Tina Birk

Fate of Food-Associated Bacteria in Pork as Affected by Marinade, Temperature, and Ultrasound

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 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 42 degrees C red wine for 15 min prior to storage at 4 degrees C or submerged in 4 degrees C red wine during the entire experiment. In the vacuum procedure, the meat was either submerged in 4 degrees C red wine for 4 h or submerged in 42 degrees C red wine for 15 min prior to vacuum packaging and storage at 4 degrees C. The most efficient antimicrobial procedure was submersion of the pork meat in 42 degrees C red wine for 15 min and subsequent storage at 4 degrees C, 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. 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.

A Comparative Study of Two Food Model Systems To Test the Survival of Campylobacter jejuni at −18°C

The survival of Campylobacter jejuni NCTC 11168 was tested at freezing conditions (-18 degrees C) over a period of 32 days in two food models that simulated either (i) the chicken skin surface (skin model) or (ii) the chicken juice in and around a broiler carcass (liquid model). In the skin model, cells were suspended in chicken juice or brain heart infusion broth (BHIB) and added to 4-cm2 skin pieces, which were subsequently stored at -18 degrees C. In the liquid model, cells were suspended in chicken juice or BHIB and stored at -18 degrees C. The decrease in the number of viable C. jejuni NCTC 11168 cells was slower when suspended in chicken juice than in BHIB. After freezing for 32 days, the reductions in the cell counts were 1.5 log CFU/ml in chicken juice and 3.5 log CFU/ml in BHIB. After the same time of freezing but when inoculated onto chicken skin, C. jejuni NCTC 11168 was reduced by 2.2 log units when inoculated in chicken juice and 3.2 log units when inoculated into BHIB. For both models, the major decrease occurred within the first 24 h of freezing. The results obtained in the liquid model with chicken juice were comparable to the reductions of Campylobacter observed for commercially processed chickens. The survival at -18 degrees C in the liquid model was also tested for three poultry isolates and three human clinical isolates of the serotypes 1.44, 2, and 4 complex. As observed for C. jejuni NCTC 11168, all the strains survived significantly better in chicken juice than in BHIB and were not notably influenced by serotype or origin. The findings indicate that the composition of the medium around the bacteria, rather than the chicken skin surface, is the major determining factor for the survival of C. jejuni at freezing conditions. The liquid model with chicken juice was therefore the best model system to study the freezing tolerance in Campylobacter strains.

Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

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.

Survival and growth of epidemically successful and nonsuccessful Salmonella enterica clones after freezing and dehydration.

The spread of epidemically successful nontyphoidal Salmonella clones has been suggested as the most important cause of salmonellosis in industrialized countries. Factors leading to the emergence of success clones are largely unknown, but their ability to survive and grow after physical stress may contribute. During epidemiological studies, a mathematical model was developed that allowed estimation of a factor (q) accounting for the relative ability of Salmonella serovars with different antimicrobial resistances to survive in the food chain and cause human disease. Based on this q-factor, 26 Salmonella isolates were characterized as successful or nonsuccessful. We studied the survival and growth of stationary- and exponential-phase cells of these isolates after freezing for up to 336 days in minced meat. We also investigated survival and growth after dehydration at 10°C and 82% relative humidity (RH) and 25°C and 49% RH for 112 days. Stationary-phase cells were reduced by less than 1 log unit during 1 year of freezing, and growth was initiated with an average lag phase of 1.7 h. Survival was lower in exponentialphase cells, but lag phases tended to be shorter. High humidity and low temperature were less harmful to Salmonella than were low humidity and high temperature. Tolerance to adverse conditions was highest for Salmonella Infantis and one Salmonella Typhimurium U292 isolate and lowest for Salmonella Derby and one Salmonella Typhimurium DT170 isolate. Dehydration, in contrast to freezing, was differently tolerated by the Salmonella strains in this study, but tolerance to freezing and dehydration does not appear to contribute to the emergence of successful Salmonella clones.

Dietary proteins extend the survival of Salmonella Dublin in a gastric acid environment.

The pH of the human stomach is dynamic and changes over time, depending on the composition of the food ingested and a number of host-related factors such as age. To evaluate the number of bacteria surviving the gastric acid barrier, we have developed a simple gastric acid model, in which we mimicked the dynamic pH changes in the human stomach. In the present study, model gastric fluid was set up to imitate pH dynamics in the stomachs of young and elderly people after ingestion of a standard meal. To model a serious foodborne pathogen, we followed the survival of Salmonella enterica serotype Dublin, and found that the addition of proteins such as pepsin, ovalbumin, and blended turkey meat to the simple gastric acid model significantly delayed pathogen inactivation compared with the control, for which no proteins were added. In contrast, no delay in inactivation was observed in the presence of bovine serum albumin, indicating that protection could be protein specific. The simple gastric acid model was validated against a more laborious and complex fermenter model, and similar survival of Salmonella Dublin was observed in both models. Our gastric acid model allowed us to evaluate the influence of food components on survival of pathogens under gastric conditions, and the model could contribute to a broader understanding of the impact of specific food components on the inactivation of pathogens during gastric passage.

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni

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.

Growth potential of exponential- and stationary-phase Salmonella Typhimurium during sausage fermentation

Raw meat for sausage production can be contaminated with Salmonella. For technical reasons, meat is often frozen prior to mincing but it is unknown how growth of Salmonella in meat prior to freezing affects its growth potential during sausage fermentation. We investigated survival of exponential- and stationary-phase Salmonella Typhimurium (DT12 and DTU292) during freezing at -18°C and their subsequent growth potential during 72h sausage fermentation at 25°C. After 0, 7 and >35d of frozen storage, sausage batters were prepared with NaCl (3%) and NaNO2 (0, 100ppm) and fermented with and without starter culture. With no starter culture, both strains grew in both growth phases. In general, a functional starter culture abolished S. Typhimurium growth independent of growth phase and we concluded that ensuring correct fermentation is important for sausage safety. However, despite efficient fermentation, sporadic growth of exponential-phase cells of S. Typhimurium was observed drawing attention to the handling and storage of sausage meat.

Cross and co resistance among Danish porcine E. coli isolates

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.