Effect of natural microbiota on growth of Salmonella spp. in fresh pork--a predictive microbiology approach.

This study was undertaken to model and predict growth of Salmonella and the dominating natural microbiota, and their interaction in ground pork. Growth of Salmonella in sterile ground pork at constant temperatures between 4 °C and 38 °C was quantified and used for developing predictive models for lag time, max. specific growth rate and max. population density. Data from literature were used to develop growth models for the natural pork microbiota. Challenge tests at temperatures from 9.4 to 24.1 °C and with Salmonella inoculated in ground pork were used for evaluation of interaction models. The existing Jameson-effect and Lotka-Volterra species interaction models and a new expanded Jameson-effect model were evaluated. F-test indicated lack-of-fit for the classical Jameson-effect model at all of the tested temperatures and at 14.1-20.2 °C this was caused by continued growth of Salmonella after the natural microbiota had reached their max. population density. The new expanded Jameson-effect model and the Lotka-Volterra model performed better and appropriately described the continued but reduced growth of Salmonella after the natural microbiota had reached their max. population density. The expanded Jameson-effect model is a new and simple species interaction model, which performed as well as the more complex Lotka-Volterra model.

Prevalence and risk factors for Salmonella in veal calves at Danish cattle abattoirs.

The study's objectives were to determine herd- and animal-level prevalence and herd-level risk factors for Salmonella in dairy-bred veal calves at slaughter in Denmark. In total, 1296 faecal samples were collected at five cattle abattoirs in Denmark during 2007-2008. The animals came from 71 randomly selected specialized veal-calf producers that delivered more than 100 animals to slaughter per year. Salmonella Dublin bacteria were isolated from 19 samples from 12 herds and Salmonella Typhimurium was isolated from one sample. The apparent prevalence of herds delivering Salmonella-shedding animals to slaughter was 18% (95% CI 9-27). The overall estimated true prevalence of shedding calves at slaughter was 1.3%. Veal-calf herds that purchased animals from herds not classified as low risk in the Danish Salmonella surveillance programme had significantly (P=0.03) higher risk of delivering Salmonella-shedding calves to slaughter. The results emphasize the importance of efforts in the dairy industry to ensure food safety for consumers.

Survival of Salmonella on cuts of beef carcasses subjected to dry aging.

AIMS: The aim of this study was to determine the survival of 15 different strains of Salmonella of selected serotypes during prolonged cold storage of beef. METHODS AND RESULTS: Fifteen strains of eight different serotypes of Salmonella were spiked onto fresh cuts beef portions, and the survival was followed during storage in a laboratory cooling system. Over a 14-day period, all strains were reduced significantly in numbers; however, strains of Salmonella Typhimurium DT104 and Salmonella Enteritidis PT4 and PT8 survived significantly longer than strains of the serovars Dublin, Derby, Infantis and Newport. For five selected strains, the observations were verified in a pilot plant cooling facility mimicking industrial cooling. No significant differences in reduction were found between the two cooling methods. CONCLUSIONS: A significant reduction in Salmonella can be obtained by dry aging of beef during cold storage but the survival is strain dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: From a hygienic point of view, cold storage of unpacked beef, which is still performed in small slaughterhouses, is a good alternative to vacuum packaging.

Horizontal transmission of antimicrobial resistance genes from E. coli to Serratia spp. in minced meat using a gfp tagged plasmid.

The transmission of antimicrobial resistance genes from enteric bacteria from the animal reservoir to indigenous bacteria in meat is a serious concern, as it can contribute to human exposure to antimicrobial resistance genes. The aim of this study was to investigate plasmid-mediated horizontal transfer of antimicrobial resistance genes from Escherichia coli to indigenous environmental bacteria in minced pork stored at 10 and 37 °C. E. coli MG1555 containing a gfp-tagged plasmid carrying tetracycline, kanamycin and streptomycin resistance genes was used as the donor with the indigenous bacteria in minced pork acting as potential recipients. The results demonstrated that enteric members of the pork meat microbiota were able to receive gfp-plasmids from the E. coli donor strain at both 10 and 37 °C. The majority of transconjugants were identified as Serratia spp. through sequencing of their 16S rRNA genes. This indicates that environmental Serratia spp. and other Enterobacteriaceae may play a role as carrier of antimicrobial resistance genes through the meat production chain to the consumer.

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.

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.

Evaluation of a cross contamination model describing transfer of Salmonella spp. and Listeria monocytogenes during grinding of pork and beef.

In a previous study, a model was developed to describe the transfer and survival of Salmonella during grinding of pork (Møller, C.O.A., Nauta, M.J., Christensen, B.B., Dalgaard, P., Hansen, T.B., 2012. Modelling transfer of Salmonella typhimurium DT104 during simulation of grinding of pork. Journal of Applied Microbiology 112 (1), 90-98). The robustness of this model is now evaluated by studying its performance for predicting the transfer and survival of Salmonella spp. and Listeria monocytogenes during grinding of different types of meat (pork and beef), using two different grinders, different sizes and different numbers of pieces of meats to be ground. A total of 19 grinding trials were collected. Acceptable Simulation Zone (ASZ), visual inspection of the data, Quantitative Microbiological Risk Assessment (QMRA), as well as the Total Transfer Potential (TTP) were used as approaches to evaluate model performance and to access the quality of the cross contamination model predictions. Using the ASZ approach and considering that 70% of the observed counts have to be inside a defined acceptable zone of ±0.5 log10CFU per portion, it was found that the cross contamination parameters suggested by Møller et al. (2012) were not able to describe all 19 trials. However, for each of the collected grinding trials, the transfer event was well described when fitted to the model structure proposed by Møller et al. (2012). Parameter estimates obtained by fitting observed trials performed at different conditions, such as size and number of pieces of meat to be ground, may not be applied to describe cross contamination of unlike processing. Nevertheless, the risk estimates, as well as the TTP, revealed that the risk of disease may be reduced when the grinding of meat is performed in a grinder made of stainless steel (for all surfaces in contact with the meat), using a well-sharpened knife and holding at room temperatures lower than 4°C.

Virulence characterization of a strain of Salmonella enterica subspecies houten (subspecies IV) with chromosomal integrated Salmonella plasmid virulence (spv) genes.

The Salmonella plasmid virulence genes (spv) are commonly found on plasmids contained in a small number of serotypes of Salmonella belonging to subspecies I, where they are important for survival within macrophages and the establishment of successful systemic infection. However, in this study, spv genes were detected by the polymerase chain reaction in the chromosome of a plasmid-free strain of S. IV 16:z4, z32:- (Salmonella subspecies IV). The full range of spv genes (spvR, spvA, spvB, spvC and spvD) was demonstrated, but a 216-bp deletion, accompanied by an insertion of 59-bp cryptic DNA, was present in spvA. S. IV 16:z4, z32:- was avirulent in mice and did not become virulent with the introduction of a fully functionally serotype-associated virulence plasmid (SAP) from S. typhimurium. By use of an spvRAB'-chloramphenicol acetyl transferase fusion gene, it was demonstrated that S. IV 16:z4, z32:- did not express the spv genes. Salmonella subspecies IV is monophasic, and in phylogenetic analyses it clusters distantly to Salmonella subspecies I, where all the serotypes that normally carry SAPs are found. The mechanisms by which spv genes have been transferred to this serotype remain unknown.

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin.

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.

Isolation of a Salmonella-specific DNA hybridization probe.

A Salmonella-specific DNA fragment of the Salmonella typhimurium LT2 chromosome has been isolated. The fragment (2.3 kilobases (kb)) was used as a probe in a colony hybridization assay, where 185 strains of 93 different Salmonella serovars were correctly identified as belonging to Salmonella. The specificity of the probe was evaluated in colony hybridization assays on pure cultures of non-Salmonella bacteria and on specimens with an indigenous flora. Sixty-three strains of 34 non-Salmonella Gram-negative species did not hybridize to the fragment. By DNA hybridization to faecal samples from calves, pigs and chickens, and samples of animal feed, three samples out of 10 positive by traditional culture methods gave negative results by hybridization, 45 samples were negative in both methods, while one sample was positive only in the hybridization assay. From this sample, Salmonella livingstone was isolated by a replica plate hybridization technique. The probe therefore proved 100% specific for the genus Salmonella. The 2.3 kb fragment may form the basis of hybridization assays for specific detection of Salmonella in food, environmental and clinical specimens.

Evaluation of a Salmonella-specific DNA probe by colony hybridization using non-isotopic and isotopic labeling.

A 2.3 kilobase (kb) Salmonella probe, JEO402-1, and two subfragments, F1214 (1.3 kb) and F1217 (0.8 kb), have been evaluated by colony hybridization using pure cultures of Salmonella serovars and non-salmonella bacteria. JEO402-1, and its subfragments, F1214 and F1217, hybridized to all of 156 different Salmonella serovars tested, while there was no reaction to 112 non-salmonella strains belonging to 19 genera and 37 species of Enterobacteriaceae. Together with previously published results, the JEO402-1 probe has now been shown to detect a total of 396 Salmonella strains belonging to 214 serovars of Salmonella subspecies I-VI. A total of 178 non-salmonella strains representing 23 genera and 51 species of Enterobacteriaceae have all tested negative with JEO402-1. The hybridization results obtained using a digoxigenin-labeled probe were similar to those obtained with 35S isotopic labeling when complete colony lysis was ensured.

Probes and polymerase chain reaction for detection of food-borne bacterial pathogens.

DNA-hybridization and the polymerase chain reaction (PCR) are techniques commonly used to detect pathogenic bacteria. In this paper, the use of these techniques for detection of Salmonella, E. coli, V. cholerae, non-O1 Vibrio, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and C. botulinum is reviewed with emphasis on application in food microbiology. In food control, DNA-techniques have most often been used in a 'culture confirmation' fashion, i.e. bacteria are enriched and sometimes even purified by traditional culture procedures and thereafter identified by the use of DNA-based methods. The most desirable approach is, however, to detect organisms directly in the food, but major problems remain to be solved before this can be routinely performed.

Mycoplasma hyosynoviae in joints with arthritis in abattoir baconers.

The occurrence of Mycoplasma hyosynoviae in synovial fluid of baconers with chronic arthritis was studied at an abattoir. Cultural examination of synovial fluid samples from diseased tarsal joints of 50 animals from 42 herds yielded M. hyosynoviae in 10 cases from 8 herds. Streptococci were found in 6 cases from 6 other herds. M. hyosynoviae antigen was found in 1 of 47 of the samples, and antibody to the mycoplasma was found in 14 of 40 of the samples by ELISA test. The presence of M. hyosynoviae in a joint was usually accompanied by the corresponding antibody. In joints with streptococcal infection antibody to M. hyosynoviae could not be found.

Salmonella in pork cuttings in supermarkets and butchers' shops in Denmark in 2002 and 2006.

The prevalence of Salmonella in fresh pork cuttings in Denmark in the years 2002 and 2006 was investigated at retail and compared with the retail supply pattern. A total of 1025 and 3473 samples were taken in 2002 from butcher's shops and supermarkets, respectively. The corresponding numbers in 2006 were 259 from butchers' shops and 628 from supermarkets. In 2002, 1.2% of all samples were positive for Salmonella; butchers' shops and supermarkets had 1.8% and 1.0% positive samples, respectively. The overall prevalence in 2006 was 4.2%, with prevalence of 8.1% and 2.6% for butchers' shops and supermarkets, respectively. Hence, increases around 3- to 5-fold were found. There was neither observed any parallel increase in Salmonella positive carcasses in Danish slaughterhouses during the study period, nor were any changes in supply routes towards slaughterhouses with higher prevalence observed, which could explain the apparent increase. We hypothesize that hygiene levels and ability to avoid cross-contamination and prevent growth of the organism, in the meat processing chain after slaughter were the most likely responsible factors. Results from this study indicate that the hygiene performance, particularly at retail, has a significant impact on the occurrence of Salmonella. This implies that there is no direct link between slaughterhouse Salmonella surveillance data and the level of Salmonella contamination at retail. To improve risk assessment of Salmonella in fresh pork meat, this study underlines the need for comprehensive retail data.

Description of extended pre-harvest pig Salmonella surveillance-and-control programme and its estimated effect on food safety related to pork.

Salmonella in pork can be combated during pre- or post-harvest. For large slaughterhouses, post-harvest measures like decontamination might be cost-effective while this is less likely with small-to-medium sized slaughterhouses. In this study, pre-harvest measures might be more relevant. We describe an extended surveillance-and-control programme for Salmonella in finisher pigs, which, to establish equivalence to the Swedish control programme, is intended for implementation on the Danish island, Bornholm. The effect of the programme on food safety was estimated by analysing Salmonella data from pig carcasses originating from herds that would have qualified for the programme during 2006-2008. Food safety was interpreted as prevalence of Salmonella on carcasses as well as the estimated number of human cases of salmonellosis related to pork produced within the programme. Data from the Danish Salmonella programme were obtained from Bornholm. We used a simulation model developed to estimate the number of human cases based on the prevalence of Salmonella on carcass swabs. Herds are only accepted in the programme if they have one or less seropositive sample within the previous 6 months. In this way, the Salmonella load is kept to a minimum. The programme is not yet in operation and pigs that qualify for the programme are currently mixed at slaughter with those that do not qualify. Therefore, we had to assess the impact on the carcass prevalence indirectly. The prevalence of Salmonella in carcass swabs among qualifying herds was 0.46% for the 3 years as a whole, with 2006 as the year with highest prevalence. According to the simulation the expected number of human cases relating to pork produced within the programme was below 10. When the programme is in operation, an extra effect of separating pigs within the programme from those outside is expected to lower the prevalence of Salmonella even further.

Research note: detection of Salmonella in minced meat by the polymerase chain reaction method.

A specific PCR assay was used to detect Salmonella in enriched broths of 48 natural samples of minced pork and 48 natural samples of minced beef. By comparison with a routine culture method, the sensitivity of the PCR method was estimated to be 92% and the specificity of the PCR method was estimated to be 99%. The sensitivity of the culture method was estimated to be 50% in this study.

Oligonucleotide probes specific for the genus Salmonella and for Salm. typhimurium.

Synthetic oligonucleotides have been deduced and synthesized based on the sequence of a salmonella-specific polynucleotide probe. Two oligonucleotide probes, ST4 and ST15rev hybridized to 93 and 92 strains respectively out of 93 strains of Salmonella analysed. ST4, however, cross hybridized to one of the 28 strains of 16 genera of Enterobacteriaceae tested. Based on sequence alignment, in 16 strains of Salmonella, of a 114 base pair region, a Salm. typhimurium specific oligonucleotide probe, ST22, was identified. In colony hybridization, this probe detected all 47 strains of Salm. typhimurium analysed without hybridization to 94 strains of other Salmonella serotypes and to 26 strains of non-Salmonella bacteria.

Salmonella identification by the polymerase chain reaction.

Polymerase chain reaction (PCR) primers for genus specific detection of Salmonella have been selected from a Salmonella-specific fragment of 2.3 kilobases (kb). Due to interserovar sequence diversity within this fragment, primer selection was based on DNA sequence alignment of sequences from 20 different Salmonella serovars. The specific PCR product of 429 base pairs (bp) was formed from 144 of 146 salmonella strains tested (116 of 118 serovars). The two false-negative strains belonged to two different serovars of the rarely isolated subspecies IIIa (monophasic S. arizonae). No product was produced in any of 86 non-Salmonella Enterobacteriacea strains tested, covering 41 species from 21 genera.

Development of an in vivo model for study of intestinal invasion by Salmonella enterica in chickens.

An in vivo loop test model for the investigation of the invasiveness of Salmonella enterica in chickens was developed. Ten jejunal loops were made in 10- to 12-week-old Lohman Brown chickens under isofluoran anaesthesia. Salmonella at 5.0 x 10(7) CFU was inoculated into each loop and left for 2 h, followed by a 1-h incubation with gentamicin in order to kill noninvading bacteria. After euthanasia, Salmonella invasiveness was measured as tissue-associated counts relative to a reference strain. The ability of Salmonella invasion was 1 log(10) CFU higher per 42-mm(2) mucosal tissue in the anterior than in the posterior part of jejunum. A statistically significant (P<0.001) sixfold difference in invasiveness was observed between a wild-type S. enterica serotype Typhimurium strain and the corresponding invH mutant. The model was shown to be able to show small differences in invasive capability and allows for comparison of strains tested in different animals, provided that the same reference strain is present in all animals.

Quantitative comparison of intestinal invasion of zoonotic serotypes of Salmonella enterica in poultry.

The aim of the present study was to compare the invasion of selected zoonotic Salmonella serotypes of poultry in an in vivo chicken intestinal loop model and also in vitro in epithelial cell cultures. Invasion was measured relative to a reference strain, Salmonella Typhimurium 4/74 invH201::TnphoA. Two serotypes demonstrated intracellular log(10) counts that differed significantly from all other serotypes tested: Salmonella Enteritidis PT4 being 1.5 log(10) colony forming units (CFU) (31-fold) higher, and Salmonella Tennessee being 0.7 log(10) CFU (fivefold) lower than the reference strain (P < or = 0.0001). A group of serotypes, which can be vertically transmitted, showed significantly higher intracellular counts (fourfold to eightfold) than the reference strain. The group included S. Typhimurium 4/74, S. Typhimurium DT104 (poultry and porcine isolates), S. Enteritidis PT1, S. Enteritidis PT6, S. Enteritidis PT8, and Salmonella Berta. The serotypes Salmonella Hadar, Salmonella Virchow, S. 4,12:b:-, S. Typhimurium DT41, and Salmonella Infantis, most of which are considered horizontally transmitted, did not show significantly different intracellular counts from the reference strain. Results from the cell culture invasion studies agreed with the in vivo data, with the exception of S. Berta and the poultry isolate of S. Typhimurium DT104.