Effects of inoculum, pH, and cations on the in vitro activity of fidaxomicin (OPT-80, PAR-101) against Clostridium difficile.

The effects of the inoculum, pH, cation concentrations, and different lots of commercial media on the in vitro susceptibility of Clostridium difficile to fidaxomicin were examined. Of the factors evaluated, only pH alterations influenced the activity of fidaxomicin against C. difficile, noticeably reducing its activity at higher pH (> or =7.9).

Typing and susceptibility of bacterial isolates from the fidaxomicin (OPT-80) phase II study for C. difficile infection.

BACKGROUND: Clostridium difficile infection (CDI) has been increasing in incidence and severity in recent years, coincident with the spread of a "hypervirulent" strain, REA type BI (ribotype 027, PFGE NAP 1). Exacerbating the problem has been the observation that metronidazole may be showing decreased effectiveness, particularly in the more severe cases. Fidaxomicin is an 18-membered macrocycle currently in phase 3 trials for the treatment of C. difficile infection (CDI). An open-label, phase II study in CDI patients has been completed and the clinical results published. C. difficile organisms were isolated from patient stool specimens and typed by restriction endonuclease analysis (REA) in order to determine the frequency and susceptibility of the C. difficile isolates and their response to treatment. METHODS: Fecal samples were plated on CCFA agar for isolation of C. difficile. These isolates were tested for susceptibility to fidaxomicin, vancomycin, and metronidazole using CLSI agar dilution methods and were typed by REA. RESULTS: C. difficile was isolated from 38 of 49 subjects and 16 (42%) were the epidemic C. difficile BI group. The BI strain was distributed approximately equally in the three dosing groups. Overall antibiotic susceptibilities were consistent with the previously reported MIC(90) values for the three antibiotics tested, but the MIC(90) of BI strains was two dilutions higher than non-BI strains for metronidazole and vancomycin (for both antibiotics, MIC(90) was 2 microg/mL vs. 0.5 microg/mL, P<0.01 for metronidazole, P=NS for vancomycin). Clinical cure for BI isolates (11/14, 79%) was not significantly different from non-BI isolates (21/22, 95%). CONCLUSION: These results underscore the high prevalence of the BI epidemic strain and demonstrate that mild to moderate CDI infection as well as severe disease can be caused by these strains. Fidaxomicin cure rates for subjects with BI and with non-BI strains are similar, although the small numbers of subjects preclude a robust statistical comparison.

Effect of transportation and acid neutralization on recovery of mycobacteria from processed specimens.

Because of the increasing incidence of tuberculosis, more rapid detection of mycobacteria has become an important issue. Realizing that not every clinical laboratory has a rapid detection system for growing mycobacteria, this study was conducted to examine the feasibility of submitting sediments of processed specimens to a reference laboratory for further testing in a radiometric system. Using N-acetyl-L-cysteine-NaOH solution, 247 respiratory specimens were processed at a diagnostic laboratory. Half of each sediment was cultured on conventional media. The remainder was kept at 4 degrees C for a period of up to 1 week before transportation to a reference laboratory for culture by BACTEC system. Both laboratories recovered 25 organisms: 15 as Mycobacterium tuberculosis (MT) and 10 as M avium complex (MAC). Additionally, mycobacteria (MT[3], MAC[6], M gordonae [4], and M fortuitum [1]) were recovered from 14 specimens by the diagnostic laboratory that were not grown by the reference laboratory. These results indicate a significant decrease in viability of mycobacteria after processing of the specimens. Acid neutralization of the digested respiratory sediments significantly improved the recovery rate of mycobacteria even after 2 days of delay in culture. This preliminary work suggests that more extensive studies will provide useful information to delineate approaches to submitting neutralized sediments for mycobacterial cultures.

Newborn piglet model for campylobacteriosis.

An in vivo model system for human campylobacteriosis has been developed in which colostrum-deprived newborn piglets are orally challenged with an invasive strain of Campylobacter jejuni. Piglets developed clinical symptoms and histopathological lesions similar to those observed in humans infected with C. jejuni. Gross lesion examination at autopsy revealed the presence of edema, hyperemia, and mucus. Histopathologic examinations by light and transmission electron microscopy demonstrated damage to surface epithelial cells with the presence of intracellular bacteria, mainly in the large intestine. Similar lesions were not demonstrated in control piglets.

Primary swine intestinal cells as a model for studying Campylobacter jejuni invasiveness.

Swine small-intestinal enterocytes were used to test the invasiveness of Campylobacter jejuni. The cells were removed from the small intestines of 6-h-old piglets by enzymatic digestion. Two clinical C. jejuni isolates invaded swine enterocytes at significantly higher frequencies than an Escherichia coli control strain. The recovered colonies of C. jejuni T13192 appeared to be highly mucoid and invaded tissue culture cells (INT 407) at higher frequency (0.14%) than the parental strain (0.003%). The data not only support the previous in vitro findings regarding the invasiveness of C. jejuni but also suggest that invasiveness of C. jejuni may be an in vivo virulence attribute.

Characterization of cross-reactive anti-DNA autoantibodies in murine lupus.

The role of crossreactive anti-DNA autoantibodies in the pathogenesis of Systemic Lupus Erythematosus (SLE) and its counterpart in the mouse (murine lupus) remains undefined. Five murine monoclonal anti-DNA autoantibodies tested in ELISA and immunofluorescence assays were found to cross-react with a variety of both nucleic acid and non-nucleic acid antigens. These included double stranded DNA (dsDNA), single stranded DNA (ssDNA), transfer RNA (tRNA), and the murine thymoma cell lines WEHI-22, WEHI-7, and EL-4. The majority of the autoantibodies reacted with all antigens tested; none of the autoantibodies reacted with only one antigen. To determine if the multiple reactivities demonstrated by these hybridoma-derived monoclonal anti-DNA autoantibodies accurately reflects the in vivo, autoimmune environment, the same assays were used to measure the reactivities of autoantibodies secreted directly from unfused autoimmune spleen cells cultured in vitro. These spleen cell-derived autoantibodies were found to display reactivities very similar to those demonstrated by the monoclonal anti-DNA autoantibodies indicating that the hybridoma process itself does not appear to select and amplify reactivities which are not present in vivo. Initial molecular characterization of F11, a monoclonal anti-DNA autoantibody crossreactive with both dsDNA and ssDNA, revealed that it utilizes the same VH gene segment as an anti-DNA autoantibody specific for ssDNA. F11 was also found to utilize similar VH, D, and JH gene segments as an antibody directed against the hapten polymer (Glutamic acid60, Alanine30, Tyrosine10)n (GAT). Thus, the same Ig gene segments used to encode crossreactive anti-DNA autoantibodies can also be utilized by anti-DNA autoantibodies displaying strict antigen specificity as well as by antibodies directed against exogenous antigens.

Invasion-related antigens of Campylobacter jejuni.

A HEp-2 cell culture model was used to investigate the antigens required for epithelial cell penetration by Campylobacter jejuni. Penetration of HEp-2 epithelial cells by C. jejuni was significantly inhibited (P less than .05) with C. jejuni lysate and a monoclonal antibody (MAb 1B4) in competitive inhibition assays. Immunogold electron microscopy revealed that MAb 1B4 bound to the flagella and cell surface of low-passage (invasive) C. jejuni M 96, whereas only the flagella of high-passage (noninvasive) C. jejuni M 96 were labeled. Western blot analysis revealed that MAb 1B4 identified an epitope on antigens of 64-44 kDa in lysates prepared from invasive and noninvasive isolates. In addition, antigens of 42-38 kDa were recognized in lysates prepared from only invasive C. jejuni strains. Proteinase K and sodium meta-periodate chemical treatment of C. jejuni M 96 lysate changed the mobility of antigens recognized by MAb 1B4. The increase in mobility demonstrated a decrease in size of molecules and suggested that antigens required for HEp-2 cell invasion by Campylobacter species may be glycoprotein in nature.