Diversity of Plasmids and Genes Encoding Resistance to Extended-Spectrum ß-Lactamase in Escherichia coli from Different Animal Sources.

Antimicrobial resistance associated with the spread of plasmid-encoded extended-spectrum ß-lactamase (ESBL) genes conferring resistance to third generation cephalosporins is increasing worldwide. However, data on the population of ESBL producing E. coli in different animal sources and their antimicrobial characteristics are limited. The purpose of this study was to investigate potential reservoirs of ESBL-encoded genes in E. coli isolated from swine, beef, dairy, and poultry collected from different regions of the United States using whole-genome sequencing (WGS). Three hundred isolates were typed into different phylogroups, characterized by BOX AIR-1 PCR and tested for resistance to antimicrobials. Of the 300 isolates, 59.7% were resistant to sulfisoxazole, 49.3% to tetracycline, 32.3% to cephalothin, 22.3% to ampicillin, 20% to streptomycin, 16% to ticarcillin; resistance to the remaining 12 antimicrobials was less than 10%. Phylogroups A and B1 were most prevalent with A (n = 92, 30%) and B1 (87 = 29%). A total of nine E. coli isolates were confirmed as ESBL producers by double-disk synergy testing and multidrug resistant (MDR) to at least three antimicrobial drug classes. Using WGS, significantly higher numbers of ESBL-E. coli were detected in swine and dairy manure than from any other animal sources, suggesting that these may be the primary animal sources for ESBL producing E. coli. These isolates carry plasmids, such as IncFIA(B), IncFII, IncX1, IncX4, IncQ1, CollRNAI, Col440I, and acquired ARGs aph(6)-Id, aph(3″)-Ib, aadA5, aph(3')-Ia, blaCTX-M-15, blaTEM-1B, mphA, ermB, catA1, sul1, sul2, tetB, dfrA17. One of the E. coli isolates from swine with ST 410 was resistant to nine antibiotics and carried more than 28 virulence factors, and this ST has been shown to belong to an international high-risk clone. Our data suggests that ESBL producing E. coli are widely distributed in different animal sources, but swine and dairy cattle may be their main reservoir.

Increased Antimicrobial and Multidrug Resistance Downstream of Wastewater Treatment Plants in an Urban Watershed.

Development and spread of antimicrobial resistance (AMR) and multidrug resistance (MDR) through propagation of antibiotic resistance genes (ARG) in various environments is a global emerging public health concern. The role of wastewater treatment plants (WWTPs) as hot spots for the dissemination of AMR and MDR has been widely pointed out by the scientific community. In this study, we collected surface water samples from sites upstream and downstream of two WWTP discharge points in an urban watershed in the Bryan-College Station (BCS), Texas area, over a period of nine months. E. coli isolates were tested for resistance to ampicillin, tetracycline, sulfamethoxazole, ciprofloxacin, cephalothin, cefoperazone, gentamycin, and imipenem using the Kirby-Bauer disc diffusion method. Antimicrobial resistant heterotrophic bacteria were cultured on R2A media amended with ampicillin, ciprofloxacin, tetracycline, and sulfamethoxazole for analyzing heterotrophic bacteria capable of growth on antibiotic-containing media. In addition, quantitative real-time polymerase chain reaction (qPCR) method was used to measure eight ARG - tetA, tetW, aacA, ampC, mecA, ermA, blaTEM, and intI1 in the surface water collected at each time point. Significant associations (p < 0.05) were observed between the locations of sampling sites relative to WWTP discharge points and the rate of E. coli isolate resistance to tetracycline, ampicillin, cefoperazone, ciprofloxacin, and sulfamethoxazole together with an increased rate of isolate MDR. The abundance of antibiotic-resistant heterotrophs was significantly greater (p < 0.05) downstream of WWTPs compared to upstream locations for all tested antibiotics. Consistent with the results from the culture-based methods, the concentrations of all ARG were substantially higher in the downstream sites compared to the upstream sites, particularly in the site immediately downstream of the WWTP effluent discharges (except mecA). In addition, the Class I integron (intI1) genes were detected in high amounts at all sites and all sampling points, and were about ∼20 times higher in the downstream sites (2.5 × 107 copies/100 mL surface water) compared to the upstream sites (1.2 × 106 copies/100 mL surface water). Results suggest that the treated WWTP effluent discharges into surface waters can potentially contribute to the occurrence and prevalence of AMR in urban watersheds. In addition to detecting increased ARG in the downstream sites by qPCR, findings from this study also report an increase in viable AMR (HPC) and MDR (E. coli) in these sites. This data will benefit establishment of improved environmental regulations and practices to help manage AMR/MDR and ARG discharges into the environment, and to develop mitigation strategies and effective treatment of wastewater.

Elevated Incidences of Antimicrobial Resistance and Multidrug Resistance in the Maumee River (Ohio, USA), a Major Tributary of Lake Erie.

Maumee River, the major tributary in the western basin of Lake Erie, serves as one of major sources of freshwater in the area, supplying potable, recreational, and industrial water. In this study we collected water samples from four sites in the Maumee River Bay between 2016-2017 and E. coli was isolated, enumerated, and analyzed for antimicrobial resistance (AMR) and multidrug resistance (MDR). Strikingly, 95% of the total isolates were found to be resistant to at least one antibiotic. A very high resistance to the drugs cephalothin (95.3%), ampicillin (38.3%), tetracycline (8.8%), gentamicin (8.2%), ciprofloxacin (4.2%), cefoperazone (4%), and sulfamethoxazole (1.5%) was observed within isolates from all four sampling sites. Percentages of AMR and MDR was consistently very high in the summer and fall months, whereas it was observed to be lowest in the winter. A remarkably high number of the isolates were detected to be MDR-95% resistant to ≥1 antibiotic, 43% resistant to ≥2 antibiotics, 15% resistant to ≥3 antibiotics, 4.9% resistant to ≥4 antibiotic and 1.2% resistant to ≥5 antibiotics. This data will serve in better understanding the environmental occurrence and dissemination of AMR/MDR in the area and assist in improving and establishing control measures.

Decomposition of poultry litter organic matter co-applied with industrial and agricultural products/by-products.

Increasing soil organic matter (SOM) is one purpose of applying manures to soils, but soil-applied manures decompose and disappear in a short time, leaving very little trace as SOM. The objective of this study was to test and identify agricultural and industrial products and by-products (PBPs) that reduce the speed of manure decomposition and, potentially, increase SOM. Raw poultry litter (PL) was amended with selected PBPs (15% fresh weight) and incubated for 1-3 mo. Unamended PL lost an average of 19% of its dry weight after 1 mo incubation and 24% of its dry weight after 3 mo. Monitoring the CO2 release during a 1-mo incubation revealed that decomposition and weight loss of unamended PL is greatest in the first 2 d. Amending PL with Al2 (SO4 )3 · 18H2 O and CaO reduced cumulative CO2 release and final dry biomass loss during the incubation period of 1-3 mo. Amending PL with Al2 (SO4 )3 · 18H2 O reduced PL temperature by up to 14 °C and pH by ∼4.0, whereas CaO elevated its temperature by up to 24 °C and pH by ∼4.0. Both products suppressed total culturable bacteria and reduced dehydrogenase activity soon after mixing. Amending PL with flue gas desulfurization gypsum, CaCO3 , cement kiln dust, or biochar either enhanced or had no effect on suppressing litter decomposition. Our results overall show that the decomposition of PL and possibly other manures may be slowed and that the soil-residence life of manure C may be increased using PBPs that raise or lower manure pH and temperature.

Electrochemical biofilm control by reconstructing microbial community in agricultural water distribution systems.

Biofilm causes considerable technical challenges in agricultural water distribution systems. Electrochemical treatment (ECT) is a potential technique for controlling biofilm in the systems. Given the limited information on how ECT performance changes of irrigation systems and microbial biofilm community shifts. In this study, the effect of anti-biofilm was assessed. Illumina Miseq high-throughput sequencing, combined with molecular ecological network analysis, were applied to detect the effects of ECT on attached biofilm microbial communities. We found that ECT effectively mitigated biofilm formation with the fixed-biofilm biomass reduced by 37.5 %-79.9 %. ECT significantly shifted the bacterial community structures in the biofilm, reduced the communities' diversity, and changed the dominant species. Molecular ecological network analysis showed that the complexity and size of bacterial networks were destabilized under ECT and decreased the interactions among bacterial species. The reconstruction in bacterial community and networks were responsible for the decline in extracellular polymer substances and biofilm biomass. However, chlorine-resistant bacteria were found increased after ECT, and higher relative abundance and low biofilm removal was identified in continuous ECT as compared with intermittent ECT. These results aimed to highlight the opportunity for biofouling mitigation by ECT for irrigation systems, and reveal the potential anti-biofilm microbial mechanisms of ECT.

A preliminary investigation of wild pig (Sus scrofa) impacts in water quality.

The United States, particularly the southern portion, has recently suffered drastic population expansion of wild pigs causing destruction of prime farmland. An associated concern, which has been understudied, is the potential transfer of nutrients and pathogens to surface water. This study aimed to identify the abiotic and biotic impacts of captive wild pigs on water quality, including nutrients, fecal indicator and pathogenic bacteria, and antimicrobial resistance. Overall, the study demonstrated that wild pigs harbored Salmonella spp., Campylobacter spp., Escherichia coli, and Clostridium perfringens, which were found in water runoff collected directly beneath the hog paddock, often 2 log10 greater than above-paddock levels. However, the impacts to downstream water quality were limited, perhaps because of a relatively large riparian buffer between the paddock and surface water. A higher rate of ammonium concentration changes over time was detected in the runoff water below the paddock; additionally, microbial releases detected in runoff were also time dependent, possibly associated with increasing pig numbers. Antibiotic resistance was generally not associated with the wild pigs. Antibiotic resistance genes were found in upstream as well as downstream surface water, suggesting that nonpoint sources of microbial contamination were present. Interestingly, intI1 levels were greater in below-paddock runoff by nearly 2 log10 . Overall, it appears that wild pigs potentially pose a threat to water quality but only if they have direct access to the water. Pathogen, fecal indicator bacteria, and some nutrient release were significantly associated with wild pigs, but riparian buffers limited water quality impairment.

Using cameras to index waterfowl abundance in winter-flooded rice fields.

Extensive wetland habitat loss across the continental United States has caused post-harvested rice fields to become an important surrogate wetland habitat for migratory waterfowl. Flooded rice fields used by waterfowl have the potential to provide agronomic benefits to soil. Increasing interest in the reciprocal relationship between birds and flooded rice fields has given rise to many studies that aim to quantify bird abundance. However, surveying large flocks of birds in open agricultural fields is challenging because traditional ground and aerial surveys can cause birds to flush or re-allocate spatially, thus biasing counts that are reflected in following management practice recommendations. To avoid this, we used camera surveys and an open-access image manipulation program to estimate 24-h bird use of rice fields. Indices of bird abundance from counts were used to estimate fecal matter input to rice fields. Camera surveys have the potential to limit biases seen in other methods because of their ability to capture bird use over a 24-h period over an entire season and the ability for multiple researchers to survey the same site.•Surveying bird flocks by traditional ground or aerial surveys can bias bird abundance estimates.•Camera surveys of waterfowl in rice fields were used to estimate bird abundance and fecal matter input.•Camera surveys reflect static bird use over 24-h which can lower bias seen in traditional methods.

Investigating the role of organic carbon amendments and microbial denitrification gene abundance in nitrogen removal from experimental agricultural drainage ditches with low-grade weirs.

Low-grade weirs placed within agricultural drainage ditches in the Lower Mississippi Alluvial Valley can be used as a management practice to enhance nitrogen removal. The addition of organic carbon amendments within ditches that contain weirs could further increase nitrogen removal. Through repeated trials, changes in NO 3 - -N concentration between inflow and outflow were variable in the ditch without weirs, while only decreases in concentration were observed in ditches with weirs. Significant differences in NO 3 - -N concentrations were observed between treatments, with greater removal of NO 3 - -N observed in dissolved organic carbon treatments compared to control and particulate organic carbon treatments. At medium- and high-flow rates, respectively, dissolved organic carbon treatments resulted in greater NO 3 - -N concentration decreases of 31.6% and 27.1% compared to 19% and 11.6% in particulate organic carbon treatments and 18.6% and 17.2% in control treatments. Significant effects of weirs and sampling date on nirS, nirK, nosZ, and 16S rRNA gene abundances were observed. Observed increases in NO 3 - -N removal with organic carbon amendments, provides support for continued investigation on improving the efficacy of organic carbon amendments as a best management practice for NO 3 - -N removal in agricultural drainage ditches. PRACTITIONER POINTS: Dissolved organic carbon amendments increased nitrate-nitrogen removal. Only decreases in nitrate-nitrogen concentration were observed in ditches with weirs. Increasing flow rate did not affect nitrate-nitrogen removal. Abundance of denitrification-performing microbes likely did not affect N removal. Lack of anaerobic soil conditions and short residence time reduced nitrate-N removal.

Bacterial Community Structure Recovery in Reclaimed Coal Mined Soil under Two Vegetative Regimes.

Coal mining can be deleterious to the soil physical and chemical makeup, but also to the soil microbial community. Effectively, the removal of nearly all organic matter from the upper soil horizons reduces the effectiveness of any soil to support vegetation, and up until recently, microbial community parameters were not considered in the successful reclamation of overburden. Thus, our study proposes to measure the uncultivated bacterial community using 16S ribosomal RNA (rRNA) high-throughput sequencing in a chronosequence of reclaimed overburden in Mississippi. The study sites comprised samplings of pasture and wooded reclamation sites consisting of 1 to 13 yr post reclamation time, as well as reference sites. Overall, the primary driver of bacterial community dynamics was vegetative cover, although time also influenced dynamics. Richness estimations for operational taxonomic units (OTUs) showed that recently reclaimed (∼1 yr) and Pasture sites were more OTU rich with levels of >1400 compared with reference site levels of ∼1000. Diversity levels also followed a similar trend. Community structure typically differed between time points and vegetative cover; however, membership was similar between sites and reference, indicating that new communities still shared some membership from the previous community. Overall, physicochemical properties trended toward more positive for soil health as time progressed, but bacterial community recovery was still not structurally recovered, although richness and diversity values exceeded reference. Overall, this study demonstrated that mine reclamation using pasture and/or wood restoration can reestablish the bacterial community to approximate reference conditions, but vegetation is still the dominating environmental factor dictating microbial community.

The influence of chlorination timing and concentration on microbial communities in labyrinth channels: implications for biofilm removal.

Chlorination is an effective method to control biofilm formation in enclosed pipelines. To date, very little is known about how to control biofilms at the mesoscale in complex pipelines through chlorination. In this study, the dynamic of microbial communities was examined under different residual chlorine concentrations on the biofilms attached to labyrinth channels for drip irrigation using reclaimed water. The results indicated that the microbial phospholipid fatty acids, extracellular polymeric substances, microbial dynamics, and the ace and Shannon microbial diversity indices showed a gradual decrease after chlorination. However, chlorination increased microbial activity by 0.5-19.2%. The increase in the relative abundances of chloride-resistant bacteria (Acinetobacter and Thermomonas) could lead to a potential risk of chlorine resistance. Thus, keeping a low chlorine concentration (0.83 mg l-1 for 3 h) is effective for controlling biofilm formation in the labyrinth channels.

Removal of fecal indicator bacteria and antibiotic resistant genes in constructed wetlands.

Wastewater discharge evidently increased bacterial diversity in the receiving waterbodies. The objective of this study was to evaluate the effectiveness of a constructed wetland in reducing fecal indicator bacteria (FIB) and antibiotic resistant genes (ARGs). We determined the prevalence and attenuation of fecal indicator bacteria including Escherichia coli and enterococci, along with ARGs, and human-associated Bacteroidales (HF183) markers by quantitative polymerase chain reaction (qPCR) method. Three types of water samples (inlet, intermediate, and outlet) from a constructed wetland were collected once a month from May to December in 2013. The overall reduction of E. coli was 50.0% based on culture method. According to the qPCR result, the overall removal rate of E. coli was only 6.7%. Enterococci were found in 62.5% of the wetland samples. HF183 genetic marker was detected in all final effluent samples with concentration ranging from 1.8 to 4.22 log10 gene copies (GC)/100 ml. Of the ARGs tested, erythromycin resistance genes (ermF) were detected in 79.2% of the wetland samples. The class 1 integrase (intI1) was detected in all water samples with concentration ranging from 0.83 to 5.54 log10 GC/100 ml. The overall removal rates of enterococci, HF183, intI1, and ermF were 84.0%, 66.6%, 67.2%, and 13.1%, respectively.

Effects of Low-Grade Weirs on Soil Microbial Communities in Agricultural Drainage Ditches.

Agricultural fertilizer application throughout the Mississippi River basin has been identified as a major source of N pollution to the Gulf of Mexico. Using best management practices, such as low-grade weirs, has been identified as a potential solution to mitigate nutrient loads in agricultural runoff. This study assessed impacts of weir implementation in four agricultural drainage ditches (three with weirs and one control site) in the Mississippi Delta. Soil samples collected from field locations in spring 2013 were analyzed for denitrifier abundance using genes (16s ribosomal RNA [rRNA] genes, , , and ) via quantitative polymerase chain reaction (qPCR), microbial community profiles via terminal-restriction fragment length polymorphism (T-RFLP) of 16s rRNA genes, soil parameters (C, N, and moisture), and vegetation presence at sample locations. Gene quantification was successful, except for , which was found below detection limits (5000 gene copies g soil). Distance from weirs was negatively correlated with 16S rRNA genes and soil moisture, and soil moisture was positively correlated with 16s rRNA and S gene abundance. Results of empirical Bayesian kriging did not exhibit obvious patterns of microbial diversity in relation to weir proximity. Preliminary assessment of seasonal trends showed genes 16s rRNA and , soil N, and mean T-RF values to be greater in fall than in spring. Results highlight that weirs had no direct impact on microbial diversity or denitrification functional gene abundance. Correlations between microbial measures and environmental parameters suggest that adequate management of N runoff from agricultural landscapes will require ecological engineering beyond weirs to optimize N mitigation.

Effects of Subsurface Banding and Broadcast of Poultry Litter and Cover Crop on Soil Microbial Populations.

Agronomic management is aimed at managing the crop environment to maximize crop yield, but soil biology is often ignored. This study aimed to compare the application of poultry litter via broadcast and subsurface banding versus standard inorganic fertilizer to cotton ( L.) and their effects on soil bacterial populations and fecal indicator bacteria. The study comprised a randomized complete block design, with fertilizer and time of application as treatment effects and cover crop as a main effect. Soil cores were collected and analyzed from 2008 to 2014. Fecal indicator bacteria were at detection limits for all treatments, where the integron 1 gene was significantly elevated in litter plots. There were few differences between litter application approaches, but both significantly increased key biogeochemical genes over control plots, whereas a cover crop only increased soil moisture and urease C. Data suggested a positive residual effect of litter application with 16S, phosphatase A, and urease C genes elevated over controls, but similar to standard fertilizer plots. High-throughput 16S ribosomal RNA analysis suggested increased diversity and enrichment indices in litter and standard fertilizer over untreated control plots. Litter and standard fertilizer effects persisted 4 and 2 yr after application, respectively, as evidenced by residual library community structures. This study demonstrated the positive effects of litter application on the soil bacterial community when compared with untreated control plots. Some differences between standard fertilization and litter practices were noted and suggest that there is a positive residual effect on soil microbial populations associated with both practices.

Antibiotic Resistant Bacteria in Municipal Wastes: Is There Reason for Concern?

Recently, there has been increased concern about the presence of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG), in treated domestic wastewaters, animal manures and municipal biosolids. The concern is whether these additional sources of ARB contribute to antibiotic resistance levels in the environment, that is, "environmental antibiotic resistance." ARB and ARG occur naturally in soil and water, and it remains unclear whether the introduction of ARB in liquid and solid municipal and animal wastes via land application have any significant impact on the background levels of antibiotic resistance in the environment, and whether they affect human exposure to ARB. In this current review, we examine and re-evaluate the incidence of ARB and ARG resulting from land application activities, and offer a new perspective on the threat of antibiotic resistance to public health via exposure from nonclinical environmental sources. Based on inputs of ARBs and ARGs from land application, their fate in soil due to soil microbial ecology principles, and background indigenous levels of ARBs and ARGs already present in soil, we conclude that while antibiotic resistance levels in soil are increased temporally by land application of wastes, their persistence is not guaranteed and is in fact variable, and often contradictory based on application site. Furthermore, the application of wastes may not produce the most direct impact of ARGs and ARB on public health. Further investigation is still warranted in agriculture and public health, including continued scrutiny of antibiotic use in both sectors.

Within-House Spatial Distribution of Fecal Indicator Bacteria in Poultry Litter.

Land application of poultry litter is often considered to be a major source of water pollutants in poultry-producing regions. However, reported levels of fecal indicator microorganisms in litter vary widely, with considerable variation possible within houses and across farms, depending on management practices. Therefore, a study was conducted to determine the levels and distribution of indicator microorganisms within 12 broiler farms representing three companies. Within each house, litter samples were collected from around the feed line, water line, north wall, cool pad end, middle, and fan end. Litter moisture content was significantly different within the houses, with the litter being driest around the feed line (19.8%) and wettest around the water line (40.7%). Mean levels of total coliforms, , enterococci, and were 3.7, 3.3, 6.4, and 4.0 log colony-forming units g dry litter, respectively. Levels of total coliforms, , and were positively correlated with litter moisture content, but enterococci levels were not. Consequently, levels of total coliforms, , and , as well as enterococci, were highest around the water line and lowest around the feed line. These results indicate that areas with higher litter water content are more likely to contain higher levels of most fecal indicator microorganisms. Approaches to reduce litter water content in these areas would not only benefit the microbial quality of litter for land application but would also likely improve in-house disease control.

Broiler Litter × Industrial By-Products Reduce Nutrients and Microbial Losses in Surface Runoff When Applied to Forages.

The inability to incorporate broiler litter (BL) into permanent hayfields and pastures leads to nutrient accumulation near the soil surface and increases the potential transport of nutrients in runoff. This study was conducted on Marietta silt loam soil to determine the effect of flue gas desulfurization (FGD) gypsum and lignite on P, N, C, and microbial concentrations in runoff. Treatments were (i) control (unfertilized) and (ii) BL at 13.4 Mg ha alone or (iii) treated with either FGD gypsum or lignite applied at 20% (w/w) (2.68 Mg ha). Rainfall simulators were used to produce a 5.6 cm h storm event sufficient in duration to cause 15 min of continuous runoff. Repeated rains were applied at 3-d intervals to determine how long FGD gypsum and lignite are effective in reducing loss of litter-derived N, P, and C from soil. Application of BL increased N, P, and C concentrations in runoff as compared to the control. Addition of FGD gypsum reduced ( < 0.05) water-soluble P and dissolved organic C concentrations in runoff by 39 and 16%, respectively, as compared to BL alone. Lignite reduced runoff total N and NH-N concentrations by 38 and 70%, respectively, as compared to BL alone. Addition of FGD gypsum or lignite failed to significantly reduce microbial loads in runoff, although both treatments reduced microbial concentration by >20%. Thus, BL treated with FGD and lignite can be considered as cost-effective management practices in the mitigation of P, N, and C and possibly microbial concentration in runoff.

Supplemental invasion of Salmonella from the perspective of Salmonella enterica serovars Kentucky and Typhimurium.

BACKGROUND: Critical to the development of Salmonellosis in humans is the interaction of the bacterium with the epithelial lining of the gastrointestinal tract. Traditional scientific reasoning held type III secretion system (T3SS) as the virulence factor responsible for bacterial invasion. In this study, field-isolated Salmonella enterica serovar Kentucky and a known human pathogen Salmonella enterica serovar Typhimurium were mutated and evaluated for the invasion of human colorectal adenocarcinoma epithelial cells. RESULTS: S. enterica serovar Kentucky was shown to actively invade a eukaryotic monolayer, though at a rate that was significantly lower than Typhimurium. Additionally, strains mutated for T3SS formation were less invasive than the wild-type strains, but the decrease in invasion was not significant in Kentucky. CONCLUSIONS: Strains mutated for T3SS formation were able to initiate invasion of the eukaryotic monolayer to varying degrees based on strain, In the case of Kentucky, the mutated strain initiated invasion at a level that was not significantly different from the wild-type strain. A different result was observed for Typhimurium as the mutation significantly lowered the rate of invasion in comparison to the wild-type strain.

Salmonella enterica Serovar Kentucky Flagella Are Required for Broiler Skin Adhesion and Caco-2 Cell Invasion.

Nontyphoidal Salmonella strains are the main source of pathogenic bacterial contamination in the poultry industry. Recently, Salmonella enterica serovar Kentucky has been recognized as the most prominent serovar on carcasses in poultry-processing plants. Previous studies showed that flagella are one of the main factors that contribute to bacterial attachment to broiler skin. However, the precise role of flagella and the mechanism of attachment are unknown. There are two different flagellar subunits (fliC and fljB) expressed alternatively in Salmonella enterica serovars using phase variation. Here, by making deletions in genes encoding flagellar structural subunits (flgK, fliC, and fljB), and flagellar motor (motA), we were able to differentiate the role of flagella and their rotary motion in the colonization of broiler skin and cellular attachment. Utilizing a broiler skin assay, we demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables Salmonella motility, but this subunit is unable to mediate tight attachment. Deletion of the flgK gene prevents proper flagellar assembly, making Salmonella significantly less adherent to broiler skin than the wild type. S Kentucky with deletions in all three structural genes, fliC, fljB, and flgK, as well as a flagellar motor mutant (motA), exhibited less adhesion and invasion of Caco-2 cells, while an fljB mutant was as adherent and invasive as the wild-type strain. IMPORTANCE: In this work, we answered clearly the role of flagella in S Kentucky attachment to the chicken skin and Caco-2 cells. We demonstrated that the presence of FliC is necessary for attachment to broiler skin. Expression of the alternative flagellar subunit FljB enables Salmonella motility, but this subunit is unable to mediate strong attachment. Deletion of the flgK gene prevents proper flagellar assembly, making Salmonella significantly less adherent to broiler skin than the wild type. S Kentucky with deletions in all three structural genes, fliC, fljB, and flgK, as well as a flagellar motor mutant (motA), exhibited less adhesion and invasion of Caco-2 cells, while an fljB mutant was as adherent and invasive as the wild-type strain. We expect these results will contribute to the understanding of the mechanisms of Salmonella attachment to food products.

Investigation of denitrifying microbial communities within an agricultural drainage system fitted with low-grade weirs.

Enhancing wetland characteristics in agricultural drainage ditches with the use of low-grade weirs, has been identified as a best management practice (BMP) to mitigate nutrient runoff from agriculture landscapes. A major objective of utilizing low-grade weirs as a BMP includes fostering environments suitable for the biogeochemical removal of nitrogen via denitrification. This study examined the spatial resolution of microbial communities involved in denitrification in agricultural drainage systems fitted with low-grade weirs. Appropriate sampling scales of microbial communities were investigated using 16S rRNA and denitrification functional genes nosZ, nirS, and nirK via quantitative polymerase chain reaction (qPCR) and terminal-restriction fragment length polymorphism (T-RFLP) analysis. Genes 16S rRNA, nosZ, and nirS were all successfully detected in soil samples, while nirK was below the detection limit throughout the study. Utilizing a combination of three sampling regimes (management, reach, catchment) was found to be effective in capturing microbial community patterns, as ANOVA results revealed nosZ gene abundance was significantly greater at the management rather than reach scale (p = 0.045; F = 3.311), although, no significant differences were observed in 16S rRNA or nirS between sampling scales (p > 0.05). A Pearson correlation matrix confirmed that 16S rRNA and nosZ gene abundances were positively correlated with soil carbon (C), nitrogen (N), and moisture, while nirS abundance was only positively correlated with soil C and soil moisture. This highlights the potential for wetland-like characteristics to be recovered in agricultural drainage systems, as weir proximity is observed to enhance soil moisture and conditions for N remediation. This study provides the basis for additional investigations of these unique environments in the Mississippi Alluvial Valley and a starting point for adaptive management to enhance agricultural drainage systems for microbial communities towards nutrient remediation goals.