Performance Equivalence and Validation of the Soleris Automated System for Quantitative Microbial Content Testing Using Pure Suspension Cultures.

Using United States Pharmacopeia-National Formulary (USP-NF) general method <1223> guidance, the Soleris(®) automated system and reagents (Nonfermenting Total Viable Count for bacteria and Direct Yeast and Mold for yeast and mold) were validated, using a performance equivalence approach, as an alternative to plate counting for total microbial content analysis using five representative microbes: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus brasiliensis. Detection times (DTs) in the alternative automated system were linearly correlated to CFU/sample (R(2) = 0.94-0.97) with ≥70% accuracy per USP General Chapter <1223> guidance. The LOD and LOQ of the automated system were statistically similar to the traditional plate count method. This system was significantly more precise than plate counting (RSD 1.2-2.9% for DT, 7.8-40.6% for plate counts), was statistically comparable to plate counting with respect to variations in analyst, vial lots, and instruments, and was robust when variations in the operating detection thresholds (dTs; ±2 units) were used. The automated system produced accurate results, was more precise and less labor-intensive, and met or exceeded criteria for a valid alternative quantitative method, consistent with USP-NF general method <1223> guidance.

Effects of quaternary-ammonium-based formulations on bacterial community dynamics and antimicrobial susceptibility.

Quaternary ammonium compounds (QACs) are widely used as adjuncts to hygiene in domestic cleaning products. Current concern that the increased use of such biocides in consumer products might contribute to the emergence of antibiotic resistance has led us to examine the effects of a QAC-containing domestic cleaning fluid on the population dynamics and antimicrobial susceptibility of domestic sink drain biofilm communities. QAC susceptibilities of numerically dominant, culturable drain bacteria (15 genera, 17 species) were determined in vitro before and after repeated QAC exposure (14 passages). A fully characterized drain microcosm was then exposed to short-term (12 days) and long-term (3 months) dosing with a QAC-containing domestic detergent (QD). QAC exposure of isolated cultures caused both increases (three species) and circa twofold decreases (six species) in QAC susceptibility. The susceptibility of Ralstonia sp. was considerably decreased following 14 consecutive QAC passages. Control drain microcosm biofilms maintained dynamic stability, as evidenced by culture and denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial population densities were largely unaffected during short-term exposure to use levels of QD, although 50% QD caused circa 10-fold viability reductions. DGGE analysis supported these observations; identified the major microcosm genera as Pseudomonas, Pseudoalteromonas, Erwinia, and Enterobacter, and showed that aeromonads increased in abundance under 10 to 50% QD. Long-term exposure of the microcosms to QD did not significantly alter the pattern of antimicrobial susceptibility. These data demonstrate the recalcitrance of domestic drain biofilms toward QAC and that although repeated QAC exposure of drain isolates in pure culture results in susceptibility change in some test bacteria, such changes do not necessarily occur within complex communities.

Effects of triclosan-containing rinse on the dynamics and antimicrobial susceptibility of in vitro plaque ecosystems.

Dental plaque microcosms were established under a feast-famine regimen within constant-depth film fermentors and exposed four times daily postfeeding to a triclosan (TR)-containing rinse (dentifrice) (TRD). This was diluted so that the antimicrobial content was 0.6 mg/ml. Microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the V2-V3 region of the eubacterial 16S rRNA gene (rDNA). Dominant isolates and PCR amplicons were identified by partial sequencing of 16S rDNA. TRD caused considerable decreases in the counts of both gram-negative organisms and total anaerobic cells, transiently lowered the numbers of streptococci and actinomycetes, and markedly increased the proportion of lactobacilli. DGGE indicated the presence of putatively unculturable bacteria and showed that a Porphyromonas sp. and Selenomonas infelix had been inhibited by TRD. Pure culture studies of 10 oral bacteria (eight genera) showed that Neisseria subflava, Prevotella nigrescens, and Porphyromonas gingivalis were highly susceptible to TR, while the lactobacilli and streptococci were the least susceptible. Clonal expansion of the lactobacilli in the pulsed microcosm could be explained on the basis of TR activity. The mean MICs of TR, chlorhexidine, erythromycin, penicillin V, and vancomycin for the population before and after 5 days of exposure to TRD showed few significant changes. In conclusion, changes in plaque microcosm populations following repeated exposure to TRD showed inhibition of the most susceptible flora and clonal expansion of less susceptible species.

Exposure of sink drain microcosms to triclosan: population dynamics and antimicrobial susceptibility.

Recent concern that the increased use of triclosan (TCS) in consumer products may contribute to the emergence of antibiotic resistance has led us to examine the effects of TCS dosing on domestic-drain biofilm microcosms. TCS-containing domestic detergent (TCSD) markedly lowered biofouling at 50% (wt/vol) but was poorly effective at use levels. Long-term microcosms were established and stabilized for 6 months before one was subjected to successive 3-month exposures to TCSD at sublethal concentrations (0.2 and 0.4% [wt/vol]). Culturable bacteria were identified by 16S rDNA sequence analysis, and their susceptibilities to four biocides and six antibiotics were determined. Microcosms harbored ca. 10 log(10) CFU/g of biofilm, representing at least 27 species, mainly gamma proteobacteria, and maintained dynamic stability. Viable cell counts were largely unaffected by TCSD exposure, but species diversity was decreased, as corroborated by denaturing gradient gel electrophoresis analysis. TCS susceptibilities ranged widely within bacterial groups, and TCS-tolerant strains (including aeromonads, pseudomonads, stenotrophomonads, and Alcaligenes spp.) were isolated before and after TCSD exposure. Several TCS-tolerant bacteria related to Achromobacter xylosoxidans became clonally expanded during dosing. TCSD addition did not significantly affect the community profiles of susceptibility to the test biocides or antibiotics. Several microcosm isolates, as well as reference bacteria, caused clearing of particulate TCS in solid media. Incubations of consortia and isolates with particulate TCS in liquid led to putative TCS degradation by the consortia and TCS solubilization by the reference strains. Our results support the view that low-level exposure of environmental microcosms to TCS does not affect antimicrobial susceptibility and that TCS is degradable by common domestic biofilms.

Effects of a chlorhexidine gluconate-containing mouthwash on the vitality and antimicrobial susceptibility of in vitro oral bacterial ecosystems.

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (P < 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in Prevotella sp. and Selenomonas infelix. Pure-culture studies of 10 oral bacteria (eight genera) showed that Actinomyces naeslundii, Veillonella dispar, Prevotella nigrescens, and the streptococci were highly susceptible to CHX, while Lactobacillus rhamnosus, Fusobacterium nucleatum, and Neisseria subflava were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (P < 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.

Development and use of a high-throughput bacterial DNA gyrase assay to identify mammalian topoisomerase II inhibitors with whole-cell anticancer activity.

A high-throughput screen (HTS) was developed and used to identify inhibitors of bacterial DNA gyrase. Among the validated hits were 53 compounds that also inhibited mammalian topoisomerase II with IC(50) values of <12.5 micro g/mL for 51 of them. Using computational methods, these compounds were subjected to cluster analysis to categorize them according to their chemical and structural properties. Nine compounds from different clusters were tested for their whole-cell inhibitory activity against 3 cancer cell lines-NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS)-at a concentration of 100 micro M. Five compounds inhibited cell growth by >50% for all 3 cell lines tested. These compounds were tested further against a panel of 53 to 57 cell lines representing leukemia, melanoma, colon, CNS, ovarian, renal, prostate, breast, and non-small cell lung cancers. In this assay, PGE-7143417 was found to be the most potent compound, which inhibited the growth of all the cell lines by 50% at a concentration range of 0.31 to 2.58 micro M, with an average of 1.21 micro M. An additional 17 compounds were also tested separately against a panel of 10 cell lines representing melanoma, colon, lung, mammary, ovarian, prostate, and renal cancers. In this assay, 4 compounds-PGE-3782569, PGE-7411516, PGE-2908955, and PGE-3521917-were found to have activity with concentrations for 50% cell growth inhibition in the 0.59 to 3.33, 22.5 to 59.1, 7.1 to >100, and 24.7 to >100 micro M range.

Microbial characterization of biofilms in domestic drains and the establishment of stable biofilm microcosms.

We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log(10) cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log(10) cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan Colpoda maupasi, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.