William J. Ritz

Inhibitory and bactericidal activities of levofloxacin, ofloxacin, erythromycin, and rifampin used singly and in combination against Legionella pneumophila

The susceptibilities of 56 Legionella pneumophila isolates (43 clinical and 15 environmental isolates) to levofloxacin, ofloxacin, erythromycin, and rifampin were studied with buffered charcoal yeast extract (BCYE) agar (inoculum, 10(4) CFU per spot), and the susceptibilities of five isolates were studied with buffered yeast extract (BYE) broth (inoculum, 10(5) CFU/ml). The MICs inhibiting 90% of strains tested on BCYE agar were 0.125, 0.25, 1.0, and < or = 0.004 micrograms/ml for levofloxacin, ofloxacin, erythromycin, and rifampin, respectively. The MICs by the BYE broth dilution method were 1 to 3, 2, 1 to 2, and 1 tube lower than those by the agar dilution method for levofloxacin, ofloxacin, erythromycin, and rifampin, respectively. The MBCs were 1 to 2 tubes higher than the broth dilution MICs for levofloxacin, 1 to 3 tubes higher than the broth dilution MICs for ofloxacin, 1 to 3 tubes higher than the broth dilution MICs for erythromycin, and the same as the broth dilution MICs for rifampin. In kinetic time-kill curve studies, at drug concentrations of 1.0 and 2.0 times the MIC, the most active drugs were levofloxacin and rifampin. At 72 h, concentrations of levofloxacin and rifampin of 2.0 times the MIC demonstrated a bactericidal effect against L. pneumophila. In contrast, at concentrations of 1.0 and 2.0 times the MICs regrowth was observed with ofloxacin and only a gradual decrease in the numbers of CFU per milliliter was observed with erythromycin. Only a minor inhibitory effect was observed with 0.25 or 0.5 time the MICs of all drugs at 24 to 48 h, with regrowth occurring at 72 h. In contrast to erythromycin or ofloxacin plus rifampin at 0.25 time the MICs, only levofloxacin plus rifampin demonstrated synergy. Thus, levofloxacin demonstrated the best inhibitory and bactericidal effects against L. pneumophila when it was studied alone or in a combination with rifampin.

Activities of Daptomycin and Comparative Antimicrobials, Singly and in Combination, against Extracellular and Intracellular Staphylococcus aureus and Its Stable Small-Colony Variant in Human Monocyte-Derived Macrophages and in Broth

ABSTRACT We investigated the antistaphylococcal activities of daptomycin, gentamicin, and rifampin against two Staphylococcus aureus strains and their stable small-colony variants, singly and in combination, in human monocyte-derived macrophages and in broth. Intracellularly, the three-drug combination and two-drug combinations with rifampin were most effective. Extracellularly, daptomycin, daptomycin plus gentamicin, gentamicin plus rifampin, and the three-drug combination had similar activities.

Antimicrobial Activities of Daptomycin, Vancomycin, and Oxacillin in Human Monocytes and of Daptomycin in Combination with Gentamicin and/or Rifampin in Human Monocytes and in Broth against Staphylococcus aureus

ABSTRACT We investigated the antistaphylococcal activity of daptomycin, vancomycin, oxacillin, gentamicin, and rifampin in human monocyte-derived macrophages. Compared with vancomycin and oxacillin, daptomycin had the most rapid and greatest antibacterial activity, but that of oxacillin was most sustained. The combination of daptomycin, gentamicin, and rifampin was most effective intracellularly, while daptomycin plus gentamicin and the three-drug combination were most effective extracellularly, completely eliminating viable Staphylococcus aureus.

Comparison of in vitro Inhibitory and Bactericidal Activities of Daptomycin (LY 146032) and Four Reference Antibiotics, Singly and in Combination, against Gentamicin-Susceptible and High-Level-Gentamicin-Resistant Enterococci

The in vitro inhibitory and bactericidal activities of daptomycin and reference antibiotics were determined by serial macrobroth dilution for 23 gentamicin-susceptible (MIC: < 2,000 mg/l) and 21 high-level-gentamicin-resistant (HLGR) Enterococcus faecalis, Enterococcus faecium, and Enterococcus avium isolates. The activities of daptomycin, vancomycin, and teicoplanin were independent of the gentamicin susceptibility profile and species tested. For all the isolates, the inhibitory activity of daptomycin (MIC90: 2 mg/l) was comparable to vancomycin (MIC90: 2 mg/l), but less than that of teicoplanin (MIC90: 0.5 mg/l). Daptomycin demonstrated excellent bactericidal activity against all enterococci tested (MBC90: 8 mg/l). In contrast, all microorganisms were tolerant to vancomycin and teicoplanin. Ampicillin and ciprofloxacin MICs and MBCs were dependent on enterococcal gentamicin resistance profile and species, with MICs and MBCs that ranged between 1 and > 64 mg/l. By time-kill curves, the combination of daptomycin plus ampicillin demonstrated synergistic bactericidal activity against gentamicin-susceptible and HLGR E. faecalis. Daptomycin, singly and in combination, may be useful in treating enterococcal infections, including those caused by HLGR microorganisms.

Vancomycin-Resistant Enterococci in Stool Specimens Submitted for Clostridium difficile Cytotoxin Assay

The prevalence of, and clinical risk factors associated with, vancomycin-resistant enterococcal colonization were investigated in patients suspected of having Clostridium difficile infection. Stools submitted for C difficile cytotoxin testing were screened for vancomycin-resistant enterococci (VRE). Isolates were speciated and characterized further by antibiotic susceptibility testing, DNA fingerprinting, and DNA:DNA hybridization for detection of specific vancomycin resistance genes. Of the 79 evaluable patients identified during a 3-month period, 16.5% were VRE-positive. The VRE isolates were genetically heterogeneous, although all carried the vanA gene. DNA fingerprinting data suggest that patient-to-patient transmission occurred, implicating colonized patients as potential reservoirs for VRE transmission. A positive C difficile cytotoxin assay and diabetes mellitus were the only identifiable risk factors associated with VRE colonization. Patients at risk for C difficile infection therefore may serve as reservoirs for VRE.

Effects of Cytokines and Fluconazole on the Activity of Human Monocytes against Candida albicans

ABSTRACT This study evaluates the effects of cytokines, used singly and in combination, on the microbicidal activity of human monocyte-derived macrophages (MDM) against intracellular Candida albicans in the presence and absence of fluconazole. In the absence of fluconazole, the addition of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), gamma interferon (IFN-γ), or IL-4 had no effect on the growth of C. albicans. In contrast, the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in decreased growth (P < 0.05), while the addition of IL-10 resulted in increased growth (P < 0.01). In the presence of fluconazole, only the addition of IFN-γ resulted in an increase in the growth of C. albicans. In the presence or absence of fluconazole, all cytokine combinations except IFN-γ plus GM-CSF caused significant decreases in growth (P < 0.01). IL-10 and IL-4 did not influence the activity of TNF-α or IL-1β. In the absence or presence of C. albicans the addition of fluconazole, all of the cytokines studied, and combinations of fluconazole and selected cytokines caused increases in nitric oxide (NO) production (P < 0.01). Similar observations were made for superoxide (O2−) only in the presence of C. albicans. The greatest concentrations of NO and O2− were produced when C. albicansalone was present in the assays. Our results demonstrate that in the presence of low concentrations of fluconazole (0.1 times the MIC), selected cytokines and their combinations significantly increase the microbicidal activity of MDM against intracellular C. albicans.

Anticandidal effects of voriconazole and caspofungin, singly and in combination, against Candida glabrata, extracellularly and intracellularly in granulocyte-macrophage colony stimulating factor (GM-CSF)-activated human monocytes

OBJECTIVES The antifungal effects of voriconazole and caspofungin, singly and in combination, were determined against Candida glabrata in time-kill curves in broth, in human monocyte-derived macrophages (MDMs) and in MDMs activated by granulocyte-macrophage colony-stimulating factor (GM-CSF). METHODS Three strains of fluconazole-resistant C. glabrata were evaluated. For intracellular studies, MDM monolayers, with or without GM-CSF activation, were infected with C. glabrata and treated with voriconazole and caspofungin at 2.5x and 5x MIC, respectively, or at 1x MIC. Extracellular studies in broth were performed using drug concentrations from 0.1 to 10x MIC. Viable yeast were enumerated at 0, 24 and 48 h. RESULTS Significantly greater killing of C. glabrata occurred with the drug combination than with either single drug, both intracellularly and extracellularly (P < 0.01). For voriconazole, the antifungal activity in MDM activated by GM-CSF was greater than that in unactivated MDM, regardless of antibiotic concentration or time of exposure. However, for caspofungin and for the two-drug combination, enhanced activity in GM-CSF-activated MDM depended on the drug concentration and time of exposure. CONCLUSIONS Our data suggest that combinations of voriconazole and caspofungin may be efficacious for the treatment of serious C. glabrata infections. With single-drug therapy, especially voriconazole, GM-CSF activation of monocytes could be considered.

In vitro activity of cefdinir (FK482) and ten other antibiotics against gram-positive and gram-negative bacteria isolated from adult and pediatric patients.

The in vitro activity of cefdinir, an oral aminothiazolyl hydroxyimino cephalosporin was compared with that of cefixime, cefpodoxime, cefaclor, cephalexin, ciprofloxacin, ofloxacin, oxacillin, ampicillin, vancomycin and trimethoprim-sulfamethoxazole against 279 gram-positive and gram-negative recent clinical isolates from adult and pediatric patients. Cefdinir was the most active drug among the cephalosporins against oxacillin-sensitive Staphylococcus aureus and coagulase-negative staphylococci, Streptococcus pneumoniae, S. pyogenes, Escherichia coli and Moraxella catarrhalis (MIC90 0.015-2 mg/l). Cefixime was the most active agent against Hemophilus influenzae, Klebsiella pneumoniae, K. oxytoca, Proteus mirabilis and P. vulgaris (MIC90 < 0.015-0.125 mg/l). The activity of cefpodoxime was better than that of cefixime against S. pneumoniae and oxacillin-sensitive staphylococci (MIC90 0.25-8 vs. 0.5-32 mg/l), similar to cefixime against S. pyogenes (MIC90 0.06 mg/l) and not as good as cefixime against H. influenzae, M. catarrhalis, Klebsiella spp. and Proteus spp. (MIC90 < 0.015-0.25 vs. 0.125-0.5 mg/l). The activity of cefdinir was greater than that of the other cephalosporins against Enterococcus faecalis (MIC90 16-32 vs. > 64 mg/l). None of the cephalosporins were active against methicillin-resistant, coagulase-positive or -negative staphylococci or Pseudomonas aeruginosa (MIC90 > 64 mg/l). Overall, the susceptibilities of adult and pediatric isolates were similar. Kinetic kill curves demonstrated rapid and similar killing at 6 h by cefdinir, cefixime, cefpodoxime and ofloxacin. At 24 h at 1 x MIC, the least regrowth was observed with cefdinir and cefpodoxime; at 2 x MIC, suppression of growth was similar with all four drugs.

Molecular epidemiology of vancomycin-resistant enterococci from 6 hospitals in New York State

BACKGROUND Vancomycin resistance among enterococci is an emerging nosocomial problem. Consequently, it is important to understand the distribution of vancomycin-resistant enterococci (VRE) within and between hospitals to implement appropriate infection control measures. METHODS In this study, 116 VRE isolates obtained from patients in 6 New York State hospitals were analyzed by antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) fingerprinting, plasmid profile analysis, vanA and vanB polymerase chain reaction, and DNA:DNA hybridization with vanA and vanB probes. RESULTS PFGE and plasmid typing generally agreed, but plasmid profiles were more variable. These analyses revealed that genetic heterogeneity among isolates from within each of the 6 hospitals varied considerably. Among 23 Enterococcus faecium isolates from one hospital, there were only 3 PFGE types, and 20 isolates had the same type. However, in another hospital, each isolate was genetically distinct. Closely related strains were not found in separate hospitals. VRE strains with vanA genes and strains with vanB genes were found in 3 hospitals. Both plasmid and chromosomal carriage of these genes was detected. CONCLUSIONS PFGE typing showed that nosocomial VRE transmission had occurred in some hospitals. However, there was no evidence for it in others. Neither was there evidence for intrahospital transmission or for emergence of an endemic strain. These observations demonstrate that it is important to evaluate genetic heterogeneity among VRE before implementation of infection control measures. PFGE is the method of choice for epidemiologic typing, but polymerase chain reaction, plasmid, and hybridization studies can provide important information concerning the presence and potential for transfer of vancomycin resistance genes.

Use of the Microbial Growth Curve in Postantibiotic Effect Studies of Legionella pneumophila

ABSTRACT Using the standard Craig and Gudmundsson method (W. A. Craig and S. Gudmundsson, p. 296-329, in V. Lorian, ed., Antibiotics in Laboratory Medicine, 1996) as a guideline for determination of postantibiotic effects (PAE), we studied a large series of growth curves for two strains of Legionella pneumophila. We found that the intensity of the PAE was best determined by using a statistically fitted line over hours 3 to 9 following antibiotic removal. We further determined the PAE duration by using a series of observations of the assay interval from hours 3 to 24. We determined that inoculum reduction was not necessarily the only predictor of the PAE but that the PAE was subject to the type and dose of the drug used in the study. In addition, there was a variation between strains. Only levofloxacin at five and ten times the minimum inhibitory concentration (MIC) resulted in a PAE duration of 4 to 10 h for both strains of L. pneumophila tested. Ciprofloxacin at five and ten times the MIC and azithromycin at ten times the MIC caused a PAE for one strain only. No PAE could be demonstrated for either strain with erythromycin or doxycycline. Using the presently described method of measuring PAE for L. pneumophila, we were able to detect differences in PAE which were dependent upon the L. pneumophila strain, the antibiotic tested, and the antibiotic concentration. We suggest the use of mathematically fitted curves for comparison of bacterial growth in order to measure PAE for L. pneumophila.