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Microbial ecology on integrated crop–livestock farms (ICLFs) can impact food safety through pathogen transfer between animals and crops. Recent reports of pathogen-contaminated products sold in local organic retail, roadside, and farmers markets highlight the need for assessment of the ecological patterns of bacterial pathogens. This study investigated the prevalence and antibiotic resistance of the virulent type of diarrheagenic E. coli in ICLFs. Over two years, 2973 samples from ICLFs and markets in Maryland and Washington DC were analyzed. Diarrheagenic E. coli was found in 4.30% (128/2973) of collected samples, with a higher isolation rate in environmental (4.42%, 59/1332) and produce (4.20%, 69/1641) samples. Overall, livestock bedding materials had the highest prevalence (8.51%, 4/47). Post-harvest produce exhibited a lower contamination rate of 1.32% (10/756), whereas pre-harvest produce had a higher incidence with 6.67% contamination (59/885), indicating the presence of E. coli. Alarmingly, 92.30% (72/78) of pathogenic E. coli isolates were resistant to common antibiotics. The findings highlight potential risks associated with integrated farming practices and emphasize the importance of safe harvesting and post-harvesting measures, particularly in the context of the growing popularity of local integrated farming. Implementing precautions at on-farm and market levels is crucial to mitigate the risk of antibiotic-resistant E. coli-related enteric illnesses, safeguarding both consumers and the integrity of integrated farming systems.

The incidence of gastrointestinal illness attributable to Salmonella enterica serovar Typhimurium (ST) remains a concern for public health worldwide, as it can progress into systemic infections mediated by the type-three secretion system (T3SS), which allows for adherence and invasion to intestinal epithelial cells. The current study evaluates the ability of gallic acid (GA), protocatechuic acid (PA), and vanillic acid (VA) to impair the adhesion and invasion abilities of ST to a human epithelial (INT-407) cell monolayer while also assessing their cytotoxicity. GA, PA, and VA inhibited detectable ST growth at specific concentrations but showed cytotoxicity against INT-407 cells (>20% reduction in viability) after 3 h of treatments. Adjusting the pH of the solutions had a neutralizing effect on cytotoxicity, though it did reduce their antimicrobial potency. Adhesion of ST was reduced significantly when the cells were treated with 4.0 mg/mL of VA, whereas invasion was reduced in all treatments, with GA requiring the lowest concentration (0.5 mg/mL). Relative gene expression of virulence genes after treatment with GA showed downregulation in the T3SS regulator and effector hilA and sipA, respectively. These findings suggest further use of phenolic acids in reducing the activity of key virulence factors critical during ST infection.

Salmonella enterica serovar Typhimurium (ST) remains a predominant zoonotic pathogen because of its colonization in poultry, survivability in the environment, and increasing antibiotic-resistance pattern. Plant-derived phenolics, gallic acid (GA), protocatechuic acid (PA), and vanillic acids (VA) have demonstrated antimicrobial activity in vitro; therefore, this study collected chicken cecal fluid and supplemented it with these phenolics to evaluate their potential for eliminating ST and mod-ulating the microbiota of complex environments. ST was quantified through plating, while micro-biome analysis was performed through pair-end 16S-rRNA gene sequencing. CFU/mL of ST in cecal fluid with GA was significantly reduced by 3.28 and 2.78 log at 24 h and 48 h, while PA only had a slight numerical decrease. VA significantly reduced ST by 4.81 and 5.20 log at 24 h and 48 h. Changes in relative abundance of major phyla were observed at 24 h for samples with GA and VA as Firmicute levels increased 8.30% and 20.90%, while Proteobacteria decreased 12.86% and 18.48%, respectively. Significant changes in major genre were observed in Acinetobacter (3.41% for GA) and Escherichia (13.53% for VA), while Bifidobacterium increased (3.44% for GA) and Lactobacillus remained unchanged. Results suggest that phenolic compounds exert different effects on certain pathogens, while supporting some commensal bacteria.

Salmonella enterica (S. enterica) is the most common foodborne pathogen worldwide, leading to massive economic loss and a significant burden on the healthcare system. The primary source of S. enterica remains contaminated or undercooked poultry products. Considering the number of foodborne illnesses with multiple antibiotic resistant S. enterica, new controlling approaches are necessary. Bacteriophage (phage) therapies have emerged as a promising alternative to controlling bacterial pathogens. However, the limitation on the lysis ability of most phages is their species-specificity to the bacterium. S. enterica has various serovars, and several major serovars are involved in gastrointestinal diseases in the USA. In this study, Salmonella bacteriophage-1252 (phage-1252) was isolated and found to have the highest lytic activity against multiple serovars of S. enterica, including Typhimurium, Enteritidis, Newport, Heidelberg, Kentucky, and Gallinarum. Whole-genome sequencing analysis revealed phage-1252 is a novel phage strain that belongs to the genus Duplodnaviria in the Myoviridae family, and consists of a 244,421 bp dsDNA, with a G + C content of 48.51%. Its plaque diameters are approximately 2.5 mm to 0.5 mm on the agar plate. It inhibited Salmonella Enteritidis growth after 6 h. The growth curve showed that the latent and rise periods were approximately 40 min and 30 min, respectively. The burst size was estimated to be 56 PFU/cell. It can stabilize and maintain original activity between 4 °C and 55 °C for 1 h. These results indicate that phage-1252 is a promising candidate for controlling multiple S. enterica serovars in food production.

As a traditional agricultural system, integrated crop-livestock farms (ICLFs) involve the production of animals and crops in a shared environment. The ICLFs in the mid-Atlantic region of the United States practice sustainable manure aging or composting processes to provide an on-farm source of soil amendment for use as natural fertilizer and soil conditioner for crop production. However, crop fertilization by soil incorporation of aged manure or compost may introduce different microbes and alter the soil microbial community. The aim of this study was to characterize the influence of aged or composted manure application on the diversity of soil bacterial community in ICLFs. Soil samples from six ICLFs in Maryland were collected before (pre-crop) and during the season (2020–2021) and used to analyze soil bacterial microbiome by 16S rDNA sequencing. Results showed that both phylum- and genus-level alterations of soil bacterial communities were associated with amendment of aged or composted manure. Particularly, Proteobacteria and Actinobacteria were enriched, while Acidobacteria, Bacteroidetes, Planctomycetes, Firmicutes, and Chloroflexi were reduced after manure product application. Meanwhile, the relative abundance of Bacillus was decreased, while two zoonotic pathogens, Salmonella and Listeria, were enriched by manure amendments. Overall, animal manure amendment of soil increased the phylogenetic diversity, but reduced the richness and evenness of the soil bacterial communities. Although manure composting management in ICLFs benefits agricultural sustainable production, the amendments altered the soil bacterial communities and were associated with the finding of two major zoonotic bacterial pathogens, which raises the possibility of their potential transfer to fresh horticultural produce crops that may be produced on the manured soils and then subsequently consumed without cooking.

Development of phage-resistant probiotic particularly Lactobacillus is an alternative approach to enhance their beneficial effects as in animal feed supplements. In this study, we developed phage-resistant Lactobacillus plantarum (LP+PR) mutant and compared their antimicrobial effects and probiotic potential against zoonotic bacterial pathogens including Salmonella enterica serovar Typhimurium, enterohemorrhagic Escherichia coli (EHEC), Staphylococcus aureus, and Listeria monocytogenes with phage-sensitive L. plantarum (LP) strain. LP+PR strain showed markedly higher growth rate than wild-type LP strain. In co-culture with LP+PR and in the presence of cell-free cultural supernatants (CFCSs) of LP+PR, the growth of S. Typhimurium, EHEC, S. aureus, and L. monocytogenes were reduced significantly (P < 0.05). The adhesion ability of LP+PR was slightly higher than the LP on human epithelial INT-407 cells. Most importantly, LP+PR strain significantly inhibited the adhesive and invasive abilities of all four zoonotic pathogens to INT-407 cells (P < 0.05). Moreover, real-time qPCR revealed that in the presence of LP+PR strain or its CFCSs, expression of virulence genes of these zoonotic bacterial pathogens were suppressed significantly (P < 0.05). These findings suggest that the LP+PR strain is capable of inhibiting major zoonotic bacterial pathogens efficiently and would be a potential candidate for industrial usage in animal production or fermentation.