Frederick C. Michel

Development and Application of Real-Time PCR Assays for Quantification of erm Genes Conferring Resistance to Macrolides-Lincosamides-Streptogramin B in Livestock Manure and Manure Management Systems

ABSTRACT Erythromycin and tylosin are commonly used in animal production, and such use is perceived to contribute to the overall antimicrobial resistance (AR) reservoirs. Quantitative measurements of this type of AR reservoir in microbial communities are required to understand AR ecology (e.g., emergence, persistence, and dissemination). We report here the development, validation, and use of six real-time PCR assays for quantifying six classes of erm genes (classes A through C, F, T, and X) that encode the major mechanism of resistance to macrolides-lincosamides-streptogramin B (MLSB). These real-time PCR assays were validated and used in quantifying the six erm classes in five types of samples, including those from bovine manure, swine manure, compost of swine manure, swine waste lagoons, and an Ekokan upflow biofilter system treating hog house effluents. The bovine manure samples were found to contain much smaller reservoirs of each of the six erm classes than the swine manure samples. Compared to the swine manure samples, the composted swine manure samples had substantially reduced erm gene abundances (by up to 7.3 logs), whereas the lagoon or the biofilter samples had similar erm gene abundances. These preliminary results suggest that the methods of manure storage and treatment probably have a substantial impact on the persistence and decline of MLSB resistance originating from food animals, thus likely affecting the dissemination of such resistance genes into the environment. The abundances of these erm genes appeared to be positively correlated with those of the tet genes determined previously among these samples. These real-time PCR assays provide a rapid, quantitative, and cultivation-independent measurement of six major classes of erm genes, which should be useful for ecological studies of AR.

Development and Application of Real-Time PCR Assays for Quantification of Genes Encoding Tetracycline Resistance

ABSTRACT We report here the development, validation, and use of three real-time PCR assays to quantify the abundance of the following three groups of tetracycline resistance genes: tet(A) and tet(C); tet(G); and tet genes encoding ribosomal protection proteins, including tet(M), tet(O), tetB(P), tet(Q), tet(S), tet(T), and tet(W). The assays were validated using known numbers of sample-derived tet gene templates added to microbiome DNA. These assays are both precise and accurate over at least 6 log tet gene copies. New tet gene variants were also identified from cloned tet amplicons as part of this study. The utility of these real-time PCR assays was demonstrated by quantifying the three tet gene groups present in bovine and swine manures, composts of swine manure, lagoons of hog house effluent, and samples from an Ekokan upflow biofilter system treating hog house effluent. The bovine manures were found to contain fewer copies of all three groups of tet genes than the swine manures. The composts of swine manures had substantially reduced tet gene abundance (up to 6 log), while lagoon storage or the upflow biofilter had little effect on tet gene abundance. These results suggest that the method of manure storage and treatment may have a substantial impact on the persistence and dissemination of tet genes in agricultural environments. These real-time PCR assays provide rapid, quantitative, cultivation-independent measurements of 10 major classes of tet genes, which should be useful for ecological studies of antibiotic resistance.

Persistence of Mycobacterium avium subsp. paratuberculosis and Other Zoonotic Pathogens during Simulated Composting, Manure Packing, and Liquid Storage of Dairy Manure

ABSTRACT Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis (Mycobacterium paratuberculosis). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated M. paratuberculosis, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55°C, manure packing at 25°C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with M. paratuberculosis at 106 CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of M. paratuberculosis was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO2, while no significant change in carbon content was observed in the liquid slurry. Escherichia coli, Salmonella, and Listeria were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55°C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days. M. paratuberculosis was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56, M. paratuberculosis was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However, M. paratuberculosis DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.

Succession of Bacterial Communities during Early Plant Development: Transition from Seed to Root and Effect of Compost Amendment

ABSTRACT Compost amendments to soils and potting mixes are routinely applied to improve soil fertility and plant growth and health. These amendments, which contain high levels of organic matter and microbial cells, can influence microbial communities associated with plants grown in such soils. The purpose of this study was to follow the bacterial community compositions of seed and subsequent root surfaces in the presence and absence of compost in the potting mix. The bacterial community compositions of potting mixes, seed, and root surfaces sampled at three stages of plant growth were analyzed via general and newly developed Bacteroidetes-specific, PCR-denaturing gradient gel electrophoresis methodologies. These analyses revealed that seed surfaces were colonized primarily by populations detected in the initial potting mixes, many of which were not detected in subsequent root analyses. The most persistent bacterial populations detected in this study belonged to the genus Chryseobacterium (Bacteroidetes) and the family Oxalobacteraceae (Betaproteobacteria). The patterns of colonization by populations within these taxa differed significantly and may reflect differences in the physiology of these organisms. Overall, analyses of bacterial community composition revealed a surprising prevalence and diversity of Bacteroidetes in all treatments.

Occurrence and persistence of erythromycin resistance genes (erm) and tetracycline resistance genes (tet) in waste treatment systems on swine farms.

Animal manure from modern animal agriculture constitutes the single largest source of antibiotic resistance (AR) owing to the use of large quantities of antibiotics. After animal manure enters the environment, the AR disseminates into the environment and can pose a potentially serious threat to the health and well-being of both humans and animals. In this study, we evaluated the efficiency of three different on-farm waste treatment systems in reducing AR. Three classes of erythromycin resistance genes (erm) genes (B, F, and X) conferring resistances to macrolide–lincosamides–streptogramin B (MLSB) and one class of tetracycline resistance genes (tet) gene (G) conferring resistance to tetracyclines were used as models. Real-time polymerase chain reaction assays were used to determine the reservoir sizes of these AR genes present in the entire microbiome. These classes of AR genes varied considerably in abundance, with erm(B) being more predominant than erm(F), erm(X), and tet(G). These AR genes also varied in persistence in different waste treatment systems. Aerobic biofiltration reduced erm(X) more effectively than other AR genes, while mesophilic anaerobic digestion and lagoon storage did not appreciably reduce any of these AR genes. Unlike chemical pollutants, some AR genes could increase after reduction in a preceding stage of the treatment processes. Season might also affect the persistence of AR. These results indicate that AR arising from swine-feeding operations can survive typical swine waste treatment processes and thus treatments that are more effective in destructing AR on farms are required.

Contrasting patterns of seed and root colonization by bacteria from the genus Chryseobacterium and from the family Oxalobacteraceae

Microbial colonization of plant seeds and roots is a highly complex process in which soil and plant type can influence the composition of the root-associated and rhizosphere microbial communities. Amendment of compost, a common agricultural technique, introduces exogenous nutrients and microorganisms to the soil–plant environment, and can further influence microbial community composition in the plant environment. Although compost amendments can strongly influence soil and rhizosphere microbial communities, there is evidence that with increasing proximity to the root, plant influences predominate over soil effects. We hypothesized that the ‘rhizosphere effect’ observed with proximity to plant surfaces does not act equally on all microorganisms. To explore this issue, we examined two bacterial taxa that reproducibly colonized seed and root surfaces in an experiment examining the influence of compost amendment on plant-associated bacterial communities. Population-specific analyses revealed striking differences in the ecology of bacteria from the genus Chryseobacterium and the family Oxalobacteraceae in potting mix and plant-associated environments. Seed- and root-colonizing Oxalobacteraceae populations were highly sensitive to plant effects, and phylogenetic analyses of root-colonizing Oxalobacteraceae revealed the presence of root-associated populations that were highly similar, regardless of treatment, and differed from the potting mix populations detected at the same sampling points. Conversely, Chryseobacterium community composition was found to be essentially invariant within treatments, but was strongly influenced by compost amendment. This persistence and stable nature of the Chryseobacterium community composition demonstrates that rhizosphere selection is not the exclusive factor involved in determining the composition of the cucumber spermosphere and rhizosphere communities.

Bioremediation of a PCB-Contaminated Soil Via Composting

Polychlorinated biphenyls (PCBs) were widely used in the past and now contaminate many industrial and natural areas. In this study, a PCB-contaminated soil from a former paper mill was mixed with a yard trimmings amendment and composted in field scale piles to determine the effect of soil to amendment ratio on PCB degradation. Temperature, oxygen concentrations, and a number of other environmental parameters that influence microbial activity during composting were monitored. The PCBs in contaminated soil had a concentration of 16 ±1 mg/kg dw and an average of 4 chlorines per biphenyl. The soil was composted with five levels of yard trimmings amendment (14% to 82% by weight) in pilot scale compost piles (25 m3) turned once per month. Results showed up to a 40% loss of PCBs with amendment levels of 60% and 82%. Congener specific PCB analysis indicated that less chlorinated PCB congeners (1-3 chlorines per biphenyl) were preferentially degraded. Bench-scale studies indicated that less than 1% of the PCBs in the contaminated soil were volatilized from composts during incubation with forced aeration at 55°C. In conclusion, PCB loss observed during the composting of the PCB-contaminated soil appeared to be largely due to biodegradation and not volatilization. Effective bioremediation of aged PCB-contaminated soils may require coupling of composting with additional remediation technologies to reduce levels of PCB congeners with greater than 4 chlorines.

Effects of Different Animal Waste Treatment Technologies on Detection and Viability of Porcine Enteric Viruses

ABSTRACT Enteric pathogens in animal waste that is not properly processed can contaminate the environment and food. The persistence of pathogens in animal waste depends upon the waste treatment technology, but little is known about persistence of porcine viruses. Our objectives were to characterize the porcine enteric viruses (porcine noroviruses [PoNoVs], porcine sapoviruses [PoSaVs], rotavirus A [RV-A], RV-B, and RV-C) in fresh feces or manure and to evaluate the effects of different candidate environmentally superior technologies (ESTs) for animal waste treatment on the detection of these viruses. Untreated manure and samples collected at different stages during and after treatment were obtained from swine farms that used conventional waste management (CWM) and five different candidate ESTs. The RNA from porcine enteric viruses was detected by reverse transcription-PCR and/or seminested PCR; PoSaV and RV-A were also detected by enzyme-linked immunosorbent assay. Cell culture immunofluorescence (CCIF) and experimental inoculation of gnotobiotic (Gn) pigs were used to determine RV-A/C infectivity in posttreatment samples. The PoSaV and RV-A were detected in pretreatment samples from each farm, whereas PoNoV and RV-C were detected in pretreatment feces from three of five and four of five farms using the candidate ESTs, respectively. After treatment, PoSaV RNA was detected only in the samples from the farm using CWM and not from the farms using the candidate ESTs. RV-A and RV-C RNAs were detected in four of five and three of four candidate ESTs, respectively, after treatment, but infectious particles were not detected by CCIF, nor were clinical signs or seroconversion detected in inoculated Gn pigs. These results indicate that only RV-A/C RNA, but no viral infectivity, was detected after treatment. Our findings address a public health concern regarding environmental quality surrounding swine production units.

Effects of different wavelengths of light on lignin peroxidase production by the white-rot fungi Phanerochaete chrysosporium grown in submerged cultures.

In this study, the effects of different wavelengths of light (UV, blue, green, yellow, red) and white light on lignin peroxidase (LiP), protein, biomass and exo-polysaccharide production and glucose uptake by Phanerochaete chrysosporium BKM-F-1767 were determined. The experiments were conducted under aerated (CS) and oxygenated (RS) culture conditions. The results showed that only green light significantly increased maximum LiP production (by 20% and 27% in CS and RS cultures respectively). Green light also increased biomass production in oxygenated cultures (RS). Blue and UV light both significantly reduced maximum LiP activity. Yellow, red and white lights had mixed effects on culture properties. This is the first time that the effects of different wavelengths of light on lignin peroxidase production and other culture properties have been investigated. The novel findings may be important in improving the yield of lignin modifying enzymes for biomass conversion processes and understanding their regulation.

Changes in diversity of cultured bacteria resistant to erythromycin and tetracycline in swine manure during simulated composting and lagoon storage.

This study investigated the impact of composting and lagoon storage on survival and change in diversity of tetracycline‐resistant (Tcr) and erythromycin‐resistant (Emr) bacteria and the resistance genes they carry in swine manure. Treatments were arranged as a 2 × 2 factorial design: composting vs lagoon storage and 0 vs 1% Surround WP Crop Protectant (a clay product) in three replicates. After 48 days of treatments, resistant bacteria were enumerated by selective plating and identified by 16S rRNA gene sequencing. The erm and the tet gene(s) carried by the resistant isolates were screened using class‐specific PCR assays. The plate counts of Tcr and Emr bacteria decreased by 4–7 logs by composting, but only by 1–2 logs by the lagoon treatment. During the treatments, Acinetobacter gave way to Pseudomonas and Providencia as the largest resistant genera. The clay product had little effect on survival or diversity of resistant bacteria. Of six classes of erm and seven classes of tet genes tested, changes in prevalence were also noted. The results indicate that composting can dramatically shift Tcr and Emr bacterial populations, and composting can be an effective and practical approach to decrease dissemination of antibiotic resistance from swine farms to the environment.