Phenomic and genomic approaches to studying the inhibition of multiresistant Salmonella enterica by microcin J25.

In livestock production, antibiotics are used to promote animal growth, control infections and thereby increase profitability. This practice has led to the emergence of multiresistant bacteria such as Salmonella, of which some serovars are disseminated in the environment. The objective of this study is to evaluate microcin J25 as an inhibitor of Salmonella enterica serovars of various origins including human, livestock and food. Among the 116 isolates tested, 37 (31.8%) were found resistant to at least one antibiotic, and 28 were multiresistant with 19 expressing the penta-resistant phenotype ACSSuT. Microcin J25 inhibited all isolates, with minimal inhibitory concentration values ranging from 0.06 µg/ml (28.4 nM) to 400 µg/ml (189 µM). Interestingly, no cross-resistance was found between microcin J25 and antibiotics. Multiple sequence alignments of genes encoding for the different proteins involved in the recognition and transport of microcin J25 showed that only ferric-hydroxamate uptake is an essential determinant for susceptibility of S. enterica to microcin J25. Examination of Salmonella strains exposed to microcin J25 by transmission electronic microscopy showed for the first-time involvement of a pore formation mechanism. Microcin J25 was a strong inhibitor of several multiresistant isolates of Salmonella and may have a great potential as an alternative to antibiotics.

Antimicrobial-Resistant E. coli from Surface Waters in Southwest Ontario Dairy Farms.

Untreated surface waters can be contaminated with a variety of bacteria, including , some of which can be pathogenic for both humans and animals. Therefore, such waters need to be treated before their use in dairy operations to mitigate risks to dairy cow health and milk safety. To understand the molecular ecology of , this study aimed to assess antimicrobial resistance (AMR) in recovered from untreated surface water sources of dairy farms. Untreated surface water samples ( = 240) from 15 dairy farms were collected and processed to isolate . A total of 234 isolates were obtained and further characterized for their serotypes and antimicrobial susceptibility. Of the 234 isolates, 71.4% were pan-susceptible, 23.5% were resistant to one or two antimicrobial classes, and 5.1% were resistant to three or more antimicrobial classes. Whole genome sequence analysis of 11 selected multidrug-resistant isolates revealed AMR genes including and that confer resistance to the critically important extended-spectrum cephalosporins, as well as a variety of plasmids (mainly of the replicon type) and class 1 integrons. Phylogenetic and comparative genome analysis revealed a genetic relationship between some of the sequenced and Shiga toxin-producing O157:H7 (STEC), which warrants further investigation. This study shows that untreated surface water sources contain antimicrobial-resistant which may serve as a reservoir of AMR that could be disseminated through horizontal gene transfer. This is another reason why effective water treatment before usage should be routinely done on dairy farm operations.

Effects of bacterial-derived antimicrobial solutions on shelf-life, microbiota and sensory attributes of raw chicken legs under refrigerated storage condition.

In the present study, bacterial-derived antimicrobial agents included 5 mM reuterin combined with either 103.91 mM lactic acid (RL) or 0.08 µM microcin J25 (RJ) were evaluated for their effects on the microbiota and sensory attributes of raw chicken legs. Peracetic acid (13.67 mM), a conventional chemical commonly used in the poultry industry, was used as a positive control to compare efficacy. The chicken legs were sprayed with antimicrobial solutions and aerobically stored at 4 °C for 10 days. The RL treatment maintained the total viable count below the limit of 7 log CFU/g until the 8th day. Therefore, compared to the nontreated group, shelf-life was extended by 3 days in the RL treated group. The RJ treatment extended the shelf-life to 7 days, which is similar to what was achieved with the use of peracetic acid. Based on culture-independent amplicon sequencing, the RL and RJ treatments affected the microbial community on the chicken legs, inducing a delay in the increase of Pseudomonas, Psychrobacter and Carnobacterium while decreasing of Shigella. Significant decreases in sensory scores occurred in the nontreated group, while slight changes occurred in the combinations treated groups over the same period. Overall, sensory property scores for chicken legs treated with RL and RJ remained higher (P < 0.05) than those treated with peracetic acid or without antimicrobial agents. The antimicrobial combinations delayed the deterioration of sensory attributes throughout the storage period. These results suggest that RL and RJ provide a promising natural-sourced antimicrobial approach to control the growth of spoilage microorganisms on chicken legs.

Effects of drinking water supplementation with Lactobacillus reuteri, and a mixture of reuterin and microcin J25 on the growth performance, caecal microbiota and selected metabolites of broiler chickens.

BACKGROUND: Since the overuse of antibiotics in animal production has led to a selection of antibiotic-resistant pathogens that affect humans and animals as well. Scientists are therefore searching for novel natural alternatives to antibiotics. In this study Lactobacillus reuteri and a combination of reuterin and microcin J25 (RJ) were evaluated as promoters of growth and modulators of the cecal microbiota and metabolite profiles in broiler chickens. One-day-old Cobb 500 male broilers were distributed to 8 treatments: negative control (without antibiotic), positive control (bacitracin), three concentrations of RJ and three doses of L. reuteri plus glycerol. The birds (2176, 34 per pen, 8 pens per treatment) were reared for 35 d. RESULTS: The body weight of the bacitracin and 5 mmol/L reuterin combined with 0.08 µmol/L microcin J25 (10RJ) treatment group was significantly higher than that of the negative control group (P < 0.05). L. reuteri had no significant effect on broiler growth. MiSeq high-throughput sequencing of 16S rRNA showed clustering of cecal microbial operational taxonomic unit diversity according to treatment. The influence of bacitracin and 10RJ on bacterial community overall structure was similar. They promoted Ruminococcaceae, Lachnospiraceae and Lactobacillaceae, increased the relative abundance of Faecalibacterium and decreased the abundance of Bacteroides and Alistipes, while the negative control condition favored Bacteroidaceae and Rikenellaceae. Furthermore, 10RJ increased the concentration of short-chain fatty acid in the cecum and changed the metabolome overall. CONCLUSIONS: These overall suggest that 10RJ can promote a host-friendly gut environment by changing the cecal microbiome and metabolome. This combination of natural antimicrobial agents in the drinking water had a positive effect on broiler growth and may be suitable as an alternative to antibiotic growth promoters.

Inhibitory Activity of Natural Synergetic Antimicrobial Consortia Against Salmonella enterica on Broiler Chicken Carcasses.

The currently most utilized antimicrobial agent in poultry processing facilities is peracetic acid, a chemical increasingly recognized as hazardous to human health. We evaluated the efficacy of mixtures of natural antimicrobial compounds, namely reuterin, microcin J25, and lactic acid, for reducing the viability of Salmonella enterica and total aerobes on broiler chicken carcasses. The compounds were compared singly and in combination with water and 0.1% peracetic acid. The minimum inhibitory concentrations of reuterin, lactic acid, and microcin J25 against S. enterica serovar Enteritidis were respectively 2 mM, 0.31%, and 0.03 µM. In vitro, the combinations of reuterin + lactic acid and reuterin + microcin J25 were synergic, making these compounds effective at four times lower concentrations than those used alone. Salmonella viable counts fell to zero within 10 min of contact with reuterin + lactic acid at 10 times the concentrations used in combination, compared to 18 h in the case of reuterin + microcin J25. Sprayed onto chilled chicken carcasses, this reuterin + lactic acid mixture reduced Salmonella spp. counts by 2.02 Log CFU/g, whereas reuterin + microcin J25 and peracetic acid reduced them by respectively 0.83 and 1.13 Log CFU/g. The synergy of reuterin with lactic acid or microcin J25 as inhibitors of bacterial growth was significant. Applied as post-chill spray, these mixtures could contribute to food safety by decreasing Salmonella counts on chicken carcasses.

Immunostimulatory Potential of Fruits and Their Extracts in Poultry.

The impact of antibiotic use for growth promotion in livestock and poultry production on the rise of antimicrobial resistance (AMR) in bacteria led to the ban of this practice in the European Union in 2006 and a restriction of antimicrobial use (AMU) in animal agriculture in Canada and the United States of America. There is a high risk of infectious diseases such as necrotic enteritis due to Clostridium perfringens, and colibacillosis due to avian pathogenic Escherichia coli in antimicrobial-free broiler chickens. Thus, efficient and cost-effective methods for reducing AMU, maintaining good poultry health and reducing public health risks (food safety) are urgently needed for poultry production. Several alternative agents, including plant-derived polyphenolic compounds, have been investigated for their potential to prevent and control diseases through increasing poultry immunity. Many studies in humans reported that plant flavonoids could modulate the immune system by decreasing production of pro-inflammatory cytokines, T-cell activation, and proliferation. Fruits, especially berries, are excellent sources of flavonoids while being rich in nutrients and other functionally important molecules (vitamins and minerals). Thus, fruit byproducts or wastes could be important resources for value-added applications in poultry production. In the context of the circular economy and waste reduction, this review summarizes observed effects of fruit wastes/extracts on the general health and the immunity of poultry.

Dynamics of antimicrobial resistance and virulence genes in Enterococcus faecalis during swine manure storage.

In this study we used 2 experimental approaches to evaluate the stability of antimicrobial resistance (AMR) phenotypes, selected AMR genes, and selected virulence genes in Enterococcus faecalis during manure storage on a commercial swine farm. Isolates of E. faecalis were obtained directly from fresh fecal material (n = 120) and from the manure storage facility (n = 85) and compared. Tetracycline resistance and the virulence genes cob, esp, eep, and ccf were detected at lower frequency in manure isolates than in fecal isolates. A second approach consisted of immersing in diffusion chambers pure cultures of E. faecalis that varied in their AMR phenotypes and virulence genotypes in the swine manure storage facility for 8 weeks, sampling periodically, and evaluating the recovered strains for changes in their genotypic or phenotypic characteristics. Enterococcus faecalis populations declined exponentially, with rate constants ranging from 0.011 to 0.022 h(-1). Among the AMR and virulence genes examined, 1 AMR gene (sat4) and 7 virulence genes (agrB(fs), cob, cpd, cylB, efaA(fs), enlA, and esp) were lost at low frequencies in the recovered strains. The AMR phenotypes were stable during the incubation, with minimal loss (P > 0.05) of the streptomycin-resistance phenotype. Overall, these results suggest that some attributes of public health significance in populations of E. faecalis decrease in frequency during manure storage.

Molecular Epidemiology of Carbapenemases in Enterobacteriales from Humans, Animals, Food and the Environment.

The Enterobacteriales order consists of seven families including Enterobacteriaceae, Erwiniaceae, Pectobacteriaceae, Yersiniaceae, Hafniaceae, Morganellaceae, and Budviciaceae and 60 genera encompassing over 250 species. The Enterobacteriaceae is currently considered as the most taxonomically diverse among all seven recognized families. The emergence of carbapenem resistance (CR) in Enterobacteriaceae caused by hydrolytic enzymes called carbapenemases has become a major concern worldwide. Carbapenem-resistant Enterobacteriaceae (CRE) isolates have been reported not only in nosocomial and community-acquired pathogens but also in food-producing animals, companion animals, and the environment. The reported carbapenemases in Enterobacteriaceae from different sources belong to the Ambler class A (blaKPC), class B (blaIMP, blaVIM, blaNDM), and class D (blaOXA-48) ß-lactamases. The carbapenem encoding genes are often located on plasmids or associated with various mobile genetic elements (MGEs) like transposons and integrons, which contribute significantly to their spread. These genes are most of the time associated with other antimicrobial resistance genes such as other ß-lactamases, as well as aminoglycosides and fluoroquinolones resistance genes leading to multidrug resistance phenotypes. Control strategies to prevent infections due to CRE and their dissemination in human, animal and food have become necessary. Several factors involved in the emergence of CRE have been described. This review mainly focuses on the molecular epidemiology of carbapenemases in members of Enterobacteriaceae family from humans, animals, food and the environment.

Antibiotic resistance in Escherichia coll and Enterococcus spp. isolates from commercial broiler chickens receiving growth-promoting doses of bacitracin or virginiamycin.

Antibacterial agents such as zinc bacitracin (ZB) and virginiamycin (VG) are used as growth promoting agents (GP) in broiler chicken production. The objective of this study was to evaluate the effect of the use of ZB and VG on the emergence of antibacterial resistance in a commercial broiler chicken farm. Three trials were conducted using 3 different diets: one without antibacterial agents, one containing VG, and one with ZB. Escherichia coli and Enterococcus spp. strains were isolated and tested for their susceptibility to various antibacterial agents. The occurrence of the resistance genes vatD, ermB, and bcrR in Enterococcus spp. isolates was determined by polymerase chain reaction (PCR). Comparative quantification of vatD and bcrR genes in total deoxyribonucleic acid (DNA) extracts from litter was done by SYBR Green Real-Time PCR (QPCR). Escherichia coli and Enterococcus spp. isolates from diet groups had different levels of resistance to various antibacterial agents over time. These GPs did not select for specific antibacterial agent resistance (AAR) in Enterococcus spp. The use of GPs seemed to lower the percentage of E. coli isolates resistant to some antibacterial agents. The presence of the bcrR gene could not explain all resistant phenotypes to ZB. Genes other than vatD and ermB might be involved in the resistance to VG in Enterococcus spp. Use of GPs was not associated with presence of the bcrR gene in DNA extracts from litter, but use of VG was associated with vatD presence.

Genomic and Proteomic Analyses of Salmonella enterica Serovar Enteritidis Identifying Mechanisms of Induced de novo Tolerance to Ceftiofur.

With the alarming proliferation of antibiotic resistance, it is important to understand the de novo development of bacterial adaptation to antibiotics in formerly susceptible lineages, in the absence of external genetic input from existing resistance pools. A strain of ceftiofur susceptible Salmonella enterica serovar Enteritidis ABB07-SB3071 (MIC = 1.0 µg/ml) was successively exposed to sub-MIC of ceftiofur to allow its adaptation for tolerance to a concentration of 2.0 µg/ml of this antibiotic. Genomic and proteomic comparative analyses of the parental strain and induced tolerant derived lineages were performed to characterize underlying mechanisms of de novo adaptation (tolerance). Expression and localization of specific drug-, heme-, sugar-, amino acid-, and sulfate-transporters were altered, as was the localization of the cell membrane stabilizing protein OsmY in the tolerant strains adapted to 2.0 µg/ml compared to the parental isolate lines. This redistribution of existing transporters acts to minimize the concentrations of ceftiofur in the periplasm, by decreasing facilitated import and increasing active efflux and cytosolic sequestration as determined by high performance liquid chromatography quantification of residual total and extracellular ceftiofur after growth. Genetic, subcellular localization, and abundance changes of specific regulators of transcription, translation, and post-translational dynamics in the derived ceftiofur tolerant lineages decrease metabolic strain on cell walls and enhance periplasmic envelop stability against stress. This produces slower growing, more tolerant populations, which deplete free ceftiofur concentrations significantly more than susceptible parental populations (P < 0.05), as measured by recoverable levels of ceftiofur from cultures of equivalent cellular density incubated with equal ceftiofur concentrations. Genetic and abundance changes to specific carbon and nitrogen metabolism enzymes, not traditionally associated with beta-lactam metabolism, establish an enzymatic framework with the potential to detoxify/degrade ceftiofur, while mutations and changes in subcellular localization in specific cell surface factors enhance the stability of the Gram-negative cell envelop despite the compromising effect of ceftiofur. The observed changes highlight generalizable mechanisms of de novo tolerance without horizontal gene transfer, and thus can inform policies to combat antibiotic tolerance and minimize induction of de novo tolerance.

Antibiotic resistance and virulence genes in commensal Escherichia coli and Salmonella isolates from commercial broiler chicken farms.

Antibiotic resistance patterns and the presence of antibiotic and virulence determinants in 74 sorbitol-negative Escherichia coli and 62 Salmonella isolates from nine different broiler chicken farms were investigated. Each farm was supplied by one of three companies that used different antimicrobial agents in feed for growth promotion. The isolates were identified by API 20E for E. coli and by serological tests for Salmonella. The susceptibility of the isolates to antibiotics was determined by Sensititre using the Clinical and Laboratory Standards Institute's breakpoints. Fifty-two E. coli isolates (70.3%) and nine Salmonella isolates (14.52%) were multiresistant to at least nine antibiotics. The multiresistant isolates were evaluated for the presence of tetracycline resistance, integron class 1, and blacMY 2 genes by PCR. Of the 74 E. coli isolates, 55 were resistant to amoxicillin and ceftiofur. Among these 55 resistant E. coli isolates, 45 (81.8%) and 22 (40.0%) were positive for blacMY-2 and qacEdeltal-Sull genes, respectively. Tetracycline resistance was found in 56 isolates (75.8%) among which 12 (21.4%) and 24 (42.9%) gave positive results for tetA and tetB, respectively. Virulence genes (iss, tsh, and traT), aerobactin operon (iucC), and the eaeA gene were detected in some E. coli strains. Among the 27 amoxicillin- and ceftiofur-resistant Salmonella isolates, the blacMY-2 gene was detected in 22 isolates. The class 1 integron gene (qacEdeltal-Sull) was not detected in any Salmonella isolates, whereas the invasin (inv) and virulence (spy) genes were found in 61 (98.4%) and 26 (42%) of the Salmonella isolates, respectively. This study indicated that multiple antibiotic-resistant commensal E. coli and Salmonella strains carrying virulence genes can be found on commercial broiler chicken farms and may provide a reservoir for these genes in chicken production facilities. Except for the presence of tetB, there was no significant effect of feed formulations on the phenotypic or genotypic characteristics of the isolates.

Genomic Analysis of Third Generation Cephalosporin Resistant Escherichia coli from Dairy Cow Manure.

The production of extended-spectrum ß-lactamases (ESBLs) conferring resistance to new derivatives of ß-lactams is a major public health threat if present in pathogenic Gram-negative bacteria. The objective of this study was to characterize ceftiofur (TIO)- or cefotaxime (FOX)-resistant Escherichia coli isolated from dairy cow manure. Twenty-four manure samples were collected from four farms and incubated under anaerobic conditions for 20 weeks at 4 °C or at 25 °C. A total of 37 TIO- or FOX-resistant E. coli were isolated from two of the four farms to determine their susceptibility to 14 antibiotics. Among the 37 resistant E. coli, 10 different serotypes were identified, with O8:H1 being the predominant serotype (n = 17). Five isolates belonged to each of serotypes O9:NM and O153:H42, respectively. All 37 cephalosporin resistant isolates were multi-resistant with the most prevalent resistance spectrum being amoxicillin-clavulanic acid-ampicillin-cefoxitin-ceftiofur-ceftriaxone-chloramphenicol-streptomycin-sulfisoxazole-tetracycline-trimethoprim-sulfamethoxazole. The genomes of 18 selected isolates were then sequenced and compared to 14 selected human pathogenic E. coli reference genomes obtained from public repositories using different bioinformatics approaches. As expected, all 18 sequenced isolates carried at least one ß-lactamase bla gene: TEM-1, TEM-81, CTX-M115, CTX-M15, OXA-1, or CMY-2. Several other antibiotic resistance genes (ARGs) and virulence determinants were detected in the sequenced isolates and all of them harbored antimicrobial resistance plasmids belonging to classic Inc groups. Our results confirm the presence of diverse ESBL producing E. coli isolates in dairy cow manure stored for a short period of time. Such manure might constitute a reservoir of resistance and virulence genes for other bacteria that share the same environment.

Characterization of Extraintestinal Pathogenic Escherichia coli isolated from retail poultry meats from Alberta, Canada.

Extraintestinal Pathogenic Escherichia coli (ExPEC) have the potential to spread through fecal waste resulting in the contamination of both farm workers and retail poultry meat in the processing plants or environment. The objective of this study was to characterize ExPEC from retail poultry meats purchased from Alberta, Canada and to compare them with 12 human ExPEC representatives from major ExPEC lineages. Fifty-four virulence genes were screened by a set of multiplex PCRs in 700 E. coli from retail poultry meat samples. ExPEC was defined as the detection of at least two of the following virulence genes: papA/papC, sfa, kpsMT II and iutA. Genetic relationships between isolates were determined using pulsed field gel electrophoresis (PFGE). Fifty-nine (8.4%) of the 700 poultry meat isolates were identified as ExPEC and were equally distributed among the phylogenetic groups A, B1, B2 and D. Isolates of phylogenetic group A possessed up to 12 virulence genes compared to 24 and 18 genes in phylogenetic groups B2 and D, respectively. E. coli identified as ExPEC and recovered from poultry harbored as many virulence genes as those of human isolates. In addition to the iutA gene, siderophore-related iroN and fyuA were detected in combination with other virulence genes including those genes encoding for adhesion, protectin and toxin while the fimH, ompT, traT, uidA and vat were commonly detected in poultry ExPEC. The hemF, iss and cvaC genes were found in 40% of poultry ExPEC. All human ExPEC isolates harbored concnf (cytotoxic necrotizing factor 1 altering cytoskeleton and causing necrosis) and hlyD (hemolysin transport) genes which were not found in poultry ExPEC. PFGE analysis showed that a few poultry ExPEC isolates clustered with human ExPEC isolates at 55-70% similarity level. Comparing ExPEC isolated from retail poultry meats provides insight into their virulence potential and suggests that poultry associated ExPEC may be important for retail meat safety. Investigations into the ability of our poultry ExPEC to cause human infections are warranted.

Antibiotic resistance and diversity of Salmonella enterica serovars associated with broiler chickens.

The objective of this study was to analyze the antibiotic resistance phenotype and genotype of Salmonella isolated from broiler production facilities. A total of 193 Salmonella isolates recovered from commercial farms in British Columbia, Canada, were evaluated. Susceptibility to antibiotics was determined with the Sensititre system. Virulence and antibiotic resistance genes were detected by PCR assay. Genetic diversity was determined by pulse-field gel electrophoresis (PFGE) typing. Seventeen serovars of Salmonella were identified. The most prevalent Salmonella serovars were Kentucky (29.0% of isolates), Typhimurium (23.8%), Enteritidis (13.5%), and Hadar (11.9%); serovars Heidelberg, Brandenburg, and Thompson were identified in 7.7, 4.1, and 3.6% of isolates, respectively. More than 43% of the isolates were simultaneously resistant to ampicillin, amoxicillin-clavulanic acid, ceftiofur, cefoxitim, and ceftriaxone. This ß-lactam resistance pattern was observed in 33 (58.9%) of the Salmonella Kentucky isolates; 2 of these isolates were also resistant to chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Genes associated with resistance to aminoglycosides (aadA1, aadA2, and strA), ß-lactams (blaCMY-2, blaSHV, and blaTEM), tetracycline (tetA and tetB), and sulfonamide (sul1) were detected among corresponding resistant isolates. The invasin gene (invA) and the Salmonella plasmid virulence gene (spvC) were found in 97.9 and 25.9% of the isolates, respectively, with 33 (71.7%) of the 46 Salmonella Typhimurium isolates and 17 (65.4%) of the 26 Salmonella Enteritidis isolates carrying both invA and spvC. PGFE typing revealed that the antibiotic-resistant serovars were genetically diverse. These data confirm that broiler chickens can be colonized by genetically diverse antibiotic-resistant Salmonella isolates harboring virulence determinants. The presence of such strains is highly relevant to food safety and public health.

Ultrastructural and cytochemical study of cell wall modification by lactoferrin, lactoferricin and penicillin G against Staphylococcus aureus.

To investigate the effect of lactoferrin or lactoferricin with or without penicillin G, light and transmission electron microscopy were performed on thin sections of two Staphylococcus aureus strains. Lactoferrin affected the ultrastructure of S. aureus and groups of undivided cells were observed after lactoferrin treatment with or without penicillin G. These results suggest that lactoferrin can affect staphylococcal cell separation and therefore prevent dissemination of daughter cells from spreading infection. After treatment with lactoferrin, S. aureus cells were less covered (P<0.05) with wheatgerm agglutinin labelled with gold, thus suggesting that lactoferrin affected the synthesis of peptidoglycan and/or the binding to N-acetyl-beta-D-glucosamine. Lactoferricin with or without penicillin G induced the lysis of many bacteria, formation of mesosomal structures and modifications of cell wall. Lactating female CD-1 mice were infected by intramammary delivery of a penicillin-resistant S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. Electron microscopy examination showed that 2 days of systemic treatments with lactoferrin affected the morphology and aggregation of S. aureus. In conclusion, lactoferrin and lactoferricin affect S. aureus morphology which may facilitate its killing by penicillin G.