Bacterial profile of pork from production to retail based on high-throughput sequencing.

Pork is a common vehicle for foodborne pathogens, including Salmonella spp. and Yersinia enterocolitica. Cross-contamination can occur at any stage of the pork production chain, from farm to market. In the present study, high-throughput sequencing was used to characterize bacterial profiles and track their changes along the whole supply chain. Tracked meat samples (pig on the farm, carcass in the slaughterhouse, unprocessed carcass and processed meat in the processing plant, and fresh pork at the local retail stores) and their associated environmental samples (e.g., water, floor, feed, feces, and workers' gloves) were collected from sequential stages (n = 96) and subjected to 16S rRNA metataxonomic analyses. At the farm, a total of 652 genera and 146 exclusive genera were identified in animal and environmental samples (pig, drain, floor, fan, and feces). Based on beta diversity analysis, it was demonstrated that the microbial composition of animal samples collected at the same processing step is similar to that of environmental samples (e.g., drain, fan, feces, feed, floor, gloves, knives, tables, and water). All animal and environmental samples from the slaughterhouse were dominated by Acinetobacter (55.37 %). At the processing plant, belly meat and neck meat samples were dominated by Psychrobacter (55.49 %). At the retail level, key bacterial players, which are potential problematic bacteria and important members with a high relative abundance in the samples, included Acinetobacter (8.13 %), Pseudomonas (6.27 %), and Staphylococcus (2.13 %). In addition, the number of confirmed genera varied by more than twice that identified in the processing plant. Source tracking was performed to identify bacterial contamination routes in pork processing. Animal samples, including the processing plant's carcass, the pig from the farm, and the unwashed carcass from the slaughterhouse (77.45 %), along with the processing plant's gloves (5.71 %), were the primary bacterial sources in the final product. The present study provides in-depth knowledge about the bacterial players and contamination points within the pork production chain. Effective control measures are needed to control pathogens and major pollutants at each stage of pork production to improve food safety.

A genetic and virulence characterization of Brazilian strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is considered the primary causative agent of porcine enzootic pneumonia (EP), a chronic contagious respiratory disease that causes economic losses. Obtaining new pathogenic isolates and studying the genome and virulence factors are necessary. This study performed a complete sequencing analysis of two Brazilian strains, UFV01 and UFV02, aiming to characterize the isolates in terms of the virulence factors and sequence type. The complete genome analysis revealed the main virulence genes (mhp385, mhp271, MHP_RS03455, p102, p97, p216, MHP_RS00555, mhp107) and ST-123, the presence of three toxin-related genes (tlyC, PLDc_2 and hcnC), and some genetic groups specific to these two isolates. Subsequently, the pathogenicity of the isolates was evaluated via an experimental infection conducted in a swine model. The study was divided into three groups, namely a negative control group (n = 4) and two test groups (n = 8), totaling 20 animals. They were challenged at 35 days of age with 107 CCU (Color Changing Units) M. hyopneumoniae via the intratracheal route. The UFV01 group showed earlier and higher seroconversion (IgG) (100%), while only 50% of the UFV02 group seroconverted. The same trend was observed when analyzing the presence of IgA in the bronchoalveolar lavage fluid (BALF) at 35 days post-infection (dpi). The UFV01 group had a mean macroscopic lesion score of 11.75% at 35 dpi, while UFV02 had 3.125%. Microscopic lesions were more severe in the UFV01 group. Based on laryngeal swab samples evaluated by qPCR, and the detection began at 14 days. The UFV01 group showed 75% positivity at 14 dpi. The UFV02 group also started excreting at 14 dpi, with a positivity rate of 37.5%. The results indicate that the UFV01 isolate exhibits higher virulence than UFV02. These findings may aid in developing new vaccines and diagnostic kits and establishing experimental models for testing.

Advancing understanding of microbial biofilms through machine learning-powered studies.

Microbial biofilms are prevalent in various environments and pose significant challenges to food safety and public health. The biofilms formed by pathogens can cause food spoilage, foodborne illness, and infectious diseases, which are difficult to treat due to their enhanced antimicrobial resistance. While the composition and development of biofilms have been widely studied, their profound impact on food, the food industry, and public health has not been sufficiently recapitulated. This review aims to provide a comprehensive overview of microbial biofilms in the food industry and their implication on public health. It highlights the existence of biofilms along the food-producing chains and the underlying mechanisms of biofilm-associated diseases. Furthermore, this review thoroughly summarizes the enhanced understanding of microbial biofilms achieved through machine learning approaches in biofilm research. By consolidating existing knowledge, this review intends to facilitate developing effective strategies to combat biofilm-associated infections in both the food industry and public health.

Transmission of antibiotic resistance at the wildlife-livestock interface.

Antibiotic-resistant microorganisms (ARMs) are widespread in natural environments, animals (wildlife and livestock), and humans, which has reduced our capacity to control life threatening infectious disease. Yet, little is known about their transmission pathways, especially at the wildlife-livestock interface. This study investigated the potential transmission of ARMs and antibiotic resistance genes (ARGs) between cattle and wildlife by comparing gut microbiota and ARG profiles of feral swine (Sus scrofa), coyotes (Canis latrans), cattle (Bos taurus), and environmental microbiota. Unexpectedly, wild animals harbored more abundant ARMs and ARGs compared to grazing cattle. Gut microbiota of cattle was significantly more similar to that of feral swine captured within the cattle grazing area where the home range of both species overlapped substantially. In addition, ARMs against medically important antibiotics were more prevalent in wildlife than grazing cattle, suggesting that wildlife could be a source of ARMs colonization in livestock.

Tracing the source and route of uterine colonization by exploring the genetic relationship of Escherichia coli isolated from the reproductive and gastrointestinal tract of dairy cows.

The source and route of bacterial colonization of the uterus are still not established. The objective was to investigate the source and route of bacterial colonization of the uterus by exploring the genetic relationship among E. coli strains isolated from the gastrointestinal and the reproductive tract of dairy cows pre- and postpartum. Secondarily, uterine health status (metritis vs. healthy) was evaluated. Cows (n = 34) had the rectoanal junction (RAJ), vulva, and vagina swabbed every three days starting six days before expected calving until nine days postpartum. The uterus was swabbed postpartum. A blood sample was collected at all time points, but cultures were negative. Whole-genome sequencing was performed on 44 isolates recovered from eight cows (four metritic and four healthy) with growth on selective E. coli media from the RAJ, vulva and/or vagina and uterus. Clonal isolates were found in the RAJ or the vulva prepartum and in the vulva, vagina or uterus postpartum. Clonal isolates were also found in the RAJ, the vulva, the vagina and the uterus postpartum. Clonal isolates were found in individual cows and different cows. Absence of clustering based on virulence factor genes and all genes indicate no strain specificity to body site or uterine health status. These findings indicate that the gastrointestinal tract is the likely source of bacteria that colonize the reproductive tract via ascending colonization of the uterus through the lower genital tract. Additionally, cow to cow transmission occurs, and strains are not specific to body site or to health status.

Biofilm-associated heat resistance of Bacillus cereus spores in vitro and in a food model, Cheonggukjang jjigae.

Bacillus cereus, a foodborne pathogen, is capable of forming spores and biofilms as methods to withstand environmental stresses. These bacterial structures are an issue for food safety as they aid the bacteria survive heat sterilisation processes of foods and food contact surfaces. This study was conducted to investigate the role of the biofilm structure in providing an extra layer of protection to spores against heat treatments. For this, heat resistance of B. cereus spores in intact biofilms was compared to that of planktonic spores in vitro and in a Cheonggukjang jjigae food model. Using methods developed in this study to measure the wet and dry heat resistance of spores in intact biofilms, it was found that B. cereus spores have significantly higher heat resistances when present in biofilms rather than as planktonic spores, and that dry heat is less effective than wet heat at killing spores in biofilms. In further detail, for wet heat treatments, spores in biofilms of the strain isolated from Cheonggukjang (Korean fermented whole soybean), B. cereus CH3, had generally higher wet heat resistances than the reference strain, B. cereus ATCC 10987, both in vitro and in the Cheonggukjang jjigae food model. However, the spores in biofilms of the two strains showed similar heat resistance to dry heat, with some exceptions, when biofilms were formed in vitro or in Cheonggukjang jjigae broth. Meanwhile, B. cereus ATCC 10987 spores in biofilms had higher or similar wet heat resistances in vitro compared to in Cheonggukjang jjigae broth. Wet heat resistances of B. cereus CH3 spores in biofilms were all statistically similar regardless of biofilm formation media (brain heart infusion and Cheonggukjang jjigae broths). For dry heat, spores in biofilms of both B. cereus strains were more heat resistant when biofilms were formed in the Cheonggukjang jjigae food model rather than in vitro. Altogether, heat resistances of spores in biofilms formed in vitro and in the food environment were found to be different depending on the tested B. cereus strain, but higher than planktonic spores in any case. This is the first study examining the heat resistance of B. cereus spores in intact biofilms matrices attached to the surface, both in vitro and in a food model. Therefore, this research is valuable to understand the protective effects of biofilms formed in food environments and to reduce the food safety risks associated with B. cereus.

Streptococcus vicugnae sp. nov., isolated from faeces of alpacas (Vicugna pacos) and cattle (Bos taurus), Streptococcus zalophi sp. nov., and Streptococcus pacificus sp. nov., isolated from respiratory tract of California sea lions (Zalophus californianus).

Four novel independent strains of Streptococcus spp. were isolated from faeces of alpaca (SL1232T), cattle (KCJ4950), and from respiratory tract of wild California sea lions (CSL7508T, CSL7591T). The strains were indole-, oxidase- and catalase-negative, non-spore-forming, non-motile Gram-positive cocci in short and long chains, facultative anaerobes. The 16S rRNA gene of SL1232T and KCJ4950 shared 99.40-99.60% nucleotide similarity to strains of S. equinus, S. lutetiensis, S. infantarius, and the 16S rRNA gene of CSL7508T and CSL7591T demonstrated 98.72 and 98.92% similarity, respectively, to S. marimammalium. All other known Streptococcus species had the 16S rRNA gene sequence similarities of ≤95%. The genomes were sequenced for the novel strains. Average nucleotide identity (ANI) analysis for strains SL1232T and KCJ4950, showed the highest similarity to S. equinus, S. lutetiensis, and S. infantarius with 85.21, 87.17, 88.47, 85.54, 87.47 and 88.89%, respectively, and strains CSL7508T and CSL7591T to S. marimammalium with 87.16 and 83.97%, respectively. Results of ANI were confirmed by pairwise digital DNA-DNA hybridization and phylogeny, which also revealed that the strains belong to three novel species of the genus Streptococcus. Phenotypical features of the novel species were in congruence with closely related members of the genus Streptococcus and gave negative reactions with the tested Lancefield serological groups (A-D, F and G). MALDI-TOF mass spectrometry supported identification of the species. Based on these data, we propose three novel species of the genus Streptococcus, for which the name Streptococcus vicugnae sp. nov. is proposed with the type strain SL1232T (=NCTC 14341T=DSM 110741T=CCUG 74371T), Streptococcus zalophi sp. nov. is proposed with the type strain CSL7508T (=NCTC 14410T=DSM 110742T=CCUG 74374T) and Streptococcus pacificus sp. nov. is proposed with the type strain CSL7591T (=NCTC 14455T=DSM 111148T=CCUG 74655T). The genome G+C content is 36.89, 34.85, and 35.34 % and draft genome sizes are 1906993, 1581094 and 1656080 bp for strains SL1232T, CSL7508T, and CSL7591T, respectively.

Diverse ß-lactam antibiotic-resistant bacteria and microbial community in milk from mastitic cows.

Intramammary bacterial infection, the most common cause of mastitis, is the most costly disease in dairy cattle in the US and reason for antibiotic usage. Ceftiofur, a third-generation cephalosporin, is generally used to treat such disease, but it has a high treatment failure rate. Though the reason is not known clearly, it is hypothesized that multiple factors are associated with the treatment failure. In this study, we analyzed 169 milk samples from cows with mastitis in two independent dairy farms (Farm A and B) in which 19.4% (Farm A) and 14.3% (Farm B) of the antibiotic treated cows were not cured. The prevalence of cephalosporin-resistant bacteria (CRB) in milk was 72.0% and 42.1% in Farm A and B, respectively. Nineteen and nine bacterial genera were identified in Farm A and B respectively, with the most abundant genus being Staphylococcus (27.1%; Farm A) and Bacillus (63.5%; Farm B). However, no strong relationship between the treatment failure rate and the CRB prevalence was observed. Furthermore, the metagenomic analysis showed no significant differences in the α- and ß-diversities of microbiota in milk samples from cured and uncured cows, suggesting that antibiotic-resistant bacteria were not the sole reason for the antibiotic treatment failure. KEY POINTS: • The mastitic milk samples had high prevalence of cephalosporin-resistant bacteria (CRB). • The CRB identified belong to diversified species. • Antibiotic treatment failure was not solely caused by the abundance of CRB.

A novel nanohybrid antimicrobial based on chitosan nanoparticles and antimicrobial peptide microcin J25 with low toxicity.

Bacterial resistance to antibiotics is a critical public health concern. Alternatives of antibiotics are needed urgently. Herein, we designed and engineered a new nano-antimicrobial, chitosan nanoparticles (CNs)-antimicrobial peptide microcin J25 (MccJ25) conjugates (CNMs). The engineered CNMs proved to be highly active against Gram-negative and Gram-positive bacteria, and the activity of CNMs and CNs was stable in various thermal and pH environments. Escherichia coli K88 strain treated with CNMs did not acquire resistance in serial passage assays over a period of 18 days. Risk assessment with cell lines showed that CNMs did not cause toxicity. Additionally, CNMs did not reduce the lifespan of C. elegans. In summary, this study demonstrated that CNMs can serve as an excellent novel antimicrobial agent against multi-drug resistance pathogens.

Characterization of the Vibriocidal Activity of Chitosan Microparticles: A Potential Therapeutic Agent for Emerging Multidrug-Resistant Cholera Infections.

Due to increasing reports of multidrug-resistant (MDR) Vibrio cholerae O1, the goal of this study was to characterize the in vitro antimicrobial activity of chitosan microparticles (CMs) to evaluate their potential as a novel therapeutic agent for cholera. We examined the antimicrobial activity of CMs against toxigenic V. cholerae O1 using direct enumeration, microscopy, and fluorescence microplate assays. Bacterial viability kinetics were measured with different concentrations of CMs, solution pH, and salt content using a live/dead staining technique. Growth inhibition of CM-exposed V. cholerae strains was conducted using a redox-sensitive stain and compared between wild-type and isogenic outer membrane (OM) mutants. CM concentrations above 0.1 wt % were sufficient to kill V. cholerae O1 suspensions with approximately 108 CFU/mL within 3 h. The nonviable cells demonstrated increased OM permeability that corresponded to gross morphological changes observed through scanning electron microscopy. CMs exhibited dose-dependent bactericidal activity that increased predictably at lower pH and decreased with salt addition. V. cholerae O1 strains lacking O-antigen were twice as susceptible to growth inhibition by CMs, whereas those with glycine modification to lipid A were ten times more resistant. We propose that CMs exert vibriocidal activity via electrostatic surface interactions between their positively charged amine groups and the negatively charged Gram-negative bacterial OM, resulting in disruption, increased permeability, decreased redox metabolism, and subsequent loss of cellular viability. Further research should be conducted in vivo to evaluate the efficacy of CMs as luminal agents to treat infections caused by MDR, toxigenic V. cholerae and other diarrheal pathogens.

The Role of Enterococcus faecium as a Key Producer and Fermentation Condition as an Influencing Factor in Tyramine Accumulation in Cheonggukjang.

The study evaluated the role of Enterococcus faecium in tyramine production and its response to fermentation temperature in a traditional Korean fermented soybean paste, Cheonggukjang. Tyramine content was detected in retail Cheonggukjang products at high concentrations exceeding the recommended limit up to a factor of 14. All retail Cheonggukjang products contained Enterococcus spp. at concentrations of at least 6 Log CFU/g. Upon isolation of Enterococcus strains, approximately 93% (157 strains) produced tyramine at over 100 µg/mL. The strains that produced the highest concentrations of tyramine (301.14-315.29 µg/mL) were identified as E. faecium through 16S rRNA sequencing. The results indicate that E. faecium is one of the major contributing factors to high tyramine content in Cheonggukjang. During fermentation, tyramine content in Cheonggukjang groups co-inoculated with E. faecium strains was highest at 45 °C, followed by 37 °C and 25 °C. The tyramine content of most Cheonggukjang groups continually increased as fermentation progressed, except groups fermented at 25 °C. At 45 °C, the tyramine content occasionally exceeded the recommended limit within 3 days of fermentation. The results suggest that lowering fermentation temperature and shortening duration may reduce the tyramine content of Cheonggukjang, thereby reducing the safety risks that may arise when consuming food with high tyramine concentrations.

Genetic and Functional Analyses of Virulence Potential of an Escherichia coli O157:H7 Strain Isolated From Super-Shedder Cattle.

Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 is an enteric pathogen that causes life-threatening disease in humans, with cattle being major natural reservoirs. A group of STEC O157:H7 with a dramatic combination of high virulence potentials and super-shedder bovine origin have been isolated. Here, an STEC O157:H7 isolate, JEONG-1266, was analyzed by comparative genomics, stx genotyping, and phenotypic analyses. The phylogenetic typing and whole-genome comparison consistently showed that JEONG-1266 is genetically close to EC4115 (one of 2006 Spinach outbreak isolates) and SS17 (an isolate from super-shedder cattle) strains, all of which belong to lineage I/II and Clade 8. Both lineage I/II and Clade 8 are known to be mostly associated with clinical strains with high virulence and severe clinical symptoms. Further, JEONG-1266, like EC4115 and SS17, harbors stx2a/stx2c genes, and carries Stx-encoding prophages, specifically the φstx2a-γ subtype. Possession of the φstx2a-γ subtype of Stx-encoding prophages and production of Stx2a have been shown to be a key signature associated with hypervirulent STEC O157:H7 strains. In silico virulence typing elucidated JEONG-1266, EC4115, and SS17 shared a highly conserved profile of key virulence genes at the nucleotide sequence level. Consistently, phenotypic data showed that JEONG-1266 expressed a high level of Stx2 toxins and had the full capacity of adhesion in vitro. Taken together, our study suggests that JEONG-1266 may represent an emerging STEC O157:H7 group, which are hypervirulent strains that originate from super-shedders, that can be a threat to food safety and public health.

Draft Genome Sequences of Bacteroides pyogenes Strains Isolated from the Uterus of Holstein Dairy Cows with Metritis.

Bacteroides pyogenes is found in the human and animal gut and is implicated in the pathogenesis of metritis in cows. We report the draft genome sequences of four Bacteroides pyogenes isolates obtained from the uterus of metritic cows. This will increase the understanding of its pathogenicity, antimicrobial resistance, and differentiation across hosts.

Animal Breed Composition Is Associated With the Hindgut Microbiota Structure and ß-Lactam Resistance in the Multibreed Angus-Brahman Herd.

Antibiotics have been widely used in livestock to treat and prevent bacterial diseases. However, use of antibiotics has led to the emergence of antibiotic resistant microorganisms (ARMs) in food animals. Due to the decreased efficacy of antibiotics, alternatives to antibiotics that can reduce infectious diseases in food animals to enhance animal health and growth performance are urgently required. Here, we show that animal genetics is associated with the hindgut microbiome, which is related to fat deposition and beta-lactam resistance in the gastrointestinal tract. We investigated the hindgut microbiota structure in 95 postweaning heifers belonging to the unique multibreed Angus-Brahman herd with breed composition ranging from 100% Angus to 100% Brahman. The hindgut microbial composition of postweaning heifers differed among breed groups. The mucin-degrading bacterium Akkermansia known for promoting energy expenditure was enriched in Brahman calves that contained less intramuscular fat content, while butyrate-producing bacterium Faecalibacterium was linearly positively correlated with Angus proportion. Moreover, the higher relative abundance of beta-lactam resistant genes including ampC gene and arcA gene was associated with the greater Brahman proportion. As the first study aimed at understanding changes in hindgut microbiota among beef cattle with linear gradient of breed composition and its association with marbling in meat, our results suggest that the effects of animal genetics on the gut microbiota structure is associated with fat deposition and potentially a factor affecting the gut antimicrobial resistance.

Draft Genome Sequences of Helcococcus ovis Strains Isolated at Time of Metritis Diagnosis from the Uterus of Holstein Dairy Cows.

Helcococcus ovis is an emerging pathogen implicated in the pathogenesis of metritis in dairy cows. Herein, we report the first draft genome sequences of four Helcococcus ovis isolates from the uterus of dairy cows with metritis. This information will enable a better understanding of the bacterium's pathogenicity and antimicrobial resistance.

Effects of Calcium and Manganese on Sporulation of Bacillus Species Involved in Food Poisoning and Spoilage.

Spores are resistant against many extreme conditions including the disinfection and sterilization methods used in the food industry. Selective prevention of sporulation of Bacillus species is an ongoing challenge for food scientists and fermentation technologists. This study was conducted to evaluate the effects of single and combined supplementation of calcium and manganese on sporulation of common pathogenic and food spoilage Bacillus species: B. cereus, B. licheniformis, B. subtilis and B. coagulans. Sporulation of Bacillus vegetative cells was induced on sporulation media supplemented with diverse concentrations of the minerals. Under the various mineral supplementation conditions, the degree of sporulation was quantified with colonies formed by the Bacillus spores. The results revealed that B. licheniformis and B. cereus displayed the weakest sporulation capabilities on media with minimal supplementation levels of calcium and manganese. The lowest sporulation of B. subtilis and B. coagulans was observed on media supplemented with the highest level of calcium and low levels of manganese. Depending on effect of supplementation on sporulation, the Bacillus species were divided into two distinct groups: B. licheniformis and B. cereus; and B. subtilis and B. coagulans. The information provides valuable insight to selectively reduce sporulation of Bacillus species undesirable in the food industry.

Draft Genome Sequences of Two Fusobacterium necrophorum Strains Isolated from the Uterus of Dairy Cows with Metritis.

A commensal in the gastrointestinal tract, Fusobacterium necrophorum is involved in the pathogenicity of abscesses, foot rot, and metritis in cattle. Here, we present the draft genomes of two Fusobacterium necrophorum isolates from the uterus of dairy cows with metritis to allow for future comparative genome studies.

High Prevalence of Cefotaxime Resistant Bacteria in Grazing Beef Cattle: A Cross Sectional Study.

Although the over-use of antibiotics during food animal production is a potential driver of antimicrobial resistant microorganisms (ARMs), a high prevalence of cefotaxime resistant bacteria (CRB) has been observed in grazing animals raised without antibiotic supplementation. In this cross-sectional study, the prevalence and concentration of CRB in beef cattle on grazing farms were investigated. Fecal samples from the recto-anal junction of cattle (n = 840) and environmental samples (n = 258) were collected from 17 farms in North and Central Florida in the United States, and a survey of farm characteristics, animal husbandry practices, and antibiotic usage was conducted. CRB were detected in fecal samples from 47.4% of all cattle, with the prevalence ranging from 21.1 to 87.5% on farms, and significantly higher (P < 0.001) in calves compared to adult cows (54.1 vs. 41.8%). Environmental samples had a higher prevalence than fecal samples (P < 0.001), with CRB detected in 88.6% of water, 98.7% of soil, and 95.7% of forage samples. Compared to the concentration (log CFU/g) of CRB in fecal samples (2.95, 95% CI: 2.89, 3.02), the concentration of CRB was higher (P < 0.001) in soil and forage samples (5.37, 95% CI: 5.16, 5.57) and lower (P < 0.001) in water samples (1.08, 95% CI: 0.82, 1.36). Soil microbiota from farms with high prevalence of CRB clustered closer together and the proportion of Phylum Proteobacteria was higher on farms with high prevalence of CRB resistance. Large farming operations were associated with a 58% higher likelihood of CRB detection in fecal samples. Regular cleaning of drinking troughs and the addition of ionophores to feed were associated with CRB reduction in fecal samples. Taken together, the widespread of CRB into both cattle seldom treated with cephalosporin antibiotics and the surrounding environment suggests the environment is a natural source of antimicrobial resistance in beef cattle.

Prevalence and Molecular Characteristics of Extended-Spectrum and AmpC ß-Lactamase Producing Escherichia coli in Grazing Beef Cattle.

The emergence of extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase producing Escherichia coli represent a contemporary public health threat. ESBL and AmpC ß-lactamase genes translocate between chromosomes and plasmids, facilitating rapid spread throughout the environment. In this study, ESBL/AmpC producing bacteria were isolated from beef cattle farms with seldom antibiotic use. Eleven farms out of 17 tested, had ESBL/AmpC producing E. coli in animals, soil, and forage samples. Fifty-nine CTX-M or CMY-2 positive E. coli isolates were further characterized with whole-genome sequencing. The isolates commonly carried CMY-2, TEM, or CTX-M genes, and over half encoded both CTX-M and TEM genes. Using comparative genomics, antimicrobial resistance genes from 12 classes of antimicrobial were identified and confirmed by antibiotic susceptibility test, revealing multidrug resistance against multiple classes of antibiotics. Virulence factors related to adherence, invasion, iron uptake, and bacterial secretion systems were shared by all isolates; some of which were identified as enteropathogenic E. coli. Phylogenetic analyses revealed a pattern of close genetic relatedness, suggesting that ESBL/AmpC producing E. coli were transmitted among farms as well as independent evolution within farms. Our results indicate that ESBL and AmpC ß-lactamases prevail in food animal production system regardless antibiotic use and have the characteristics for zoonotic transmission.

Engineering of chitosan-derived nanoparticles to enhance antimicrobial activity against foodborne pathogen Escherichia coli O157:H7.

Chitosan is an abundant and natural polymer derived from chitin, which presents a wide variety of properties, including antimicrobial activity. The raising of antibiotic resistant bacteria has increased the interest in finding alternatives to traditional antibiotics. Many studies have assessed the antimicrobial activity of chitosan itself, but a few have performed comparisons among different chitosan nanoparticle synthesis, which will be of particular interest for further applications. In this study, the effects of two types of cross-linking agents, sodium sulfate vs. tripolyphosphate, along with molecular weight (Mw) of chitosan, low vs. high Mw, and different sonication treatments, time and power, were assessed to determine the optimal conditions to enhance antimicrobial activity against bacterial pathogens. Physiochemical characteristics of the engineered chitosan nanoparticles were determined. It was observed that 20 min sonication time, low Mw of chitosan, Sodium sulfate as cross-linker, and particle size smaller than <300 nm, showed the greatest antimicrobial activity. Chitosan nanoparticles generated at this condition completely killed pathogenic E. coli O157:H7 without raising resistant bacteria, providing great insights into potential use as alternative antimicrobial agents.