Castration alters the ileum microbiota of Holstein bulls and promotes beef flavor compounds.

BACKGROUND: In the beef industry, bull calves are usually castrated to improve flavor and meat quality; however, this can reduce their growth and slaughter performance. The gut microbiota is known to exert a significant influence on growth and slaughter performance. However, there is a paucity of research investigating the impact of castration on gut microbiota composition and its subsequent effects on slaughter performance and meat flavor. RESULT: The objective of this study was to examine the processes via which castration hinders slaughter productivity and enhances meat quality. Bull and castrated calves were maintained under the same management conditions, and at slaughter, meat quality was assessed, and ileum and epithelial tissue samples were obtained. The research employed metagenomic sequencing and non-targeted metabolomics techniques to investigate the makeup of the microbiota and identify differential metabolites. The findings of this study revealed the Carcass weight and eye muscle area /carcass weight in the bull group were significantly higher than those in the steer group. There were no significant differences in the length, width, and crypt depth of the ileum villi between the two groups. A total of 53 flavor compounds were identified in the two groups of beef, of which 16 were significantly higher in the steer group than in the bull group, and 5 were significantly higher in the bull group than in the steer group. In addition, bacteria, Eukaryota, and virus species were significantly separated between the two groups. The lipid metabolism pathways of α-linolenic acid, linoleic acid, and unsaturated fatty acids were significantly enriched in the Steers group. Compared with the steer group, the organic system pathway is significantly enriched in the bull group. The study also found that five metabolites (LPC (0:0/20:3), LPC (20:3/0:0), LPE (0:0/22:5), LPE (22:5/0:0), D-Mannosamine), and three species (s_Cloning_vector_Hsp70_LexA-HP1, s_Bacteroides_Coprophilus_CAG: 333, and s_Clostridium_nexile-CAG: 348) interfere with each other and collectively have a positive impact on the flavor compounds of beef. CONCLUSIONS: These findings provide a basic understanding that under the same management conditions, castration does indeed reduce the slaughter performance of bulls and improve the flavor of beef. Microorganisms and metabolites contribute to these changes through interactions.

Associations of plant-based foods, red and processed meat, and dairy with gut microbiome in Finnish adults.

PURPOSE: Population-based studies on the associations of plant-based foods, red meat or dairy with gut microbiome are scarce. We examined whether the consumption of plant-based foods (vegetables, potatoes, fruits, cereals), red and processed meat (RPM) or dairy (fermented milk, cheese, other dairy products) are related to gut microbiome in Finnish adults. METHODS: We utilized data from the National FINRISK/FINDIET 2002 Study (n = 1273, aged 25-64 years, 55% women). Diet was assessed with 48-hour dietary recalls. Gut microbiome was analyzed using shallow shotgun sequencing. We applied multivariate analyses with linear models and permutational ANOVAs adjusted for relevant confounders. RESULTS: Fruit consumption was positively (beta = 0.03, SE = 0.01, P = 0.04), while a dairy subgroup including milk, cream and ice-creams was inversely associated (beta=-0.03, SE 0.01, P = 0.02) with intra-individual gut microbiome diversity (alpha-diversity). Plant-based foods (R2 = 0.001, P = 0.03) and dairy (R2 = 0.002, P = 0.01) but not RPM (R2 = 0.001, P = 0.38) contributed to the compositional differences in gut microbiome (beta-diversity). Plant-based foods were associated with several butyrate producers/cellulolytic species including Roseburia hominis. RPM associations included an inverse association with R. hominis. Dairy was positively associated with several lactic producing/probiotic species including Lactobacillus delbrueckii and potentially opportunistic pathogens including Citrobacter freundii. Dairy, fermented milk, vegetables, and cereals were associated with specific microbial functions. CONCLUSION: Our results suggest a potential association between plant-based foods and dairy or their subgroups with microbial diversity measures. Furthermore, our findings indicated that all the food groups were associated with distinct overall microbial community compositions. Plant-based food consumption particularly was associated with a larger number of putative beneficial species.

A New Peptide Nucleic Acid Fluorescence In Situ Hybridization Probe for the Specific Detection of Salmonella Species in Food Matrices.

Salmonella spp. is among the most central etiological agents in foodborne bacterial disorders. To identify Salmonella spp., numerous new molecular techniques have been developed conversely to the traditional culture-based methods. In this work, a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for the specific detection of Salmonella species, allowing a faster analysis compared with the traditional methods (ISO 6579-1: 2017). The method was optimized based on a novel PNA probe (SalPNA1692) combined with a blocker probe to detect Salmonella in food samples through an assessment of diverse-rich and selective enrichment broths. Our findings indicated that the best outcome was obtained using a 24-h pre-enrichment step in buffered peptone water, followed by RambaQuick broth selective enrichment for 16 h. For the enrichment step performance validation, fresh ground beef was artificially contaminated with two ranges of concentration of inoculum: a low level (0.2-2 colony-forming units [CFUs]/25 g) and a high level (2-10 CFUs/25 g). The new PNA-FISH method presented a specificity of 100% and a detection limit of 0.5 CFU/25 g of food sample, which confirms the great potential of applying PNA probes in food analysis.

Development of a Sensitive Colorimetric Indicator for Detecting Beef Spoilage in Smart Packaging.

This study aimed to fabricate and characterize a novel colorimetric indicator designed to detect ammonia (NH3) and monitor meat freshness. The sensing platform was constructed using electrospun nanofibers made from polylactic acid (PLA), which were then impregnated with anthocyanins as a natural pH-sensitive dye, extracted from red cabbage. This research involved investigating the relationship between the various concentrations of anthocyanins and the colorimetric platform's efficiency when exposed to ammonia vapor. Scanning electron microscope (SEM) results were used to examine the morphology and structure of the nanofiber mats before and after the dip-coating process. The study also delved into the selectivity of the indicator when exposed to various volatile organic compounds (VOCs) and their stability under extreme humidity levels. Furthermore, the platform's sensitivity was evaluated as it encountered ammonia (NH3) in concentrations ranging from 1 to 100 ppm, with varying dye concentrations. The developed indicator demonstrated an exceptional detection limit of 1 ppm of MH3 within just 30 min, making it highly sensitive to subtle changes in gas concentration. The indicator proved effective in assessing meat freshness by detecting spoilage levels in beef over time. It reliably identified spoilage after 10 h and 7 days, corresponding to bacterial growth thresholds (107 CFU/mL), both at room temperature and in refrigerated environments, respectively. With its simple visual detection mechanism, the platform offered a straightforward and user-friendly solution for consumers and industry professionals alike to monitor packaged beef freshness, enhancing food safety and quality assurance.

Enhancing the Shelf Life of Sous-Vide Red Deer Meat with Piper nigrum Essential Oil: A Study on Antimicrobial Efficacy against Listeria monocytogenes.

Using sous-vide technology in combination with essential oils offers the potential to extend the preservation of food items while preserving their original quality. This method aligns with the growing consumer demand for safer and healthier food production practices. This study aimed to assess the suitability of minimal processing of game meat and the effectiveness of vacuum packaging in combination with Piper nigrum essential oil (PNEO) treatment to preserve red deer meat samples inoculated with Listeria monocytogenes. Microbial analyses, including total viable count (TVC) for 48 h at 30 °C, coliform bacteria (CB) for 24 h at 37 °C, and L. monocytogenes count for 24 h at 37 °C, were conducted. The cooking temperature of the sous-vide was from 50 to 65 °C and the cooking time from 5 to 20 min. Additionally, the study monitored the representation of microorganism species identified through mass spectrometry. The microbiological quality of red deer meat processed using the sous-vide method was monitored over 14 days of storage at 4 °C. The results indicated that the TVC, CB, and L. monocytogenes counts decreased with the temperature and processing time of the sous-vide method. The lowest counts of individual microorganism groups were observed in samples treated with 1% PNEO. The analysis revealed that PNEO, in combination with the sous-vide method, effectively reduced L. monocytogenes counts and extended the shelf life of red deer meat. Kocuria salsicia, Pseudomonas taetrolens, and Pseudomonas fragi were the most frequently isolated microorganism species during the 14-day period of red deer meat storage prepared using the sous-vide method.

Revealing the spoilage characteristics of refrigerated prepared beef steak by advanced bioinformatics tools.

BACKGROUND: Although microorganisms are the main cause of spoilage in prepared beef steaks, very few deep spoilage mechanisms have been reported so far. Aiming to unravel the mechanisms during 12 days of storage at 4 °C affecting the quality of prepared beef steak, the present study investigated the changes in microbial dynamic community using a combined high-throughput sequencing combined and bioinformatics. In addition, gas chromatography-mass spectrometry combined with multivariate statistical analysis was utilized to identify marker candidates for prepared steaks. Furthermore, cloud platform analysis was applied to determine prepared beef steak spoilage, including the relationship between microbiological and physicochemical indicators and volatile compounds. RESULTS: The results showed that the dominant groups of Pseudomonas, Brochothrix thermosphacta, Lactobacillus and Lactococcus caused the spoilage of prepared beef steak, which are strongly associated with significant changes in physicochemical properties and volatile organic compounds (furan-2-pentyl-, pentanal, 1-octanol, 1-nonanol and dimethyl sulfide). Metabolic pathways were proposed, among which lipid metabolism and amino acid metabolism were most abundant. CONCLUSION: The present study is helpful with respect to further understanding the relationship between spoilage microorganisms and the quality of prepared beef steak, and provides a reference for investigating the spoilage mechanism of dominant spoilage bacteria and how to extend the shelf life of meat products. © 2024 Society of Chemical Industry.

The pathogenic and spoilage bacteria associated with red meat and application of different approaches of high CO2 packaging to extend product shelf-life.

With the fast-global development of packaging techniques, the potential antimicrobial effect of CO2, as a safe, cheap and readily available gas, makes it the integral component for packaging of meat products. The associated spoilage and/or pathogenic bacteria on raw meat may respond in different ways to elevated CO2 concentrations. The growth of some aerobic Gram-negative bacteria such as Pseudomonas spp. is significantly inhibited but some LAB bacteria may be allowed to grow faster and dominate the product. The antimicrobial efficacy of enriched CO2 packaging is attributed to the rate of CO2 solubility in the product which is itself affected by the level of headspace CO2, product pH, temperature and the ratio of headspace gas to product (G:P). This review, first, explores the varied range of beef and sheep meat spoilage and pathogenic bacteria and the intrinsic and extrinsic parameters that may influence the pattern of microbial growth and meat spoilage rate during storage. Then, the antimicrobial mechanism of elevated CO2 packaging will be discussed and the different approaches of achieving enriched CO2 packaging i.e. the traditional technique of flushing a desired gas mixture and/or using the new commercially developed CO2 emitters will then be compared in terms of their strengths, limitations and technical mode of action.

Quantitative microbiological risk assessment of nontyphoidal Salmonella in ground pork in households in Chengdu, China.

Foodborne disease caused by nontyphoidal Salmonella (NTS) is one of the most important food safety issues worldwide. The objectives of this study were to carry out microbial monitoring on the prevalence of NTS in commercial ground pork, investigate consumption patterns, and conduct a quantitative microbiological risk assessment (QMRA) that considers cross-contamination to determine the risk caused by consuming ground pork and ready-to-eat food contaminated during food handling in the kitchen in Chengdu, China. The food pathway of ground pork was simplified and assumed to be several units according to the actual situation and our survey data, which were collected from our research or references and substituted into the QMRA model for simulation. The results showed that the prevalence of NTS in ground pork purchased in Chengdu was 69.64% (95% confidence interval [CI], 60.2-78.0), with a mean contamination level of -0.164 log CFU/g. After general cooking, NTS in ground pork could be eliminated (contamination level of zero). The estimated probability of causing salmonellosis per day was 9.43E-06 (95% CI: 8.82E-06-1.00E-05), while the estimated salmonellosis cases per million people per year were 3442 (95% CI: 3218-3666). According to the sensitivity analysis, the occurrence of cross-contamination was the most important factor affecting the probability of salmonellosis. To reduce the risk of salmonellosis caused by NTS through ground pork consumption, reasonable hygiene prevention and control measures should be adopted during food preparation to reduce cross-contamination. This study provides valuable information for household cooking and food safety management in China.

Countrywide multi-serotype outbreak of Salmonella Bovismorbificans ST142 and monophasic Salmonella Typhimurium ST34 associated with dried pork sausages in France, September 2020* to January 2021.

The French National Reference Centre for Escherichia coli, Shigella and Salmonella (FNRC-ESS) detected two human clusters of 33 cases (median age: 10 years; 17 females) infected by Salmonella enterica serotype Bovismorbificans, ST142, HC5_243255 (EnteroBase HierCC­cgMLST scheme) in September-November 2020 and of 11 cases (median age: 11 years; seven males) infected by S. enterica serotype 4,12:i:-, ST34, HC5_198125 in October-December 2020. Epidemiological investigations conducted by Santé publique France linked these outbreaks to the consumption of dried pork sausages from the same manufacturer. S. Bovismorbificans and S. 4,12:i:- were isolated by the National Reference Laboratory from different food samples, but both strains were identified in a single food sample only by qPCR. Three recalls and withdrawals of dried pork products were issued by the French general directorate of food of the French ministry for agriculture and food in November 2020, affecting eight supermarket chains. A notification on the European Rapid Alert System for Food and Feed and a European urgent enquiry on the Epidemic Intelligence Information System for Food and Waterborne Diseases and Zoonoses (EPIS-FWD) were launched. No cases were reported outside France. Outbreaks caused by multiple serotypes of Salmonella may go undetected by protocols in standard procedures in microbiology laboratories.

Antibacterial Pattern of Rosa roxburghii Tratt Pomace Crude Extract Against Staphylococcus aureus and Its Application in Preservation of Cooked Beef.

Staphylococcus aureus is a common foodborne pathogen and spoilage bacterium in meat products. To develop a natural preservative for meat products, this study revealed the antibacterial activity and mechanism of Rosa roxburghii Tratt pomace crude extract (RRPCE) against S. aureus, and applied RRPCE to the preservation of cooked beef. The diameter of inhibition zone, minimum inhibitory concentration (MIC), and minimum bactericide concentration of RRPCE against S. aureus were 15.85 ± 0.35 to 16.21 ± 0.29 mm, 1.5 mg/mL, and 3 mg/mL, respectively. The growth curve of S. aureus was completely stalled by treatment with RRPCE at 2 MIC. RRPCE results in the decrease of intracellular adenosine 5'-triphosphate (ATP) content, depolarization of cell membrane, leakage of cell fluid including nucleic acid and protein, and destruction of cell membrane integrity and cell morphology. During storage, RRPCE significantly reduced S. aureus viable counts, pH, and total volatile basic nitrogen of cooked beef compared with untreated samples (p < 0.05). In addition, RRPCE could significantly increase the redness (a*) value, decrease lightness (L*) and yellowness (b*) values, and slow down the color change of cooked beef (p < 0.05). These findings suggest that RRPCE can effectively inhibit S. aureus, and has the potential as a natural preservative for the preservation of cooked beef.

Polydopamine nanospheres-assisted direct PCR for rapid detection of Escherichia coli O157:H7.

Polymerase chain reaction (PCR) is one of the most common methods for rapid monitoring of foodborne pathogens; however, it requires purified nucleic acid as a template. Conventional nucleic acid purification is a time-consuming and laborious process. To overcome this, we developed polydopamine nanospheres (PDA NPs)-assisted direct PCR for detecting Escherichia coli O157:H7 (E. coli O157:H7). PDA NPs significantly enhanced PCR efficiency because of their strong interaction with PCR reagents, including polymerase and primers, thereby enabling regulation of the PCR performance. The optimal concentration and diameter for PDA NPs were 0.10 µg/µL and 504 nm, respectively. The PDA NPs-assisted direct PCR exhibited high sensitivity in E.coli O157:H7 detection. The detection limit of PDA NPs-assisted direct PCR was 6.7 × 104 CFU/mL, which was 10-fold lower than that of direct PCR (6.7 × 105 CFU/mL). Moreover, the sensor demonstrated excellent selectivity against E. coli O157:H7, with a negative reaction to eight other common pathogens. Most importantly, the PDA NPs-assisted direct PCR detected the order of 104-5 CFU/mL E.coli O157:H7 in milk, beef, and watermelon samples. No cultural enrichment was required, with the whole process taking <3 h. Therefore, PDA NPs-assisted direct PCR has tremendous potential in the rapid and sensitive detection of pathogens.

Characterization of cinnamon essential oil and its application in Malva sylvestris seed mucilage edible coating to the enhancement of the microbiological, physicochemical and sensory properties of lamb meat during storage.

AIMS: Firstly, Cinnamomum zeylani essential oil (CZEO) was isolated and characterized. Secondly, CZEO was used in Malva sylvestris mucilage (MSM) coating and its antioxidant and antimicrobial effects on lamb meat slices were evaluated in 10 days at 4°C. METHODS AND RESULTS: The main chemical compounds and functional groups of the CZEO were identified and quantified by a gas chromatograph coupled to a mass spectrometer and by an Fourier transform infrared spectrometer respectively. The total phenol and flavonoid contents of CZEO were determined by the Folin-Ciocalteu reagent-based and aluminium chloride methods respectively. Various microbiological, physicochemical analyses and sensory evaluations were also utilized regarding the coated lamb meat slices. CZEO contains benzyl benzoate (40.93%), caryophyllene oxide (26.07%) and (E)-cinnamaldehyde (13.01%), with strong radical scavenging activity and antibacterial effect against investigated pathogenic microorganisms. The CZEO-loaded MSM edible coating greatly postponed the growth of microorganisms and extended the product life (>10 days). The pH value, moisture content and hardness of the samples were also preserved more efficiently when high concentrations of the essential oil were incorporated into the edible coating (p < 0.05). The CZEO-rich MSM coating was also able to possess considerable activity against lipid oxidation in lamb meat samples, and significantly decreased the production of primary and secondary oxidation products (p < 0.05). Moreover, sensory parameters of the samples were preserved more efficiently during cold storage when the CZEO-enriched edible coating, particularly MSM + 2% CZEO was used. CONCLUSIONS: The use of edible coating based on MSM and CZEO is therefore effective in reducing microbial growth and chemical reactions in lamb meat during the storage period. SIGNIFICANCE AND IMPACT OF THE STUDY: The importance of the results of this study is in order to increase the use of natural preservatives, maintain food safety and of course the health of the people in the community.

Unknown cold-tolerant Clostridium spp.: Characteristics and potential to cause meat spoilage.

Clostridium spp. are ubiquitous bacteria and often found in foods and animals. Some species are pathogenic, others food spoiling or commensals. In this study, 65 cold-tolerant Clostridium spp. strains isolated from variable samples (beef, lamb, venison, feces/skin of wild boars) were investigated. Fifty strains were lecithinase positive; six additionally produced ß-hemolysis. By applying specific qPCR, 16S rRNA gene analysis, RFLP method, and MALDI-TOF MS, they were classified into two major groups: 29 strains were identified as C. tagluense-like, while the other 36 remained unidentified. Subsequently, twenty-two vacuum-packed beef samples were spiked with a single strain from both groups and stored at 4 °C for 8 weeks. The odor of challenged samples was variable (from unchanged, sour/musty, to sulfurous), while color, meat consistency and drip loss were similar to the control group. The ability to produce gas of all tested strains was lower than of C. estertheticum. Even though both groups of cold-tolerant clostridia exhibited similar 16S rRNA genes and biochemical activities, RFLP methods and MALDI-TOF MS are sufficient to differentiate them. In terms of food safety, strains producing lecithinase and hemolysin should be further investigated for their potential to produce substances affecting human and animal health.

Exploring the microbiological quality and safety of dry-aged beef: A cross-sectional study of loin surfaces during ripening and dry-aged beef steaks from commercial meat companies in Belgium.

A cross-sectional survey was undertaken in Belgian beef producing companies to study the current practices and the microbiological load of dry-aged loins (during production) and trimmed steaks (final product). In each company, the temperature and relative humidity of the ripening chamber were measured, and two loins (each in a different stage of the ripening process) were sampled. From the surface of each loin, a lean meat and adipose tissue sample was analysed separately, and different groups of bacteria, yeasts and moulds were enumerated. The average relative humidity in the ripening chambers was 72 ± 13% and the temperature ranged between 0.0 °C and 5.9 °C. During the drying process, most of the lean meat and adipose tissue samples showed high numbers of total psychrotrophic aerobic bacteria, Pseudomonas spp., psychrotrophic lactic acid bacteria, and yeasts, but the variation between loins was high. The microbiological load on freshly cut dry-aged steaks was generally lower than on loin surfaces, but both psychrotrophic aerobic and anaerobic bacteria were present inside several steaks. The water activity inside dry-aged beef steaks was high (aw ≥ 0.98), which could allow growth of psychrotrophic pathogens, though more in-depth studies are necessary to determine potential growth during the storage of (trimmed) steaks or even inside loins during the dry-aging process.

Spoilage potential of bacterial species from chilled vacuum-packed lamb.

The objective of this study was to establish whether specific organisms play important roles in the spoilage rate of vacuum-packed (VP) lamb at low storage temperatures. The spoilage potential of representative organisms (n = 13) of the spoilage community of VP lamb were investigated through a series of shelf-life challenge trials. Each isolate was individually inoculated onto sterile (irradiated) and non-sterile (i.e., containing natural microbial community) VP lamb meat. Meat quality was assessed over time by measuring sensorial qualities, bacterial growth and pH. Among all test organisms, Clostridium spp. had the highest spoilage potential and had a major effect on the spoilage rate of VP lamb (based on sensory assessment). C. estertheticum caused premature 'blown pack' spoilage; however, the spoilage was delayed in a community setting. C. putrefaciens and C. algidicarnis caused premature spoilage of VP lamb independently and in a community setting. In contrast, all facultative anaerobes and Pseudomonas sp. tested were not capable of spoiling meat independently or within a community, expect for Carnobacterium divergens and Serratia spp., which spoiled meat prematurely when present in a community. Overall, these results highlight that Clostridium could be one of the main taxa driving the faster rate of quality loss of chilled VP lamb compared to beef. This research can help to inform opportunities for shelf-life extension by targeting organisms with 'high' spoilage potential, such as Clostridium.

Tracing Clostridium perfringens strains from beef processing of slaughter house by pulsed-field gel electrophoresis, and the distribution and toxinotype of isolates in Shaanxi province, China.

The purpose of this study was to investigate the distribution and specify the transmission and cross-contamination of Clostridium perfringens (C. perfringens) in the beef slaughtering and butchering process. The prevalence of 21.2% (150/708) yielded 208 isolates of C. perfringens, including 80.8% type A and 19.2% type D, 0.4% (3/708) samples carried both type A and D strains, and 72.5% type D isolates carried both cpe and atyp.cpb2 genes. C. perfringens were identified through the whole slaughtering process but no type F (cpe and cpa isolates) was found. 69 isolates were further analyzed and classified into 28 PFGE genotypes and clade I contained 94.2% isolates and 24 PFGE genotypes, which showed the genetic diversity and epidemic correlation. Our study traced C. perfringens contamination along the handling processes and showed a gradually ascending contamination rate during the whole process, revealing widespread cross-contamination from the feces and hides of slaughtered cattle to the carcass in the slaughtering workshop, so as from tools and personnel to meat of the cutting workshops. Strains from different slaughterhouses (regions) have high homology, and type A is the predominant toxinotype. It is necessary to monitor and control several key points of cross-contamination during slaughtering process to reduce a risk of C. perfringens infection.

Establishment and Application of a Predictive Growth Kinetic Model of Salmonella with the Appearance of Two Other Dominant Background Bacteria in Fresh Pork.

To better guide microbial risk management and control, growth kinetic models of Salmonella with the coexistence of two other dominant background bacteria in pork were constructed. Sterilized pork cutlets were inoculated with a cocktail of Salmonella Derby (S. Derby), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli), and incubated at various temperatures (4-37 °C). The predictive growth models were developed based on the observed growth data. By comparing R2 of primary models, Baranyi models were preferred to fit the growth curves of S. Derby and P. aeruginosa, while the Huang model was preferred for E. coli (all R2 ≥ 0.997). The secondary Ratkowsky square root model can well describe the relationship between temperature and µmax (all R2 ≥ 0.97) or Lag (all R2 ≥ 0.98). Growth models were validated by the actual test values, with Bf and Af close to 1, and MSE around 0.001. The time for S. Derby to reach a pathogenic dose (105 CFU/g) at each temperature in pork was predicted accordingly and found to be earlier than the time when the pork began to be judged nearly fresh according to the sensory indicators. Therefore, the predictive microbiology model can be applied to more accurately predict the shelf life of pork to secure its quality and safety.

Biofilm-Forming Capacity of Escherichia coli Isolated from Cattle and Beef Packing Plants: Relation to Virulence Attributes, Stage of Processing, Antimicrobial Interventions, and Heat Tolerance.

Despite the importance of biofilm formation in the contamination of meat by pathogenic Escherichia coli at slaughter plants, drivers for biofilm remain unclear. To identify selection pressures for biofilm, we evaluated 745 isolates from cattle and 700 generic E. coli isolates from two beef slaughter plants for motility, the expression of curli and cellulose, and biofilm-forming potential. Cattle isolates were also screened for serogroup, stx1, stx2, eae, and rpoS. Generic E. coli isolates were compared by source (hide of carcass, hide-off carcass, and processing equipment) before and after the implementation of antimicrobial hurdles. The proportion of E. coli isolates capable of forming biofilms was lowest (7.1%; P < 0.05) for cattle isolates and highest (87.3%; P < 0.05) from equipment. Only one enterohemorrhagic E. coli (EHEC) isolate was an extremely strong biofilm former, in contrast to 73.4% of E. coli isolates from equipment. Isolates from equipment after sanitation had a greater biofilm-forming capacity (P < 0.001) than those before sanitation. Most cattle isolates were motile and expressed curli, although these traits along with the expression of cellulose and the detection of rpoS were not necessary for biofilm formation. In contrast, isolates capable of forming biofilms on equipment were almost exclusively motile and able to express curli. The results of the present study indicate that cattle rarely carry EHEC capable of making strong biofilms in slaughter plants. However, if biofilm-forming EHEC contaminates equipment, current sanitation procedures may not eliminate the most robust biofilm-forming strains. Accordingly, new and effective antibiofilm hurdles for meat-processing equipment are required to reduce future instances of foodborne disease. IMPORTANCE As the majority of enterohemorrhagic E. coli (EHEC) isolates are not capable of forming biofilms, sources were undetermined for biofilm-forming EHEC isolated from "high-event periods" in beef slaughter plants. This study demonstrated that sanitation procedures used on beef-processing equipment may inadvertently lead to the survival of robust biofilm-forming strains of E. coli. Cattle only rarely carry EHEC capable of forming strong biofilms (1/745 isolates evaluated), but isolates with greater biofilm-forming capacity were more likely (P < 0.001) to survive equipment sanitation. In contrast, chilling carcasses for 3 days at 0°C reduced (P < 0.05) the proportion of biofilm-forming E. coli. Consequently, an additional antibiofilm hurdle for meat-processing equipment, perhaps involving cold exposure, is necessary to further reduce the risk of foodborne disease.

Interplay between Bacterial Clones and Plasmids in the Spread of Antibiotic Resistance Genes in the Gut: Lessons from a Temporal Study in Veal Calves.

Intestinal carriage of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing, and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over 6 months. We characterized the bacterial clones and plasmids carrying blaESBL genes with a combination of genotyping methods, whole genome sequencing, and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [blaCTX-M-1 (64.7%), blaCTX-M-14 (33.5%), or blaCTX-M-15 (1.8%)] were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and blaCTX-M-carrying plasmids, few blaCTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario "clone versus plasmid spread" depended on the farm. Thus, epistatic interactions between resistance genes, plasmids, and bacterial clones contribute to optimize fitness in specific environments. IMPORTANCE The gut microbiota is the epicenter of the emergence of resistance. Considerable amount of knowledge on the molecular mechanisms of resistance has been accumulated, but the ecological and evolutionary forces at play in nature are less studied. In this context, we performed a field work on temporal intestinal carriage of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli in veal farms. Veal calves are animals with one of the highest levels of ESBL producing E. coli fecal carriage, due to early high antibiotic exposure. We were able to show that calves were colonized successively by different ESBL-producing E. coli clones, and that two main scenarios were at play in the spread of blaCTX-M genes among calves: efficient colonization of several calves by a few bacterial clones and successful plasmid spread in various bacterial clones. Such knowledge should help develop new strategies to fight the emergence of antibiotic-resistance.

Genomic and Phenotypic Analysis of Heat and Sanitizer Resistance in Escherichia coli from Beef in Relation to the Locus of Heat Resistance.

The locus of heat resistance (LHR) can confer heat resistance to Escherichia coli to various extents. This study investigated the phylogenetic relationships and the genomic and phenotypic characteristics of E. coli with or without LHR recovered from beef by direct plating or from enrichment broth at 42°C. LHR-positive E. coli isolates (n = 24) were subjected to whole-genome sequencing by short and long reads. LHR-negative isolates (n = 18) from equivalent sources as LHR-positive isolates were short-read sequenced. All isolates were assessed for decimal reduction time at 60°C (D60°C) and susceptibility to the sanitizers E-SAN and Perox-E. Selected isolates were evaluated for growth at 42°C. The LHR-positive and -negative isolates were well separated on the core genome tree, with 22/24 positive isolates clustering into three clades. Isolates within clade 1 and 2, despite their different D60°C values, were clonal, as determined by subtyping (multilocus sequence typing [MLST], core genome MLST, and serotyping). Isolates within each clade are of one serotype. The LHR-negative isolates were genetically diverse. The LHR-positive isolates had a larger (P < 0.001) median genome size by 0.3 Mbp (5.0 versus 4.7 Mbp) and overrepresentation of genes related to plasmid maintenance, stress response, and cryptic prophages but underrepresentation of genes involved in epithelial attachment and virulence. All LHR-positive isolates harbored a chromosomal copy of LHR, and all clade 2 isolates had an additional partial copy of LHR on conjugative plasmids. The growth rates at 42°C were 0.71 ± 0.02 and 0.65 ± 0.02 log(OD) h-1 for LHR-positive and -negative isolates, respectively. No meaningful difference in sanitizer susceptibility was noted between LHR-positive and -negative isolates. IMPORTANCE Resistant bacteria are serious food safety and public health concerns. Heat resistance conferred by the LHR varies largely among different strains of E. coli. The findings in this study show that genomic background and composition of LHR, in addition to the presence of LHR, play an important role in the degree of heat resistance in E. coli and that strains with certain genetic backgrounds are more likely to acquire and maintain the LHR. Also, caution should be exercised when recovering E. coli at elevated temperatures, as the presence of LHR may confer growth advantages to some strains. Interestingly, the LHR-harboring strains seem to have evolved further from their primary animal host to adapt to their secondary habitat, as reflected by fewer genes involved in virulence and epithelial attachment. The phylogenetic relationships among the isolates point toward multiple mechanisms for acquisition of LHR by E. coli, likely prior to its being deposited on meat.