Impact of temperature and hydraulic retention time on pathogen and nutrient removal in woodchip bioreactors

Woodchip denitrification bioreactors are an important edge-of-field practice for treating agricultural drainage; however, their ability to filter microbial pollutants has primarily been explored in the context of wastewater treatment. Upflow column reactors were constructed and tested for E. coli, Salmonella, NO3-N, and dissolved reactive phosphorus (DRP) at hydraulic retention times (HRTs) of 12 and 24 h and at controlled temperatures of 10 and 21.5 °C. Influent solution was spiked to 30 mg L−1 NO3-N, 2–8 × 105 E. coli and Salmonella, and 0.1 mg L−1 DRP. Microbial removal was consistently observed with removal ranging from 75 to 78% reduction at 10 °C and 90–96% at 21.5 °C. The concentration reduction ranged from 2.75 to 9.03 × 104 for both organisms. HRT had less impact on microbial removal than temperature and thus further investigation of removal under lower HRTs is warranted. Nitrate concentrations averaged 96% reduction (with load removal of 14.6 g N m−3 d−1) from 21.5 °C columns at 24 HRT and 29% reduction (with load removal of 8.8 g N m−3 d−1) from 10 °C columns at 12 HRT. DRP removal was likely temporary due to microbial uptake. While potential for removal of E. coli and Salmonella by woodchip bioreactors is demonstrated, system design will need to be considered. High concentrations of these microbial contaminants are likely to occur during peak flows, when bypass flow may be occurring. The results of this study show that woodchip bioreactors operated for nitrate removal have a secondary benefit through the removal of enteric bacteria

Genomic and phenotypic characterization of multidrug-resistant Salmonella enterica serovar Reading isolates involved in a turkey-associated foodborne outbreak

Salmonella is a global bacterial foodborne pathogen associated with a variety of contaminated food products. Poultry products are a common source of Salmonella-associated foodborne illness, and an estimated 7% of human illnesses in the United States are attributed to turkey products. From November 2017 to March 2019, the Centers for Disease Control and Prevention reported a turkey-associated outbreak of multidrug-resistant (MDR; resistant to ≥3 antimicrobial classes) Salmonella enterica serovar Reading (S. Reading) linked to 358 human infections in 42 US states and Canada. Since S. Reading was seldom linked to human illness prior to this outbreak, the current study compared genomic sequences of S. Reading isolates prior to the outbreak (pre-outbreak) to isolates identified during the outbreak period, focusing on genes that were different between the two groups but common within a group. Following whole-genome sequence analysis of five pre-outbreak and five outbreak-associated turkey/turkey product isolates of S. Reading, 37 genes located within two distinct chromosomal regions were identified only in the pre-outbreak isolates: (1) an ~5 kb region containing four protein-coding genes including uidA which encodes beta-glucuronidase, pgdA encoding peptidoglycan deacetylase, and two hypothetical proteins and (2) an ~28 kb region comprised of 32 phage-like genes and the xerC gene, which encodes tyrosine recombinase (frequently associated with phage genes). The five outbreak isolates also had a deletional event within the cirA gene, introducing a translational frame shift and premature stop codon. The cirA gene encodes a protein with dual receptor functions: a siderophore receptor for transport of dihydroxybenzoylserine as well as a colicin Ia/b receptor. Significant differences for the identified genetic variations were also detected in 75 S. Reading human isolates. Of the 41 S. Reading isolates collected before or in 2017, 81 and 90% of the isolates contained the uidA and pgdA genes, respectively, but only 24% of the isolates collected after 2017 harbored the uidA and pgdA genes. The truncation event within the cirA gene was also significantly higher in isolates collected after 2017 (74%) compared to before or in 2017 (5%). Phenotypic analysis of the S. Reading isolates for colicin and cefiderocol sensitivities (CirA) and β-methyl-D-glucuronic acid utilization (UidA and accessory proteins) supported the genomic data. Overall, a similar genome reduction pattern was generally observed in both the turkey and human isolates of S. Reading during the outbreak period, and the genetic differences were present in genes that could potentially promote pathogen dissemination due to variation in Salmonella colonization, fitness, and/or virulence

Microbial Community and Chemical Characteristics of Swine Manure during Maturation

Swine diet formulations have the potential to lower animal emissions, including odor and ammonia (NH3). The purpose of this study was to determine the impact of manure storage duration on manure chemical and microbial properties in swine feeding trials. Three groups of 12 pigs were fed a standard corn–soybean meal diet over a 13-wk period. Urine and feces were collected at each feeding and transferred to 12 manure storage tanks. Manure chemical characteristics and headspace gas concentrations were monitored for NH3, hydrogen sulfide (H2S), volatile fatty acids, phenols, and indoles. Microbial analysis of the stored manure included plate counts, community structure (denaturing gradient gel electrophoresis), and metabolic function (Biolog). All odorants in manure and headspace gas concentrations were significantly (p \u3c 0.01) correlated for length of storage using quadratic equations, peaking after Week 5 for all headspace gases and most manure chemical characteristics. Microbial community structure and metabolic utilization patterns showed continued change throughout the 13-wk trial. Denaturing gradient gel electrophoresis species diversity patterns declined significantly (p \u3c 0.01) with time as substrate utilization declined for sugars and certain amino acids, but functionality increased in the utilization of short chain fatty acids as levels of these compounds increased in manure. Studies to assess the effect of swine diet formulations on manure emissions for odor need to be conducted for a minimum of 5 wk. Efforts to determine the impact of diets on greenhouse gas emissions will require longer periods of study (\u3e13 wk)

Identification of Salmonella enterica Serovar Typhimurium Genes Important for Survival in the Swine Gastric Environment

Since the stomach is a first line of defense for the host against ingested microorganisms, an ex vivo swine stomach contents (SSC) assay was developed to search for genes important for Salmonella enterica serovar Typhimurium survival in the hostile gastric environment. Initial characterization of the SSC assay (pH 3.87) using previously identified, acid-sensitive serovar Typhimurium mutants revealed a 10-fold decrease in survival for a phoP mutant following 20 min of challenge and no survival for mutants of rpoS or fur. To identify additional genes, a signature-tagged mutagenesis bank was constructed and screened in the SSC assay. Nineteen mutants were identified and individually analyzed in the SSC and acid tolerance response assays; 13 mutants exhibited a 10-fold or greater sensitivity in the SSC assay compared to the wild-type strain, but only 3 mutants displayed a 10-fold or greater decrease in survival following pH 3.0 acidic challenge. Further examination determined that the lethal effects of the SSC are pH dependent but that low pH is not the sole killing mechanism(s). Gas chromatography analysis of the SSC revealed lactic acid levels of 126 mM. Upon investigating the effects of lactic acid on serovar Typhimurium survival in a synthetic gastric fluid, not only was a concentration- and time-dependent lethal effect observed, but the phoP, rpoS, fur, and pnp genes were identified as involved in protection against lactic acid exposure. These studies indicate a role in gastric survival for several serovar Typhimurium genes and imply that the stomach environment is defined by more than low pH

Chloramphenicol and tetracycline decrease motility and increase invasion and attachment gene expression in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium is one of the most common serovars isolated from humans and livestock, and over 35% of these isolates are resistant to three or more antibiotics. Multidrug-resistant (MDR) Salmonella is a public health concern as it is associated with increased morbidity in patients compared to antibiotic sensitive strains, though it is unknown how the antibiotic resistant isolates lead to a more severe infection. Cellular invasion is temporally regulated in Salmonella and normally occurs during late-log and stationary growth. However, our previous work determined that a 30 minute exposure to a sub-inhibitory concentration of tetracycline can induce the full invasion phenotype during early-log growth in certain MDR S. Typhimurium isolates. The current study examined whether sub-inhibitory concentrations of other antibiotics could also induce the invasiveness in the same set of isolates. Ampicillin and streptomycin had no effect on invasion, but certain concentrations of chloramphenicol were found to induce invasion in a subset of isolates. Two of the isolates induced by chloramphenicol were also inducible by tetracycline. RNA-seq analyses demonstrated that chloramphenicol and tetracycline both down-regulated motility gene expression, while up-regulating genes associated with attachment, invasion, and intracellular survival. Eleven fimbrial operons were up-regulated, which is notable as only three fimbrial operons were thought to be inducible in culture; six of these up-regulated operons have been reported to play a role in Salmonella persistence in mice. Overall, these data show that the normal progression of the genetic pathways that regulate invasion can be expedited to occur within 30 minutes due to antibiotic exposure. This altered invasion process due to antibiotics may play a role in the increased intensity and duration of infection observed in patients with MDR Salmonella

An rfaH mutant of Salmonella enterica serovar Typhimurium is attenuated in swine and reduces intestinal colonization, fecal shedding and disease severity due to virulent Salmonella Typhimurium

Swine are often asymptomatic carriers of Salmonella spp., and interventions are needed to limit colonization of swine to enhance food safety and reduce environmental contamination. We evaluated the attenuation and potential vaccine use in pigs of a Salmonella enterica serovar Typhimurium mutant of rfaH, the gene encoding the RfaH antiterminator that prevents premature termination of long mRNA transcripts. Pigs inoculated with wild-type S. Typhimurium exhibited a significant elevation in average body temperature (fever) at 1 and 2 days post-inoculation; rfaH-inoculated pigs did not (n=5/group). During the 7 day trial, a significant reduction of Salmonella in the feces, tonsils, and cecum were observed in the rfaH-inoculated pigs compared to wild-type inoculated pigs. To determine whether vaccination with the rfaH mutant could provide protection against wild-type S. Typhimurium challenge, two groups of pigs (n=14/group) were intranasally-inoculated with either the rfaH mutant or a PBS placebo at 6- and 8-weeks of age and challenged with the parental, wild-type S. Typhimurium at 11-weeks of age. The average body temperature was significantly elevated in the mock-vaccinated pigs at 1 and 2 days post-challenge, but not in the rfaH-vaccinated pigs. Fecal shedding at 2 and 3 days post-challenge and colonization of intestinal tract tissues at 7 days post-challenge by wild-type S. Typhimurium was significantly reduced in the rfaH-vaccinated pigs compared to mock-vaccinated pigs. Serological analysis using the IDEXX HerdChek Swine Salmonella Test Kit indicated that vaccination with the rfaH mutant did not stimulate an immune response against LPS. These results indicate that vaccination of swine with the attenuated rfaH mutant confers protection against challenge with virulent S. Typhimurium but does not interfere with herd level monitoring for Salmonella spp., thereby allowing for differentiation of infected from vaccinated animals (DIVA)

Chlortetracycline and florfenicol induce expression of genes associated with pathogenicity in multidrug-resistant Salmonella enterica serovar Typhimurium

Abstract Background Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Typhimurium) is a serious public health threat as infections caused by these strains are more difficult and expensive to treat. Livestock serve as a reservoir for MDR Salmonella, and the antibiotics chlortetracycline and florfenicol are frequently administrated to food-producing animals to treat and prevent various diseases. Therefore, we evaluated the response of MDR S. Typhimurium after exposure to these two antibiotics. Results We exposed four MDR S. Typhimurium isolates to sub-inhibitory concentrations of chlortetracycline (16 and 32 µg/ml) or florfenicol (16 µg/ml) for 30 min during early-log phase. Differentially expressed genes following antibiotic treatment were identified using RNA-seq, and genes associated with attachment and those located within the Salmonella pathogenicity islands were significantly up-regulated following exposure to either antibiotic. The effect of antibiotic exposure on cellular invasion and motility was also assessed. Swimming and swarming motility were decreased due to antibiotic exposure. However, we observed chlortetracycline enhanced cellular invasion in two strains and florfenicol enhanced invasion in a third isolate. Conclusions Chlortetracycline and florfenicol exposure during early-log growth altered the expression of nearly half of the genes in the S. Typhimurium genome, including a large number of genes associated with virulence and pathogenesis; this transcriptional alteration was not due to the SOS response. The results suggest that exposure to either of these two antibiotics may lead to the expression of virulence genes that are typically only transcribed in vivo, as well as only during late-log or stationary phase in vitro

Characterization of a Multidrug-Resistant Salmonella enterica Serovar Heidelberg Outbreak Strain in Commercial Turkeys: Colonization, Transmission, and Host Transcriptional Response

In recent years, multidrug-resistant (MDR) Salmonella enterica serovar Heidelberg (S. Heidelberg) has been associated with numerous human foodborne illness outbreaks due to consumption of poultry. For example, in 2011, an MDR S. Heidelberg outbreak associated with ground turkey sickened 136 individuals and resulted in 1 death. In response to this outbreak, 36 million pounds of ground turkey were recalled, one of the largest meat recalls in U.S. history. To investigate colonization of turkeys with an MDR S. Heidelberg strain isolated from the ground turkey outbreak, two turkey trials were performed. In experiment 1, 3-week-old turkeys were inoculated with 108 or 1010 CFU of the MDR S. Heidelberg isolate, and fecal shedding and tissue colonization were detected following colonization for up to 14 days. Turkey gene expression in response to S. Heidelberg exposure revealed 18 genes that were differentially expressed at 2 days following inoculation compared to pre-inoculation. In a second trial, 1-day-old poults were inoculated with 104 CFU of MDR S. Heidelberg to monitor transmission of Salmonella from inoculated poults (index group) to naive penmates (sentinel group). The transmission of MDR S. Heidelberg from index to sentinel poults was efficient with cecum colonization increasing 2 Log10 CFU above the inoculum dose at 9 days post-inoculation. This differed from the 3-week-old poults inoculated with 1010 CFU of MDR S. Heidelberg in experiment 1 as Salmonella fecal shedding and tissue colonization decreased over the 14-day period compared to the inoculum dose. These data suggest that young poults are susceptible to colonization by MDR S. Heidelberg, and interventions must target turkeys when they are most vulnerable to prevent Salmonella colonization and transmission in the flock. Together, the data support the growing body of literature indicating that Salmonella establishes a commensal-like condition in livestock and poultry, contributing to the asymptomatic carrier status of the human foodborne pathogen in our animal food supply