Michael J. Rothrock

Long-term preservation of anammox bacteria

Deposit of useful microorganisms in culture collections requires long-term preservation and successful reactivation techniques. The goal of this study was to develop a simple preservation protocol for the long-term storage and reactivation of the anammox biomass. To achieve this, anammox biomass was frozen or lyophilized at two different freezing temperatures (−60°C and in liquid nitrogen (−200°C)) in skim milk media (with and without glycerol), and the reactivation of anammox activity was monitored after a 4-month storage period. Of the different preservation treatments tested, only anammox biomass preserved via freezing in liquid nitrogen followed by lyophilization in skim milk media without glycerol achieved stoichiometric ratios for the anammox reaction similar to the biomass in both the parent bioreactor and in the freshly harvested control treatment. A freezing temperature of −60°C alone, or in conjunction with lyophilization, resulted in the partial recovery of the anammox bacteria, with an equal mixture of anammox and nitrifying bacteria in the reactivated biomass. To our knowledge, this is the first report of the successful reactivation of anammox biomass preserved via sub-zero freezing and/or lyophilization. The simple preservation protocol developed from this study could be beneficial to accelerate the integration of anammox-based processes into current treatment systems through a highly efficient starting anammox biomass.

Recovery of ammonia from poultry litter using flat gas permeable membranes.

The use of flat gas-permeable membranes was investigated as components of a new process to capture and recover ammonia (NH3) in poultry houses. This process includes the passage of gaseous NH3 through a microporous hydrophobic membrane, capture with a circulating dilute acid on the other side of the membrane, and production of a concentrated ammonium (NH4) salt. Bench- and pilot-scale prototype systems using flat expanded polytetrafluoroethylene (ePTFE) membranes and a sulfuric acid solution consistently reduced headspace NH3 concentrations from 70% to 97% and recovered 88% to 100% of the NH3 volatilized from poultry litter. The potential benefits of this technology include cleaner air inside poultry houses, reduced ventilation costs, and a concentrated liquid ammonium salt that can be used as a plant nutrient solution.

Antibiotic Resistance Patterns of Major Zoonotic Pathogens from All-Natural, Antibiotic-Free, Pasture-Raised Broiler Flocks in the Southeastern United States.

The use of antibiotics in agroecosystems has been implicated in the rise in antibiotic resistance (AR), which can affect environmental, animal, and human health. To determine the environmental impact of antibiotic use in agroecosystems, appropriate background levels of AR in agricultural environments in the absence of antibiotic application must be determined. Therefore, to determine background levels of AR in broiler production, four target microbes (, , , and ) were isolated from 15 all-natural, antibiotic-free, pasture-raised broiler flocks from six farms within the southeastern United States. The AR profiles of these isolates were characterized using the CDC National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), and these resistance patterns were compared across target microbes and farms and throughout the life cycle of the flocks along the farm-to-fork continuum. Antibiotic resistances were most prevalent in and and least prevalent in . Although and were isolated from the same farms and characterized using the same NARMS plates, they exhibited distinct AR profiles, with demonstrating clear farm-specific resistance patterns. Multidrug resistance rates (three or more antibiotics), in order of prevalence, were (63.9%), (36.0%), (12.7%), and (1.4%). The results of this study demonstrate the variability in background AR among major food safety-related microbes, even when isolated from similar production and processing samples from the same farms, and indicate the need for the proper design of future broiler production studies to account for this highly dynamic background AR.

How Should We Be Determining Background and Baseline Antibiotic Resistance Levels in Agroecosystem Research

Although historically, antibiotic resistance has occurred naturally in environmental bacteria, many questions remain regarding the specifics of how humans and animals contribute to the development and spread of antibiotic resistance in agroecosystems. Additional research is necessary to completely understand the potential risks to human, animal, and ecological health in systems altered by antibiotic-resistance-related contamination. At present, analyzing and interpreting the effects of human and animal inputs on antibiotic resistance in agroecosystems is difficult, since standard research terminology and protocols do not exist for studying background and baseline levels of resistance in the environment. To improve the state of science in antibiotic-resistance-related research in agroecosystems, researchers are encouraged to incorporate baseline data within the study system and background data from outside the study system to normalize the study data and determine the potential impact of antibiotic-resistance-related determinants on a specific agroecosystem. Therefore, the aims of this review were to (i) present standard definitions for commonly used terms in environmental antibiotic resistance research and (ii) illustrate the need for research standards (normalization) within and between studies of antibiotic resistance in agroecosystems. To foster synergy among antibiotic resistance researchers, a new surveillance and decision-making tool is proposed to assist researchers in determining the most relevant and important antibiotic-resistance-related targets to focus on in their given agroecosystems. Incorporation of these components within antibiotic-resistance-related studies should allow for a more comprehensive and accurate picture of the current and future states of antibiotic resistance in the environment.

Listeria Occurrence in Poultry Flocks: Detection and Potential Implications

Foodborne pathogens such as Salmonella, Campylobacter, Escherichia coli, and Listeria are a major concern within the food industry due to their pathogenic potential to cause infection. Of these, Listeria monocytogenes, possesses a high mortality rate (approximately 20%) and is considered one of the most dangerous foodborne pathogens. Although the usual reservoirs for Listeria transmission have been extensively studied, little is known about the relationship between Listeria and live poultry production. Sporadic and isolated cases of listeriosis have been attributed to poultry production and Listeria spp. have been isolated from all stages of poultry production and processing. Farm studies suggest that live birds may be an important vector and contributor to contamination of the processing environment and transmission of Listeria to consumers. Therefore, the purpose of this review is to highlight the occurrence, incidence, and potential systemic interactions of Listeria spp. with poultry.

Urea Hydrolysis and Calcium Carbonate Precipitation in Gypsum-Amended Broiler Litter

Broiler () litter is subject to ammonia (NH) volatilization losses. Previous work has shown that the addition of gypsum to broiler litter can increase nitrogen mineralization and decrease NH losses due to a decrease in pH, but the mechanisms responsible for these effects are not well understood. Therefore, three laboratory studies were conducted to evaluate the effect of gypsum addition to broiler litter on (i) urease activity at three water contents, (ii) calcium carbonate precipitation, and (iii) pH. The addition of gypsum to broiler litter increased ammonium concentrations ( < 0.0033) and decreased litter pH by 0.43 to 0.49 pH units after 5 d ( < 0.0001); however, the rate of urea hydrolysis in treated litter only increased on Day 0 for broiler litter with low (0.29 g HO g) and high (0.69 g HO g) water contents, and on Day 3 for litter with medium (0.40 g HO g) water content ( < 0.05). Amending broiler litter with gypsum also caused an immediate decrease in litter pH (0.22 pH units) due to the precipitation of calcium carbonate (CaCO) from gypsum-derived calcium and litter bicarbonate. Furthermore, as urea was hydrolyzed, more urea-derived carbon precipitated as CaCO in gypsum-treated litter than in untreated litter ( < 0.001). These results indicate that amending broiler litter with gypsum favors the precipitation of CaCO, which buffers against increases in litter pH that are known to facilitate NH volatilization.

Supplementation of protected sodium butyrate alone or in combination with essential oils modulated the cecal microbiota of broiler chickens challenged with coccidia and Clostridium perfringens

The objective of this study was to determine the effects of protected sodium butyrate (SB), and protected sodium butyrate plus essential oils (carvacrol and ginger; SBEO) on the cecal microbiota of broilers challenged with Eimeria maxima and Clostridium perfringens. Birds were assigned to 4 treatments (8 replicates pens of 58 birds/pen): unchallenged control; challenged control; challenged and supplemented with SB; challenged and supplemented with SBEO. On d 13, challenged birds were orally inoculated with ~5,000 Eimeria maxima oocysts. On d 18-19, the same birds were exposed to Clostridium perfringens via drinking water (~ 8 log CFU/ml). Cecal excreta was collected at d 12, 18, 21, and 28 for microbiome analysis through 16s rRNA sequencing using Illumina MiSeq platform and analyzed using QIIME v. 1.9.1 The cecal microbiota was analyzed over time within each experimental group. The inclusion of SB alone or in combination with EO contributed to larger variations in the cecal microbiota over time than the unsupplemented treatments, as shown by the diversity indices. The community structure and abundance of the cecal microbiota were significantly different across ages, especially in the groups supplemented with SB and SBEO. As shown in the PCoA analysis, the supplementation of SB led to a more stable microbial community and lower between-sample variability over time. In the unchallenged control birds, Ruminococcus decreased (p = 0.006), whereas Bacteroides and Clostridiales increased (p ≤ 0.02) as the birds aged. In the challenged control group, however, the frequency of Coprococcus and Blautia decreased as birds aged (p ≤ 0.01), and, Clostridiales did not increase. Supplementation of SB, but not SBEO, increased the frequency of Lactobacillus (p = 0.01) on d 12 compared to d 18 and d 28, and prevented the reduction in the frequency of Blautia as the birds aged. Nevertheless, supplementation of SB and SBEO contributed to unique changes in the predicted functions of the cecal microbiota over time, which was not observed in the unsupplemented birds. SB and SBEO modulated the diversity, composition, and predictive function of the cecal microbiota which may have lowered the negative impact of necrotic enteritis (NE).