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The emergence of extended-spectrum cephalosporin (ESC)-resistant Escherichia coli is a global concern. This study aimed to assess the prevalence and transmission of ESC-resistant E. coli in the Danish broiler production system. Samples from two vertically integrated Production Systems (1 and 2) and two slaughterhouses (A and B) were analyzed (n = 943) for the occurrence of ESC-resistant E. coli from 2015 to 2018. ESC-resistant E. coli isolates were whole-genome sequenced (WGS) for characterization of the multi-locus sequence type (MLST), antibiotic resistance genes, virulence genes, and plasmid replicon types. An ad hoc core genome (cg) MLST based on 2513 alleles was used to examine the genetic relatedness among isolates. The prevalence of ESC-resistant E. coli in the conventional Production System 1 was 2.7%, while in Production System 2 the prevalence was 26.7% and 56.5% for samples from the conventional and organic production, respectively. The overall prevalence of ESC-resistant E. coli in broiler thigh and fecal samples ranged from 19.3% in Slaughterhouse A to 22.4% in Slaughterhouse B. In total, 162 ESC-resistant E. coli were isolated and shown to belong to 16 different sequence types (STs). The most prevalent STs were ST2040 (n = 85) and ST429 (n = 22). Seven ESC resistance genes were detected: blaCMY-2 (n = 119), blaTEM-52B (n = 16), blaCTX-M-1 (n = 5), blaTEM-52C (n = 3), blaCTX-M-14 (n = 1), blaSHV-12 (n = 1), and up-regulation of ampC (n = 16), with an unknown resistance gene in one isolate (n = 1). The carriage of blaCMY-2 in 119 isolates was primarily associated with IncI1 (n = 87), and IncK plasmids (n = 31). Highly similar blaCMY-2 carrying E. coli isolates from ST429 were found in production systems as well as in slaughterhouses. In conclusion, findings

Køleopbevarede spiseklare og ready-to-cook (RTC) produkter kan være af rå eller varmebehandlet karakter. Også blandinger af rå og varmebehandlede fødevarer forekommer. Fælles for alle spiseklare produkter er, at de spises uden forudgående opvarmning. Derimod kræver RTC produkter af rå fødevarer varmebehandling inden brug, og RTC produkter af varmebehandlede fødevarer kræver genopvarmning inden brug. Denne forskel betyder, at der er variationer i hvilke fødevarebårne risikofaktorer, der vil være begrænsende for holdbarheden. I alt er fem fødevarebårne og sygdomsfremkaldende mikroorganismer, der kan vokse under 10 °C, udpeget som mulige holdbarhedsbegrænsende risikofaktorer for køleopbevarede spiseklare og RTC fødevarer. Disse er Listeria monocytogenes for alle spiseklare produkter, Yersinia enterocolitica for spiseklare produkter bestående af råt svinekød, Bacillus cereus for alle spiseklare produkter, ikke-proteolytiske C. botulinum for alle spiseklare samt RTC produkter samt histamin-producerende bakterier for spiseklare samt RTC produkter bestående af rå fisk.

Cross and co-resistance to antimicrobials are presented for 765 Danish Escherichia coli isolates of porcine origin from 2009 to 2013. All isolates and data originate from the DANMAP surveillance but have not previously been used to describe the occurrence of cross and co- resistance. Data presented here clearly indicate the ability of low classified antimicrobials as ampicillin to uphold resistance to critical important antimicrobials for human treatment.

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular Delta kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile Delta motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.

Food Microbiology is an interdisciplinary 12.5 ETCS second-year) course in a CDIO-based Bachelor of Engineering program in Food Science at The Technical University of Denmark (DTU). The course was first offered in 2011. Each session in the Food Microbiology course combines theory and practice in order to strengthen the students’ application-oriented competences and engagement. In this paper the results from the evaluation of the course will be presented and a discussion will be carried out about how the students responded to the multidisciplinary, real-life projects and how it affects student learning. The aims of this study were to test 1) the students’ perception combining theory with small laboratory exercises and 2) the students’ perception of how the course collaborates with and combines theories and practices from other current semester courses. The students evaluated the course in general using the Course Experience Questionnaire (Ramsden, 1991) and by answering a questionnaire concerning the collaboration between the other courses. It can be concluded that the combination of: theory/laboratory exercises/report writing stimulated the students’ motivation and that collaboration between other mandatory semester courses mainly was rated positively by the students.

Food composition, buffer capacity, and fat and protein content have been shown to effect the gastric acid survival of pathogens (Waterman & Small 1998). In this study, simple food-model substances with different buffer capacities were investigated for their ability to support survival of stationary phase Salmonella Typhimurium during simulated gastric acid passage. We used a computer-controlled fermentor to employ pH changes in synthetic gastric fluid, mimicking the dynamic pH during gastric passage. In order to minimise variation, Salmonella enterica serovar Typhimurium was contained in dialysis tubes, enabling simultaneous testing of biological triplicates under varying conditions. Surprisingly, we found that less buffered media provided higher protection of Salmonella, compared to media with high buffer capacity. By investigating the relative gene expression of rpoS and ompR encoding for two major stationary phase ATR regulators, we found an approx. four-fold increase in expression of ompR and an approx. three-fold increase of rpoS in saline and buffered saline, respectively, after 15 min of gastric acid challenge. The relative expression of these genes, were significantly lower in Brain Heart Infusion Broth having a higher buffer capacity. We suggest this to be associated with a varying ability of Salmonella Typhimurium to mount a stationary phase acid tolerance response (ATR) depending on the buffer capacity of the food vehicle.

BACKGROUND: During the transmission route from poultry to the human host, the major foodborne pathogen C. jejuni may experience many types of stresses, including low pH caused by different acids. However, not all strains are equally sensitive to the stresses. The aim of this study was to investigate the response to acid stress of three sequenced C. jejuni strains with different acid tolerances using HCl and acetic acid. RESULTS: Two-dimensional gel electrophoresis was used for proteomic analysis and proteins were radioactively labelled with methionine to identify proteins only related to acid exposure. To allow added radioactive methionine to be incorporated into induced proteins, a modified chemically defined broth was developed with the minimal amount of methionine necessary for satisfactory growth of all strains. Protein spots were analyzed using image software and identification was done with MALDI-TOF-TOF. The most acid-sensitive isolate was C. jejuni 327, followed by NCTC 11168 and isolate 305 as the most tolerant. Overall, induction of five proteins was observed within the pI range investigated: 19 kDa periplasmic protein (p19), thioredoxin-disulfide (TrxB), a hypothetical protein Cj0706 (Cj0706), molybdenum cofactor biosynthesis protein (MogA), and bacterioferritin (Dps). Strain and acid type dependent differences in the level of response were observed. For strain NCTC 11168, the induced proteins and the regulator fur were analysed at the transcriptomic level using qRT-PCR. In this transcriptomic analysis, only up-regulation of trxB and p19 was observed. CONCLUSIONS: A defined medium that supports the growth of a range of Campylobacter strains and suitable for proteomic analysis was developed. Mainly proteins normally involved in iron control and oxidative stress defence were induced during acid stress of C. jejuni. Both strain and acid type affected sensitivity and response.

The aim of this study was to determine whether marination of chicken meat in different food ingredients can be used to reduce populations of Campylobacter jejuni. C. jejuni strains were exposed to different organic acids (tartaric, acetic, lactic, malic, and citric acids) and food marinating ingredients at 4°C in broth and on chicken meat. The organic acids (0.5%) reduced populations of C. jejuni in broth (chicken juice and brain heart infusion broth) by 4 to 6 log units (after 24 h); tartaric acid was the most efficient treatment. Large strain variation was observed among 14 C. jejuni isolates inoculated in brain heart infusion broth containing 0.3% tartaric acid. On chicken meat medallions, reductions of C. jejuni were 0.5 to 2 log units when tartaric acid solutions (2, 4, 6, and 10%) were spread onto the meat. Analysis of acidic food ingredient (e.g., vinegar, lemon juice, pomegranate syrup, and soya sauce) revealed that such ingredients reduced counts of C. jejuni by at least 0.8 log units on meat medallions. Three low pH marinades (pH < 3) based on pomegranate syrup, lemon juice, and white wine vinegar were prepared. When applied to whole filets, these marinades resulted in a reduction of approximately 1.2 log units after 3 days of storage. Taste evaluations of chicken meat that had been marinated and then fried were graded positively for flavor and texture. Thus, success was achieved in creating a marinade with an acceptable taste that reduced the counts of C. jejuni.

The aim of this study was to test the effect of ultrasound, red wine, and yogurt marination on Brochotrix thermosphacta, Carnobacterium maltaromaticum, Listeria monocytogenes, and Campylobacter jejuni on pork meat. Two different marinationand on pork meat. Two different marination procedures of the pork medallions were tested: (i) submersion of meat medallions in red wine during the entire experiment and (ii) vacuum packaging of meat medallions after different forms of marination. In the submersion procedure, the meat was either submerged in 42C red wine for 15 min prior to storage at 4C or submerged in 4C red wine during the entire experiment. In the vacuum procedure, the meat was either submerged in 4C red wine for 4 h or submerged in 42C red wine for 15 min prior to vacuum packaging and storage at 4C. The most efficient antimicrobial procedure was submersion of thepork meat in 42C red wine for 15 min and subsequent storage at 4C, still submerged in red wine. After 3 days, C. maltaromaticum, L. monocytogenes, B. thermosphacta, and C. jejuni were reduced approximately 1.5, 2, 3, and 6 log, respectively.and were reduced approximately 1.5, 2, 3, and 6 log, respectively. The remarkable acid sensitivity of C. jejuni compared with the other bacteria was confirmed in an experiment with yogurt as a marinade. Ultrasound treatment in combination with red wine enhanced the antibacterial effect compared with ultrasound alone for L. monocytogenes, B. thermosphacta, and C. jejuni and resulted in approximately a 1-log reduction after 10 min. This synergistic effect of ultrasound and red wine was not observed for C. maltaromaticum.

Campylobacter jejuni is a micro-aerobic foodborne pahtogenic bacteria generally regarded as one of the major causes of gastroenteritis in humans worlwoide. Consumption and handling of fresh poultry meat are considered the primary sources of campylobacteriosis in Denmark.