmitted genital ulcer disease caused by Haemophilus ducreyi. Previously, we developed diagnostic DNA probes for H. ducreyi (L. M. Parsons, M. Shayegani, A. L. Waring, and L. H. Bopp, J. Clin. Microbiol. 27:1441-1445, 1989). In the present study, DNA sequencing of one of the diagnostic probes revealed two adjacent open reading frames (ORFs). These H. ducreyi ORFs and the encoded proteins show significant homology with the groE genes and GroES and GroEL heat shock proteins from several bacterial pathogens and with conserved eukaryotic 60-kDa heat shock proteins. The first H. ducreyi ORF (groES) is preceded by sequences similar to those of the Escherichia coli consensus heat shock promoters and is 288 nucleotides long and is capable of encoding a protein of 10.3 kDa. The second ORF (groEL) is 1,641 nucleotides long and is capable of encoding a protein of 57.8 kDa. Northern (RNA blot) analysis demonstrated the presence of a high level of groE mRNA in exponential-phase H. ducreyi grown in hemin broth at the organism's optimal growth temperature (33 degrees C), with increased levels seen following heat shock. Heat shock also increased the thermostability of the organisms, since stressed cells were more resistant to the lethal effects of rapid chilling. Electrophoretic analysis and immunoblots demonstrated that the predominant protein produced by exponential-phase H. ducreyi was a heat-inducible, immunoreactive protein of approximately 60 kDa (GroEL). Also, H. ducreyi groE mRNA and GroEL were expressed and inducible by heat in E. coli. This is the first report describing the cloning, sequencing, and expression of H. ducreyi protein-encoding genes.

Molecular relatedness of Staphylococcus epidermidis isolates obtained during a platelet transfusion-associated episode of sepsis.

Staphylococcus epidermidis was isolated from the blood of a 25-year-old pregnant woman following the administration of eight units of platelets. She had developed chills and a fever of 41.4 degrees C soon after the transfusions were completed. S. epidermidis was also obtained from all eight platelet units, as well as from the packed-erythrocyte unit associated with the first unit of platelets. The isolation of the same organism from these epidemiologically related sources provided us with the opportunity to phenotypically and genetically characterize the isolates. Several typing methods, including four molecular techniques, were used to increase our chances of finding any differences between the isolates under investigation. Phenotypic analyses demonstrated that S. epidermidis isolates from the patient, platelet units, and erythrocyte unit reacted in exactly the same manner in 15 biochemical tests, exhibited slime production, and had the same antibiotic susceptibility pattern. Genetic analyses, which included plasmid profiles, plasmid cross-hybridization, field inversion gel electrophoresis, and ribotyping, substantiated the relationships between the S. epidermidis isolates from the patient, platelet units, and erythrocyte unit. Eight S. epidermidis control strains unrelated to the case were found to differ significantly from the platelet-related strain.

Garlic-in-oil associated botulism: episode leads to product modification.

In February 1989, three cases of botulism occurred in persons who consumed garlic bread made from a garlic-in-oil product. Testing of leftover garlic-in-oil showed it to have a pH of 5.7 and to contain high concentrations of Clostridium botulinum organisms and toxin. This was the second episode of botulism associated with a low acid garlic-in-oil product which needs constant refrigeration. In response, the Food and Drug Administration has taken steps to prevent a recurrence by requiring that microbial inhibitors or acidifying agents be added to such products.

DNA probes for the identification of Haemophilus ducreyi.

Haemophilus ducreyi ATCC 33922, a virulent, well-characterized strain, was used to construct a genomic library in a bacteriophage expression vector. Three DNA fragments were selected for use as probes on the basis of their ability to encode H. ducreyi-specific proteins, as demonstrated by reactivity with rabbit polyclonal antiserum. With DNA-DNA hybridization, the three probes, labeled with 32P, reacted strongly with 16 strains of H. ducreyi obtained from a variety of sources. Thirty-seven other bacterial isolates, representing 33 different species and including organisms likely to be encountered in the urogenital tract, were also tested with the three probes. Twenty-eight of these isolates, including the genital pathogen Neisseria gonorrhoeae, showed no hybridization with the probes. In addition, herpes simplex virus-infected tissue culture cells and Treponema pallidum-infected rabbit testicular fluid were also completely nonreactive. Nine isolates, six belonging to other Haemophilus species and three belonging to Pasteurella species, reacted weakly with the probes when approximately 3.0 x 10(7) to 6.0 x 10(7) CFU was tested. When 10(5) to 10(6) CFU of these organisms was tested, the weak reactions could no longer be seen. Yet this number of H. ducreyi still reacted strongly. In fact, the three probes consistently detected 10(4) CFU of H. ducreyi in pure and mixed cultures and even produced a weak signal when only 10(3) CFU was present. It is clear from our results that use of these probes will greatly facilitate the laboratory diagnosis of this genital pathogen.

Cluster of Haemophilus influenzae type b infections in adults.

Haemophilus influenzae type b commonly causes illness in young children, among whom transmission is known to occur. Most adults are believed to be immune to H influenzae type b and outbreaks of disease among adults appear to be uncommon. From July 14 to Aug 12, 1985, a cluster of six cases of acute febrile illness with cultures positive for H influenzae, biotype II (five cases) or untyped H influenzae (one case), occurred among adults in a nursing home and an adjoining hospital. All six case-patients had personal contact with at least one other case-patient. Among the 46 nursing home residents, men were more likely than women to become ill (44% vs 0%). This cluster of disease suggests that elderly adults may be more susceptible to H influenzae infection than is generally recognized and that outbreaks among adults may result from person-to-person transmission.

Bacteremia in patients undergoing oral procedures. Study following parenteral antimicrobial prophylaxis as recommended by the American Heart Association, 1977.

The type and rate of bacteremia following dental extractions, dental cleaning, or other dental/oral surgical procedures were studied in 124 patients with valvular heart disease following parenteral antibiotic prophylaxis (penicillin G potassium with or without streptomycin sulfate, or vancomycin hydrochloride) as recommended by the American Heart Association in 1977. Generally, under penicillin G prophylaxis with or without streptomycin, detection of bacteremia in blood culture media containing no penicillinase was low (14.7% to 16.1% at five minutes and 3.1% to 9.0% at 30 minutes after the procedure). The number and types of organisms recovered from patients who received penicillin prophylaxis alone or with streptomycin were similar. Anaerobes were recovered twice as frequently as aerobes. Polymicrobial bacteremia was rare and only one patient had streptococci detected in the blood culture. Addition of penicillinase to one blood culture medium, however, and comparing it with a similar medium without penicillinase was accompanied with a sixfold greater recovery from patients of both aerobic and anaerobic bacteria, including six patients with streptococcal bacteremia. Vancomycin prophylaxis was accompanied with bacteremia in only one patient.

Comparative in-vitro activities of A-56268 (TE-031) and erythromycin against 306 clinical isolates.

The inhibitory (MIC) and bactericidal (MBC) activities of a new macrolide A-56268 (TE-031) against 306 clinical aerobic bacterial isolates was compared with that of erythromycin. The MIC90/MBC90 ratios for A-56268 were: Campylobacter jejuni 4/16, Haemophilus influenzae 8/8-16, H. parainfluenzae 8/8-16, Legionella pneumophila 0.06/0.5, methicillin-sensitive isolates of Staphylococcus aureus 0.5/1, and coagulase negative staphylococci 1/8, methicillin resistant isolates of Staph. aureus and coagulase negative staphylococci greater than 16/ greater than 16, Streptococcus pneumoniae 0.06/0.125, streptococcus Group A 0.06/2-4, streptococcus Group B 0.06/8- greater than 16, streptococcus Groups C and G 0.125/8 and Str. faecalis 4/64. Compared with erythromycin, A-56268 had greater inhibitory and bactericidal activity against isolates of L. pneumophila, with an MIC90 16-fold less and an MBC90 eight-fold less than that of erythromycin. Except for enterococci, A-56268 showed inhibitory activity equal to or greater than that of penicillin G against isolates of streptococci and an MIC two-fold less than that of erythromycin. For other strains tested, the inhibitory and bactericidal activities of A-56268 and erythromycin were similar. The clinical importance of the differences between these two macrolides will depend on the pharmacokinetic and tissue penetration properties of the new compound.

A community hospital outbreak of legionellosis. Transmission by potable hot water.

Seven cases of nosocomial legionellosis occurred between February and September 1982 in a small community hospital in Upstate New York. All seven were cases of Legionella pneumophila serogroup 1; six were hospital patients and one a hospital employee. None of the cases died. During the peak of the outbreak, the incidence of nosocomial legionellosis was 1.2 cases per 100 patient discharges. An epidemiologic comparison of the six patient cases with 21 matched patient controls suggested that longer hospital stay (chi 1(2) = 24.2, p less than 0.001) and the proximity of patients' rooms to ward showers (chi 1(2) = 4.4, p less than 0.04) were significant risk factors for acquiring legionellosis. An environmental investigation demonstrated that the ward showers and the hospital hot water system were contaminated with L. pneumophila serogroup 1. Monoclonal antibody subtyping performed on isolates obtained during the outbreak investigation confirmed that the hot water system and patient isolates had an identical pattern of reactivity. The outbreak demonstrates that legionellosis can be a significant cause of nosocomial pneumonia in a community hospital and that transmission can occur from contaminated potable hot water sources, potentially via shower aerosols.

Serotyping of 1,458 pneumococcal isolates with analysis of bacteremia in 84 patients.

During a 61-month period, 1,458 pneumococcal isolates (including 87 bacteremia strains) were collected at the Albany Veterans Administration Medical Center and serotyped with the use of the typing system of the New York Department of Health Laboratories. Fifty percent of all isolates were of types in the 14-valent vaccine, while 68% were in 23-valent vaccine. Types 3, 6, 9, and 34 were the most prevalent types. Sixty-two percent of blood isolates were of types in the 14-valent vaccine, while 77% were in the 23-valent vaccine. Fifty-six percent of bacteremias were nosocomial and 44% were community acquired with a fatality rate of 41% and 18%, respectively. The fatality rate was greatest with risk factors such as asplenia and azotemia. These data support the recommendation for the use of 23-valent pneumococcal polysaccharide vaccine in high-risk patients, especially those who might acquire bacteremia in a nosocomial setting.