Evolutionary genomics of host-use in bifurcating demes of RNA virus phi-6.

BACKGROUND: Viruses are exceedingly diverse in their evolved strategies to manipulate hosts for viral replication. However, despite these differences, most virus populations will occasionally experience two commonly-encountered challenges: growth in variable host environments, and growth under fluctuating population sizes. We used the segmented RNA bacteriophage ϕ6 as a model for studying the evolutionary genomics of virus adaptation in the face of host switches and parametrically varying population sizes. To do so, we created a bifurcating deme structure that reflected lineage splitting in natural populations, allowing us to test whether phylogenetic algorithms could accurately resolve this 'known phylogeny'. The resulting tree yielded 32 clones at the tips and internal nodes; these strains were fully sequenced and measured for phenotypic changes in selected traits (fitness on original and novel hosts). RESULTS: We observed that RNA segment size was negatively correlated with the extent of molecular change in the imposed treatments; molecular substitutions tended to cluster on the Small and Medium RNA chromosomes of the virus, and not on the Large segment. Our study yielded a very large molecular and phenotypic dataset, fostering possible inferences on genotype-phenotype associations. Using further experimental evolution, we confirmed an inference on the unanticipated role of an allelic switch in a viral assembly protein, which governed viral performance across host environments. CONCLUSIONS: Our study demonstrated that varying complexities can be simultaneously incorporated into experimental evolution, to examine the combined effects of population size, and adaptation in novel environments. The imposed bifurcating structure revealed that some methods for phylogenetic reconstruction failed to resolve the true phylogeny, owing to a paucity of molecular substitutions separating the RNA viruses that evolved in our study.

A meta-analysis of the published literature on the effectiveness of antimicrobial soaps.

The goal of this research was to conduct a systematic quantitative analysis of the existing data in the literature in order to determine if there is a difference between antimicrobial and nonantimicrobial soaps and to identify the methodological factors that might affect this difference. Data on hand washing efficacy and experimental conditions (sample size, wash duration, soap quantity, challenge organism, inoculum size, and neutralization method) from published studies were compiled and transferred to a relational database. A total of 25 publications, containing 374 observations, met the study selection criteria. The majority of the studies included fewer than 15 observations with each treatment and included a direct comparison between nonantimicrobial soap and antimicrobial soap. Although differences in efficacy between antimicrobial and nonantimicrobial soap were small (∼0.5-log CFU reduction difference), antimicrobial soap produced consistently statistically significantly greater reductions. This difference was true for any of the antimicrobial compounds investigated where n was >20 (chlorhexidine gluconate, iodophor, triclosan, or povidone). Average log reductions were statistically significantly greater (∼2 log CFU) when either gram-positive or gram-negative transient organisms were deliberately added to hands compared with experiments done with resident hand flora (∼0.5 log CFU). Our findings support the importance of using a high initial inoculum on the hands, well above the detection limit. The inherent variability in hand washing seen in the published literature underscores the importance of using a sufficiently large sample size to detect differences when they occur.

Evolution of mutational robustness in an RNA virus.

Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage phi6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.

Monte Carlo simulation of pathogen behavior during the sprout production process.

Food-borne disease outbreaks linked to the consumption of raw sprouts have become a concern over the past decade. A Monte Carlo simulation model of the sprout production process was created to determine the most-effective points for pathogen control. Published literature was reviewed, and relevant data were compiled. Appropriate statistical distributions were determined and used to create the Monte Carlo model with Analytica software. Factors modeled included initial pathogen concentration and prevalence, seed disinfection effectiveness, and sampling of seeds prior to sprouting, sampling of irrigation water, or sampling of the finished product. Pathogen concentration and uniformity of seed contamination had a large effect on the fraction of contaminated batches predicted by the simulation. The model predicted that sprout sampling and irrigation water sampling at the end of the sprouting process would be more effective in pathogen detection than seed sampling prior to production. Day of sampling and type of sample (sprout or water) taken had a minimal effect on rate of detection. Seed disinfection reduced the proportion of contaminated batches, but in some cases it also reduced the ability to detect the pathogen when it was present, because cell numbers were reduced below the detection limit. Both the amount sampled and the pathogen detection limit were shown to be important variables in determining sampling effectiveness. This simulation can also be used to guide further research and compare the levels of effectiveness of different risk reduction strategies.

Co-infection weakens selection against epistatic mutations in RNA viruses.

Co-infection may be beneficial in large populations of viruses because it permits sexual exchange between viruses that is useful in combating the mutational load. This advantage of sex should be especially substantial when mutations interact through negative epistasis. In contrast, co-infection may be detrimental because it allows virus complementation, where inferior genotypes profit from superior virus products available within the cell. The RNA bacteriophage phi6 features a genome divided into three segments. Co-infection by multiple phi6 genotypes produces hybrids containing reassorted mixtures of the parental segments. We imposed a mutational load on phi6 populations by mixing the wild-type virus with three single mutants, each harboring a deleterious mutation on a different one of the three virus segments. We then contrasted the speed at which these epistatic mutations were removed from virus populations in the presence and absence of co-infection. If sex is a stronger force, we predicted that the load should be purged faster in the presence of co-infection. In contrast, if complementation is more important we hypothesized that mutations would be eliminated faster in the absence of co-infection. We found that the load was purged faster in the absence of co-infection, which suggests that the disadvantages of complementation can outweigh the benefits of sex, even in the presence of negative epistasis. We discuss our results in light of virus disease management and the evolutionary advantage of haploidy in biological populations.

Analysis of published sprout seed sanitization studies shows treatments are highly variable.

Consumption of raw sprouts has caused many foodborne illness outbreaks in the last decade, and most outbreaks have been linked to contaminated seeds. Many seed sanitization treatments have been studied as a means to reduce the risk of illness associated with sprouts. Published data on seed sanitization were analyzed collectively to identify factors that influenced the efficacy of seed sanitization and to determine the variability associated with various sanitization processes. Temperature and duration of the sanitization treatment were found to produce a negligible effect on log microbial reductions. Salmonella, Escherichia coli O157:H7, and total aerobic microorganisms were all inactivated at similar rates. Data were fit to triangular or uniform distributions for 16 different chemical treatments. Among the most effective treatments were 8% hydrogen peroxide (uniform distribution [2.5, 4.5]), 20,000 ppm of chlorine (triangular distribution [1, 2.5, 6.5]), and 1% Ca(OH)2 (triangular distribution [0.5, 4, 5]). Chemical treatments where more published data were available showed more variability.

Statistical distributions describing microbial quality of surfaces and foods in food service operations.

Data on the microbial quality of food service kitchen surfaces and ready-to-eat foods were collected over a period of 10 years in Rutgers University dining halls. Surface bacterial counts, total aerobic plate counts, and total and fecal coliform counts were determined using standard methods. Analysis was performed on foods tested more than 50 times (primarily lunch meats and deli salads) and on surfaces tested more than 500 times (36 different surfaces types, including pastry brushes, cutting boards, and countertops). Histograms and statistical distributions were determined using Microsoft Excel and Palisades Bestfit, respectively. All data could be described by lognormal distributions, once data above and below the lower and upper limits of detection were considered separately. Histograms for surfaces counts contained one peak near 1 CFU/4 cm2. Surfaces with higher levels of contamination tended to be nonmetal, with the exception of buffalo chopper bowls, which commonly had high counts. Mean counts for foods ranged from 2 to 4 log CFU/g, with shrimp salad, roast beef, and bologna having higher means. Coleslaw, macaroni salad, and potato salad (all commercially processed products, not prepared in the dining halls) had lowest overall means. Coliforms were most commonly found in sealeg salad (present in 61% of samples) and least commonly found in coleslaw (present in only 7% of samples). Coliform counts (when present) were highest on average in shrimp salad and lowest in coleslaw. Average coliform counts for most products were typically between 1 and 2 log most probable number per gram. Fecal coliforms were not typically found in any deli salads or lunch meats.

Risk assessment of hand washing efficacy using literature and experimental data.

This study simulated factors that influence the levels of bacteria on foodservice workers' hands. Relevant data were collected from the scientific literature and from laboratory experiments. Literature information collected included: initial bacterial counts on hands and water faucet spigots, bacterial population changes during hand washing as effected by soap type, sanitizing agent, drying method, and the presence of rings. Experimental data were also collected using Enterobacter aerogenes as a surrogate for transient bacteria. Both literature and experimental data were translated into appropriate discrete or probability distribution functions. The appropriate statistical distribution for each phase of the hand washing process was determined. These distributions were: initial count on hands, beta (2.82, 2.32, 7.5); washing reduction using regular soap, beta (3.01, 1.91, -3.00, 0.60); washing reduction using antimicrobial soap, beta (4.19, 2.99, -4.50, 1.50); washing reduction using chlorhexidine gluconate (CHG), triangular (-4.75, -1.00, 0); reductions from hot air drying, beta (3.52, 1.92, -0.20, 1.00); reduction from paper towel drying, triangular (-2.25, -0.75, 0); reduction due to alcohol sanitizer, gamma (-1.23, 4.42) -5.8; reduction due to alcohol-free sanitizer, gamma (2.22, 5.38) -5.00; and the effect of rings, beta (8.55, 23.35, 0.10, 0.45). Experimental data were fit to normal distributions (expressed as log percentage transfer rate): hand-to-spigot transfer, normal (-0.80, 1.09); spigot to hand, normal (0.36, 0.90). Soap with an antimicrobial agent (in particular, CHG) was observed to be more effective than regular soap. Hot air drying had the capacity to increase the amount of bacterial contamination on hands, while paper towel drying caused a slight decrease in contamination. There was little difference in the efficacy of alcohol and alcohol-free sanitizers. Ring wearing caused a slight decrease in the efficacy of hand washing. The experimental data validated the simulated combined effect of certain hand washing procedures based on distributions derived from reported studies. The conventional hand washing system caused a small increase in contamination on hands vs. the touch-free system. Sensitivity analysis revealed that the primary factors influencing final bacteria counts on the hand were sanitizer, soap, and drying method. This research represents an initial framework from which sound policy can be promulgated to control bacterial transmission via hand contacts.