Lawrence H. Bopp

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.

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.

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.

Pseudomonas aeruginosa cytotoxin as a pathogenicity factor in a systemic infection of leukopenic mice

The effect of Pseudomonas aeruginosa cytotoxin was assessed in leukopenic outbred Swiss male mice (20 g) using a high cytotoxin-producing strain (PA158) and its cytotoxin-deficient isogenic mutant (PA114F5) generated by Tn7::Tn5 transposon mutagenesis of PA158. Leukopenia was induced by intraperitoneal (i.p.) administration of cyclophosphamide (150 micrograms/g). Anesthetized mice were infected via a 4 mm incision on the shaved back with 300 CFU/mouse (9 LD50; expected death rate 85%). Precleared mouse cytotoxin-specific heat inactivated rabbit polyclonal antibody (RPA) was administered i.p. (0.2 ml) 24 hr before challenge. Controls received i.p. normal rabbit serum, RPA, cyclophosphamide alone, or a sham procedure. Challenge with the high cytotoxin-producing strain PA158 caused earlier and a significantly greater mortality than that observed with a cytotoxin-deficient strain PA114F5 (P < 0.01). Cytotoxin-specific polyclonal antibody was protective. Pretreatment with antibody decreased the mortality rate following challenge with PA158 from 88.9% to 27.8% (P < 0.01). Pretreatment with antibody decreased the mortality rate following challenge with PA114F5 from 27.8% to 5.6% (P < 0.05). These results demonstrate that P. aeruginosa cytotoxin contributes to the pathogenicity of the organism and that cytotoxin antibody is protective in a systemic P. aeruginosa infection in leukopenic mice.

Inhibitory and bactericidal effects of telithromycin (HMR 3647, RU 56647) and five comparative antibiotics, used singly and in combination, against vancomycin-resistant and vancomycin-susceptible enterococci.

The inhibitory and bactericidal effects of telithromycin (HMR 3647, RU 66647) were compared with those of gentamicin, ampicillin, erythromycin, azithromycin and vancomycin against 74 strains of enterococci (34 Enterococcus faecalis and 40 Enterococcus faecium) by agar dilution, broth dilution, time kill assays and postantibiotic effect (PAE). The telithromycin MIC90 for vancomycin-sensitive (VSE) E. faecalis strains tested using the agar dilution method was 8 µg/ml. For a different group of VSE E. faecalis strains tested using the broth dilution method it was 0.06 µg/ml The telithromycin MIC90s for vancomycin-resistant (VRE) and VSE E. faecium strains, determined using the agar dilution method, were 4 and 8 µg/ml, respectively, while for a different set of VRE and VSE E. faecium strains tested using the broth macrodilution method, they were 32 and 16 µg/ml, respectively. Telithromycin MBC90s for E. faecalis were 4–6 tubes higher and for E. faecium 3–5 tubes higher, respectively, than the MIC90s. In time kill assays, telithromycin had bactericidal activity against only 1 of 7 E. faecium strains; for all other E. faecium and E. faecalis strains, only inhibitory activity was demonstrated. Neither synergy nor drug interference was observed when telithromycin was used in combination with ampicillin, vancomycin or gentamicin. At 10 times the MIC, the PAE of telithromycin against E. faecalis was 2.8 h, while for E. faecium it was 1.6 h. Telithromycin should be evaluated for therapy of enterococcal infections, including those caused by VRE organisms. However, because of the strain-to-strain variability in susceptibility to telithromycin, MIC determinations are important, especially for erythromycin-resistant strains.

In Vitro Activities of Garenoxacin and Levofloxacin against Chlamydia pneumoniae Are Not Affected by Presence of Mycoplasma DNA

ABSTRACT We studied 20 Chlamydia pneumoniae isolates obtained from respiratory sites and atheroma tissue of patients from various geographic areas to determine the susceptibilities of these isolates to a new des-fluoroquinolone, garenoxacin, and to levofloxacin. In addition, we assessed the cultures with these isolates by PCR for the presence or absence of Mycoplasma sp. DNA. Both the MIC at which 90% of isolates are inhibited (MIC90) and the minimal bactericidal concentration at which 90% of isolates are killed (MBC90) for garenoxacin were 0.06 μg/ml, and both the MIC90 and the MBC90 for levofloxacin were 2.0 μg/ml. The activity of garenoxacin against C. pneumoniae was 32-fold greater than that of levofloxacin. Mycoplasma sp. DNA was detected by PCR in 17 of 20 cultures. Mycoplasma amplicons from five Mycoplasma DNA-positive C. pneumoniae cultures were sequenced and found to represent four Mycoplasma species. Our data demonstrate that C. pneumoniae cultures frequently contain Mycoplasma DNA and that its presence in C. pneumoniae cultures does not appear to affect the susceptibility results for the two fluoroquinolones that we tested.

Levofloxacin Kills Chlamydia pneumoniae and Modulates Interleukin 6 Production by HEp-2 Cells

Background:Chlamydia pneumoniae is known to cause acute respiratory infection and more recently it has been studied as a pathogen causing inflammatory changes in chronic diseases such as atherosclerosis. This study addresses the antichlamydial effect of levofloxacin and its role in modulation of a proinflammatory cytokine IL-6 production by uninfected and infected HEp-2 cells. Methods: HEp-2 cell monolayers were infected with previously prepared and frozen aliquots of C. pneumoniae [1 × 103 inclusion-forming units (IFU)/ml] by centrifugation for 30 min and incubation at 37°C for 1 h. Infected monolayers were treated with levofloxacin (3 or 8 µg/ml) immediately after infection (0 h) or 24 h after infection. Monolayers were examined daily for 96 h after infection by counting inclusions with fluorescently labeled antichlamydial monoclonal antibody. Aliquots of disrupted monolayers were titrated to determine the numbers of viable C. pneumoniae IFU/ml. IL-6 concentrations in cell supernatants were determined by ELISA assays. Results: Infected HEp-2 cells produced IL–6. Noninfected HEp-2 cells demonstrated modulation of IL-6 production by levofloxacin. No viable C. pneumoniae were detected in infected HEp-2 cells when the monolayer was treated with levofloxacin immediately after infection (0 h). In contrast, when cells were treated 24 h after infection, a gradual decline in the number of viable C. pneumoniae occurred; by 96 h into the assay ≧98% of C. pneumoniae were killed. IL-6 concentrations were similar in the supernatants of levofloxacin-treated and nontreated HEp-2 cells. Conclusions: (1) Levofloxacin is effective in eliminating C. pneumoniae from infected HEp-2 cells; (2) although levofloxacin modulates the production of IL-6 in untreated HEp-2 cells, no evidence for such modulation was observed in HEp-2 cells infected with C. pneumoniae. (3) Presence of viable C. pneumoniae may not be necessary for IL-6 production by infected and treated HEp-2 cells.