Impact of Raised without Antibiotics Measures on Antimicrobial Resistance and Prevalence of Pathogens in Sow Barns.

The growing concern over the emergence of antimicrobial resistance (AMR) in animal production as a result of extensive and inappropriate antibiotic use has prompted many swine farmers to raise their animals without antibiotics (RWA). In this study, the impact of implementing an RWA production approach in sow barns on actual on-farm antibiotic use, the emergence of AMR, and the abundance of pathogens was investigated. Over a 13-month period, fecal and nasopharynx samples were collected at 3-month intervals from sows raised in RWA barns and sows in conventional barns using antibiotics in accordance with the new regulations (non-RWA). Whole genome sequencing (WGS) was used to determine the prevalence of AMR and the presence of pathogens in those samples. Records of all drug use from the 13-month longitudinal study indicated a significant reduction in antimicrobial usage in sows from RWA barns compared to conventional non-RWA barns. Antifolates were commonly administered to non-RWA sows, whereas ß-lactams were widely used to treat sows in RWA barns. Metagenomic analyses demonstrated an increased abundance of pathogenic Actinobacteria, Firmicutes, and Proteobacteria in the nasopharynx microbiome of RWA sows relative to non-RWA sows. However, WGS analyses revealed that the nasal microbiome of sows raised under RWA production exhibited a significant increase in the frequency of resistance genes coding for ß-lactams, MDR, and tetracycline.

Piglet Gut and in-Barn Manure from Farms on a Raised without Antibiotics Program Display Reduced Antimicrobial Resistance but an Increased Prevalence of Pathogens.

In response to new stringent regulations in Canada regarding the use of antibiotics in animal production, many farms have implemented practices to produce animals that are raised without antibiotics (RWA) from birth to slaughter. This study aims to assess the impact of RWA production practices on reducing the actual total on-farm use of antibiotics, the occurrence of pathogens, and the prevalence of antimicrobial resistance (AMR). A 28-month longitudinal surveillance of farms that adopted the RWA program and conventional farms using antibiotics in accordance with the new regulations (non-RWA) was conducted by collecting fecal samples from 6-week-old pigs and composite manure from the barn over six time points and applying whole-genome sequencing (WGS) to assess the prevalence of AMR genes as well as the abundance of pathogens. Analysis of in-barn drug use records confirmed the decreased consumption of antibiotics in RWA barns compared to non-RWA barns. WGS analyses revealed that RWA barns had reduced the frequency of AMR genes in piglet feces and in-barn manure. However, metagenomic analyses showed that RWA barns had a significant increase in the frequency of pathogenic Firmicutes in fecal samples and pathogenic Proteobacteria in barn manure samples.

A health metadata-based management approach for comparative analysis of high-throughput genetic sequences for quantifying antimicrobial resistance reduction in Canadian hog barns.

New Canadian regulations have required that all use of antibiotics in livestock animal production should be under veterinary prescription and oversight, while the prophylactic use and inclusion of these agents in animal feed as growth promoters are also banned. In response to this new rule, many Canadian animal producers have voluntarily implemented production practices aimed at producing animals effectively while avoiding the use of antibiotics. In the swine industry, one such program is the 'raised without antibiotics' (RWA) program. In this paper, we describe a comprehensive investigative methodology comparing the effect of the adoption of the RWA approach with non-RWA pig production operations where antibiotics may still be administered on animals as needed. Our experimental approach involves a multi-year longitudinal investigation of pig farming to determine the effects of antibiotic usage on the prevalence of antimicrobial resistance (AMR) and pathogen abundance in the context of the drug exposures recorded in the RWA versus non-RWA scenarios. Surveillance of AMR and pathogens was conducted using whole-genome sequencing (WGS) in conjunction with open source tools and data pipeline analyses, which inform on the resistome, virulome and bacterial diversity in animals and materials associated with the different types of barns. This information was combined and correlated with drug usage (types and amounts) over time, along with animal health metadata (stage of growth, reason for drug use, among others). The overarching goal was to develop a set of interconnected informatic tools and data management procedures wherein specific queries could be made and customized, to reveal statistically valid cause/effect relationships. Results demonstrating possible correlations between RWA and AMR would support the Canadian pig industry, as well as regulatory agencies in new efforts, focused on reducing overall antibiotics use and in curbing the development and spread of AMR related to animal agriculture.

Microscale and molecular analyses of river biofilm communities treated with microgram levels of cerium oxide nanoparticles indicate limited but significant effects.

Cerium oxide (CeO2) nanoparticles are used as in-fuel catalysts and in manufacturing processes, creating a potential for release to aquatic environments. Exposures at 1 and 10 µg/L CeO2-nanoparticles were made to assess effects during the development of river biofilm communities. Scanning transmission x-ray microscopy (STXM) indicated extensive sorption of nanoparticles to the community and co-localization with lipid moieties. Following 8 weeks of development, polycarbonate coupons were removed from the reactors and used for molecular analyses, denaturing gradient gel electrophoresis analysis (DGGE-16S rRNA) and 16S rRNA amplicon sequencing. Microscopic imaging of the biofilm communities (bacterial, photosynthetic biomass, exopolymer composition, thickness, protozoan numbers), as well as carbon substrate utilization fingerprinting was performed. There was a trend toward reduced photosynthetic biomass, but no significant effects of CeO2 exposure were found on photosynthetic and bacterial biomass or biofilm thickness. Sole carbon source utilization analyses indicated increased utilization of 10 carbon sources in the carbohydrate, carboxylic acid and amino acids categories related to CeO2 exposures; however, predominantly, no significant effects (p < 0.05) were detected. Measures of microbial diversity, lectin binding affinities of exopolymeric substances and results of DGGE analyses, indicated significant changes to community composition (p < 0.05) with CeO2 exposure. Increased binding of the lectin Canavalia ensiformis was observed, consistent with changes in bacterial-associated polymers. Whereas, no significant changes were observed in binding to residues associated with algal and cyanobacterial exopolymers. 16S rRNA amplicon sequencing of community DNA indicated changes in diversity and shifts in community composition; however, these did not trend with increasing CeO2 exposure. Counting of protozoans in the biofilm communities indicated no significant effects on this trophic level. Thus, based on biomass and functional measures, CeO2 nanoparticles did not appear to have significant effects; however, there was evidence of selection pressure resulting in significant changes in microbial community composition.

Role of CpxR in Biofilm Development: Expression of Key Fimbrial, O-Antigen and Virulence Operons of Salmonella Enteritidis.

Salmonella Enteritidis is a non-typhoidal serovar of great public health significance worldwide. The RpoE sigma factor and CpxRA two-component system are the major regulators of the extracytoplasmic stress response. In this study, we found that the CpxR has highly significant, but opposite effects on the auto-aggregation and swarming motility of S. Enteritidis. Auto-aggregation was negatively affected in the ∆cpxR mutant, whereas the same mutant significantly out-performed its wild-type counterpart with respect to swarming motility, indicating that the CpxR plays a role in biofilm-associated phenotypes. Indeed, biofilm-related assays showed that the CpxR is of critical importance in biofilm development under both static (microtiter plate) and dynamic (flow cell) media flow conditions. In contrast, the RpoE sigma factor showed no significant role in biofilm development under dynamic conditions. Transcriptomic analysis revealed that the cpxR mutation negatively affected the constitutive expression of the operons critical for biosynthesis of O-antigen and adherence, but positively affected the expression of virulence genes critical for Salmonella-mediated endocytosis. Conversely, CpxR induced the expression of curli csgAB and fimbrial stdAC operons only during biofilm development and flagellar motAB and fliL operons exclusively during the planktonic phase, indicating a responsive biofilm-associated loop of the CpxR regulator.

Growth and Virulence of Salmonella Typhimurium Mutants Deficient in Iron Uptake.

The present study investigated the effects of iron, iron chelators, and mutations of tonB or iroN fepA genes on the growth and virulence of Salmonella Typhimurium. Results indicated that organic iron (ferric citrate and ferrous-l-ascorbate) supported better growth of Salmonella compared to inorganic iron. Among tested chelators, 2,2'-bipyridyl at 500 µM showed the highest inhibition of Salmonella growth with 5 µM ferrous sulfate. Deletion of genes (tonB- and iroN- fepA- ) in the iron uptake system attenuated Salmonella invasion of Caco-2 cells and its ability to damage the epithelial monolayer. The expression of all tested host genes in Caco-2 was not affected under the iron-poor condition. However, claudin 3, tight junction protein 1, tumor necrosis factor α (TNF-α), and interleukin-8 (IL-8) were altered under the iron-rich condition depending on individual mutations. In Caenorhabditis elegans, a significant down-regulation of ferritin 1 expression was observed when the nematode was infected by the wild-type (WT) strain.

Regulation of waaH by PhoB during Pi Starvation Promotes Biofilm Formation by Escherichia coli O157:H7.

In open environments such as water, enterohemorrhagic Escherichia coli O157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. This activates the phosphate-specific transport (Pst) system that contains a high-affinity Pi transporter. In the Δpst mutant, PhoB is constitutively activated and regulates the expression of genes in the Pho regulon. Here, we show that Pi starvation and deletion of the pst system enhance E. coli O157:H7 biofilm formation. Among differentially expressed genes of EDL933 grown under Pi starvation conditions and in the Δpst mutant, we have found that a member of the PhoB regulon, waaH, predicted to encode a glycosyltransferase, was highly expressed. Interestingly, WaaH contributed to biofilm formation of E. coli O157:H7 during both Pi starvation and in the Δpst mutant. In the Δpst mutant, the presence of waaH was associated with lipopolysaccharide (LPS) R3 core type modifications, whereas in E. coli O157:H7, waaH overexpression had no effect on LPS structure during Pi starvation. Therefore, waaH participates in E. coli O157:H7 biofilm formation during Pi starvation, but its biochemical role remains to be clarified. This study highlights the importance of the Pi starvation stress response to biofilm formation, which may contribute to the persistence of E. coli O157:H7 in the environment.IMPORTANCE Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen that causes bloody diarrhea that can result in renal failure. Outside of mammalian hosts, E. coli O157:H7 survives for extended periods of time in nutrient-poor environments, likely as part of biofilms. In E. coli K-12, the levels of free extracellular Pi affect biofilm formation; however, it was unknown whether Pi influences biofilm formation by E. coli O157:H7. Our results show that upon Pi starvation, PhoB activates waaH expression, which favors biofilm formation by E. coli O157:H7. These findings suggest that WaaH is a target for controlling biofilm formation. Altogether, our work demonstrates how adaptation to Pi starvation allows E. coli O157:H7 to occupy different ecological niches.

Staphylococcus aureus Inhibits IL-8 Responses Induced by Pseudomonas aeruginosa in Airway Epithelial Cells.

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam3CysSK4 but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.

Shiga toxins decrease enterohaemorrhagic Escherichia coli survival within Acanthamoeba castellanii.

Enterohaemorrhagic Escherichia coli (EHEC) are zoonotic pathogens transmitted to humans through contaminated water or bovine products. One of the strategies used by pathogenic bacteria to survive in aquatic environments is using free-living amoebae as hosts. Acanthamoeba castellanii is an amoeba known to host several waterborne pathogens. This study investigates the survival of EHEC with A. castellanii, which could contribute to its spread and transmission to humans. We used a gentamicin protection assay as well as fluorescence and electron microscopy to monitor the intra-amoebae survival of EHEC O157:H7 over 24 h. The results showed that EHEC were able to survive within A. castellanii and that this survival was reduced by Shiga toxins (Stx) produced by EHEC. A toxic effect mediated by Stx was demonstrated by amoebae mortality and LDH release during co-culture of EHEC and amoeba. This work describes the ability of EHEC to survive within A. castellanii, and this host-pathogen interaction is partially controlled by the Stx. Thus, this ubiquitous amoeba could represent an environmental niche for EHEC survival and transmission.