Plant-Scale Validation of Physical Heat Treatment of Poultry Litter Composts Using Surrogate and Indicator Microorganisms for Salmonella.

This study selected and used indicator and surrogate microorganisms for Salmonella to validate the processes for physically heat-treated poultry litter compost in litter processing plants. Initially laboratory validation studies indicated that 1.2- to 2.7-log or more reductions of desiccation-adapted Enterococcus faecium NRRL B-2354 were equivalent to > 5-log reductions of desiccation-adapted Salmonella Senftenberg 775/W in poultry litter compost, depending on treatment conditions and compost types. Plant validation studies were performed in one turkey litter compost processor and one laying hen litter compost processor. E. faecium was inoculated at ca.7 log CFU g-1 into the turkey litter compost and at ca. 5 log CFU g-1 into laying hen litter compost with respectively targeted moisture contents. The thermal processes in the two plants yielded 2.8 - > 6.4 log CFU g-1 (> 99.86%) reductions E. faecium of the inoculated. Similarly, for the processing control samples, reductions of presumptive indigenous enterococci were in the order of 1.8-3.7 log CFU g-1 (98.22% to 99.98%) of the total naturally present. In contrast, there were less reductions of indigenous mesophiles (1.7-2.9 log CFU) and thermophiles (0.4-3.2 log CFU g-1). More indigenous enterococci were inactivated in the presence of higher moisture in the poultry litter compost. Based on the data collected under the laboratory conditions, the processing conditions in both plants were adequate to reduce any potential Salmonella contamination of processed poultry litter compost by at least 5 logs, even though the processing conditions varied among trials and plants.IMPORTANCE Poultry litter compost, commonly used as a biological soil amendment, is subjected to a physical heat-treatment in industry setting to reduce pathogenic bacteria such as Salmonella and produce a dry product. According to the FDA Food Safety Modernization Act (FSMA) Produce Safety Rule, the thermal process for poultry litter compost should be scientifically validated to satisfy the microbial standard requirement. To the best of our knowledge, this is the first validation study in commercial poultry litter compost processing plants, and our results indicated that Salmonella levels, if present, could be reduced by at least 5 logs based on the reductions of surrogate and indicator microorganisms, even though the processing conditions in these commercial plants varied greatly. Furthermore, both indicator and surrogate microorganisms along with the custom-designed sampler can serve as practical tools for poultry litter compost processors to routinely monitor or validate their thermal processes without introducing pathogens into the industrial environments.

Isolation of Toxigenic Clostridium difficile from Animal Manure and Composts Being Used as Biological Soil Amendments.

The well-known nosocomial pathogen Clostridium difficile has recently been recognized as a community-associated pathogen. As livestock animals carry and shed C. difficile in their feces, animal manure-based composts may play an important role in disseminating toxigenic C. difficile strains into the agricultural environment. The present study surveyed C. difficile contamination of commercially available composts and animal manure. Presumptive C. difficile isolates were confirmed by testing for the tpi housekeeping gene in addition to Gram staining. The confirmed C. difficile isolates were further tested for toxigenicity, PCR ribotype, and susceptibilities to selected antibiotics. C. difficile was found in 51 out of 142 samples (36%). A total of 58 C. difficile strains were isolated from those 51 positive compost/manure samples. The presence of C. difficile in compost did not significantly correlate (P > 0.05) with the physical and most microbiological parameters, including the presence of fecal coliforms. The majority of C. difficile isolates were toxigenic, with 63.8% positive for the toxin A gene (tcdA) and 67.2% positive for the toxin B gene (tcdB). Only 3 isolates (5.17%) were positive for the binary toxins. There were 38 different PCR ribotypes among the 58 C. difficile isolates, and ribotype 106 was the most prevalent, followed by ribotypes 020 and 412. All C. difficile isolates were susceptible to the selected antibiotics, but >50% of the isolates had reduced susceptibility to clindamycin by the agar dilution method. This study indicates that compost may be a reservoir of toxigenic C. difficile strains.IMPORTANCEClostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhea in health care facilities in developed countries. Extended hospital stays and recurrences severely increase the cost of treatments and the high mortality rate that is observed among the elderly. Community-associated CDI cases that occur without any recent contact with the hospital environment are increasing. Studies have reported the isolation of virulent C. difficile strains from water, soil, meat, vegetables, pets, livestock animals, and animal manure. The objective of this study was to isolate and characterize C. difficile strains from animal manure and commercially available compost products. Our results demonstrate that not only unprocessed animal manure but also finished composts made of different feedstocks can serve as a reservoir for C. difficile as well. Most importantly, our study revealed that properly processed compost is a potential source of toxigenic and clindamycin-resistant C. difficile isolates.

Efficacy of Silver Dihydrogen Citrate and Steam Vapor against a Human Norovirus Surrogate, Feline Calicivirus, in Suspension, on Glass, and on Carpet.

Carpets and other soft surfaces have been associated with prolonged and reoccurring human norovirus (HuNoV) outbreaks. Environmental hygiene programs are important to prevent and control HuNoV outbreaks. Despite our knowledge of HuNoV transmission via soft surfaces, no commercially available disinfectants have been evaluated on carpets. Our aim was to adapt a current standardized method for virucidal testing by assessing two disinfection technologies, silver dihydrogen citrate (SDC) and steam vapor, against one HuNoV surrogate, feline calicivirus (FCV), on wool and nylon carpets. First, we evaluated the effect of both technologies on the appearance of carpet. Next, we evaluated the efficacy of SDC in suspension and the efficacy of SDC and steam vapor against FCV on a glass surface, each with and without serum. Lastly, we tested both technologies on two types of carpet, wool and nylon. Both carpets exhibited no obvious color changes; however, SDC treatments left a residue while steam vapor left minor abrasions to fibers. SDC in suspension and on glass reduced FCV by 4.65 log10 and >4.66 log10 PFU, respectively, but demonstrated reduced efficacy in the presence of serum. However, SDC was only efficacious against FCV on nylon (3.62-log10 PFU reduction) and not wool (1.82-log10 PFU reduction). Steam vapor reduced FCV by >4.93 log10 PFU on glass in 10 s and >3.68 log10 PFU on wool and nylon carpet carriers in 90 s. There was a limited reduction of FCV RNA under both treatments compared to that of infectivity assays, but RNA reductions were higher in samples that contained serum.IMPORTANCE Human noroviruses (HuNoV) account for ca. 20% of all diarrheal cases worldwide. Disease symptoms may include diarrhea and vomit, with both known to contribute to transmission. The prevention and control of HuNoV are difficult because they are environmentally resilient and resistant to many disinfectants. Several field studies have linked both hard and soft surfaces to HuNoV outbreaks. However, many disinfectants efficacious against HuNoV surrogates are recommended for hard surfaces, but no commercially available products have demonstrated efficacy against these surrogates on soft surfaces. Our research objectives were to evaluate liquid and steam-based technologies in suspension and on hard surface carriers in addition to adapting and testing a protocol for assessing the virucidal effects of disinfection technologies on carpet carriers. These results will inform both the government and industry regarding a standard method for evaluating the virucidal effects of disinfectants on carpet while demonstrating their efficacy relative to suspension and hard-surface tests.

Prevalence of Human Noroviruses in Commercial Food Establishment Bathrooms.

Although transmission of human norovirus in food establishments is commonly attributed to consumption of contaminated food, transmission via contaminated environmental surfaces, such as those in bathrooms, may also play a role. Our aim was to determine the prevalence of human norovirus on bathroom surfaces in commercial food establishments in New Jersey, Ohio, and South Carolina under nonoutbreak conditions and to determine characteristics associated with the presence of human norovirus. Food establishments (751) were randomly selected from nine counties in each state. Four surfaces (underside of toilet seat, flush handle of toilet, inner door handle of stall or outer door, and sink faucet handle) were swabbed in male and female bathrooms using premoistened macrofoam swabs. A checklist was used to collect information about the characteristics, materials, and mechanisms of objects in bathrooms. In total, 61 (1.5%) of 4,163 swabs tested were presumptively positive for human norovirus, 9 of which were confirmed by sequencing. Some factors associated with the presence of human norovirus included being from South Carolina (odd ratio [OR], 2.4; 95% confidence interval [CI], 1.2 to 4.9; P < 0.05) or New Jersey (OR, 1.7; 95% CI, 0.9 to 3.3; 0.05 < P < 0.10), being a chain establishment (OR, 1.9; 95% CI, 1.1 to 3.3; P < 0.05), being a unisex bathroom (versus male: OR, 2.0; 95% CI, 0.9 to 4.1; 0.05 < P < 0.10; versus female: OR, 2.6; 95% CI, 1.2 to 5.7; P < 0.05), having a touchless outer door handle (OR, 3.3; 95% CI, 0.79 to 13.63; 0.05 < P < 0.10), and having an automatic flush toilet (OR, 2.5, 95% CI, 1.1 to 5.3; 0.05 < P < 0.10). Our findings confirm that the presence of human norovirus on bathroom surfaces in commercial food establishments under nonoutbreak conditions is a rare event. Therefore, routine environmental monitoring for human norovirus contamination during nonoutbreak periods is not an efficient method of monitoring norovirus infection risk.

Improving culture media for the isolation of Clostridium difficile from compost.

This study was to optimize the detection methods for Clostridium difficile from the animal manure-based composts. Both autoclaved and unautoclaved dairy composts were inoculated with a 12-h old suspension of a non-toxigenic C. difficile strain (ATCC 43593) and then plated on selected agar for vegetative cells and endospores. Six types of enrichment broths supplemented with taurocholate and l-cysteine were assessed for detecting a low level of artificially inoculated C. difficile (ca. 5 spores/g) from dairy composts. The efficacy of selected enrichment broths was further evaluated by isolating C. difficile from 29 commercial compost samples. Our results revealed that using heat-shock was more effective than using ethanol-shock for inducing endospore germination, and the highest endospore count (p < 0.05) was yielded at 60 °C for 25 min. C. difficile agar base, supplemented with 0.1% l-cysteine, 7% defibrinated horse blood, and cycloserine-cefoxitin (CDA-CYS-H-CC agar) was the best medium (p < 0.05) for recovering vegetative cells from compost. C. difficile endospore populations from both types of composts enumerated on both CDA-CYS-H-CC agar supplemented with 0.1% sodium taurocholate (CDA-CYS-H-CC-T agar) and brain heart infusion agar supplemented with 0.5% yeast extract, 0.1% l-cysteine, cycloserine-cefoxitin, and 0.1% sodium taurocholate (BHIA-YE-CYS-CC-T agar) media were not significantly different from each other (p > 0.05). Overall, enrichment of inoculated compost samples in broths containing moxalactam-norfloxacin (MN) produced significantly higher (p < 0.05) spore counts than in non-selective broths or broths supplemented with CC. Enrichment in BHIB-YE-CYS-MN-T broth followed by culturing on an agar containing 7% horse blood and 0.1% taurocholate provided a more sensitive and selective combination of media for detecting a low population of C. difficile from environmental samples with high background microflora.

Persistence of Non-O157 Shiga Toxin-Producing Escherichia coli in Dairy Compost during Storage.

Dairy compost with 20, 30, or 40% moisture content (MC) was inoculated with a mixture of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serovars at a final concentration of 5.1 log CFU/g and then stored at 22 and 4°C for 125 days. Six storage conditions-4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC-were investigated for the persistence of non-O157 STEC in the dairy compost. During the entire storage, fluctuations in indigenous mesophilic bacterial levels were observed within the first 28 days of storage. After inoculation, the non-O157 STEC population increased 0.69 and 0.79 log CFU/g in the dairy compost with 30 and 40% MC at 22°C within the first day, respectively; for all other storage conditions, the pathogen population decreased rapidly. After the 125-day storage, the reductions of non-O157 STEC for 4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC storage conditions were >4.52, >4.55, 3.89, >4.61, 3.60, and 3.17 log CFU/g, respectively. All the survival curves showed an extensive tail, indicating non-O157 STEC can survive at least for 125 days in the dairy compost. The survival data were analyzed with log-linear with tailing and Weibull models. Compared with the log-linear with tailing model, the Weibull model was found to be a better choice for predicting the survival of non-O157 STEC in dairy compost owing to a high overall R2 value (0.8738 to 0.9909). The decay rate of non-O157 STEC was higher in dairy compost stored at 4°C compared with at 22°C, and the same trend was found for the compost with 40% MC versus 20% MC. In addition, two non-O157 STEC serotypes (STEC O145 and O45) were detected on the last day of the longitudinal study and may deserve special attention in the Big 6 STEC group. Our results have provided scientific data for risk assessment of the microbiological safety of dairy compost to control non-O157 STEC during subsequent storage of dairy compost.

Refrigerated Shelf Life of a Coconut Water-Oatmeal Mix and the Viability of Lactobacillus Plantarum Lp 115-400B.

Non-dairy probiotic products have the advantage of being lactose-free and can be manufactured to sustain the growth of probiotics. In this study, coconut water and oatmeal were used with the probiotic, Lactobacillus plantarum Lp 115-400B (L. plantarum) as a starter culture. Two separate treatments were carried out probiotic (P) and probiotic and prebiotic (PP) added. In both treatments, oatmeal-coconut water matrix was inoculated with 7 log CFU/g of L. plantarum and fermented at 27 °C for 10 h. For the PP treatment, 1 g of inulin/100 mL of the product was added additionally. The fermented products were then refrigerated (4 °C) and the viability of L. plantarum, pH, total acidity, and apparent viscosity of the matrix were monitored at selected time intervals. The shelf life to reach was defined by maintenance of L. plantarum count of 7 log CFU/g product. Refrigerated shelf life was determined to be seven-weeks for the P treatment and five-weeks for PP treatment. A significant reduction of pH was observed at the end of the considered shelf life; conversely, the apparent viscosity of the product did not change significantly.

Rapid identification of Campylobacter jejuni from poultry carcasses and slaughtering environment samples by real-time PCR.

The objective of this study was to develop a real-time PCR assay for rapid identification of Campylobacter jejuni and to apply the method in analyzing samples from poultry processing. A C. jejuni-specific primer set targeting a portion of the C. jejuni hippuricase gene was developed. The specificity of the newly designed primer pair was verified using 5 C. jejuni strains and 20 other bacterial strains. Sensitivity was determined to be as low as 1 genome copy per reaction. A total of 73 samples were collected at different sites along the processing line during 2 visits to a poultry slaughterhouse and were examined by direct plating onto modified charcoal cefoperazone deoxycholate agar or after enrichment in Bolton broth followed by plating on modified charcoal cefoperazone deoxycholate agar. The newly developed real-time PCR assay was used to identify the presumptive colonies as belonging to C. jejuni. A real-time PCR assay targeting 16S ribosomal RNA was also applied to determine Campylobacter spp. prevalence. Results from the real-time PCR analysis indicated considerable variability in Campylobacter contamination, with incidence rates of 72.7 and 27.6% for sampling days A and B, respectively. Campylobacter was isolated from 100% of prescalded and preeviscerated carcasses on sampling day A. In contrast, on sampling day B, the highest number of Campylobacter-positive carcasses was recovered after evisceration (60%). The chilling process significantly reduced (P < 0.05) Campylobacter population, but the percentage of positive samples on sampling day A increased to 80%. All samples collected from the processing environment, except scalding tank 3 and the prechiller and chiller tanks, were 100% positive on day A, whereas no campylobacters were isolated from machinery on sampling day B. Our results revealed the widespread of C. jejuni in poultry processing and proved that the newly developed real-time PCR assay is a simple, specific, and inexpensive method for rapid C. jejuni identification. The newly developed PCR method can be easily used in laboratories for reliable and unambiguous identification of C. jejuni in poultry samples.

Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter.

Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter was investigated in comparison with that in a nonadapted control to examine potential cross-tolerance of desiccation-adapted cells to heat treatment. A mixture of four Salmonella serovars was inoculated into the finished compost with 20, 30, 40, and 50% moisture contents for a 24-h desiccation adaptation. Afterwards, the compost with desiccation-adapted cells was inoculated into the aged chicken litter with the same moisture content for heat treatments at 70, 75, 80, 85, and 150°C. Recovery media were used to allow heat-injured cells to resuscitate. A 5-log reduction in the number of the desiccation-adapted cells in aged chicken litter with a 20% moisture content required >6, >6, ∼4 to 5, and ∼3 to 4 h of exposure at 70, 75, 80, and 85°C, respectively. As a comparison, a 5-log reduction in the number of nonadapted control cells in the same chicken litter was achieved within ∼1.5 to 2, ∼1 to 1.5, ∼0.5 to 1, and <0.5 h at 70, 75, 80, and 85°C, respectively. The exposure time required to obtain a 5-log reduction in the number of desiccation-adapted cells gradually became shorter as temperature and moisture content were increased. At 150°C, desiccation-adapted Salmonella cells survived for 50 min in chicken litter with a 20% moisture content, whereas control cells were detectable by enrichment for only 10 min. Our results demonstrated that the thermal resistance of Salmonella in aged chicken litter was increased significantly when the cells were adapted to desiccation. This study also validated the effectiveness of thermal processing being used for producing chicken litter free of Salmonella contamination.