Subtyping of Listeria monocytogenes isolates recovered from retail ready-to-eat foods, processing plants and listeriosis patients in Sweden 2010.

Identification and prioritisation of food safety interventions requires an understanding of the relationship between food, pathogens and cases. Such understanding can be gained through different approaches, e.g. microbial subtyping to attribute cases of foodborne disease to food vehicles or other sources of illness. In this study, Listeria monocytogenes isolates (n=166) from (i) three categories of ready-to-eat (RTE) foods, (ii) food processing plant environments, and (iii) human listeriosis cases, all sampled during 2010 in Sweden, were subtyped. In addition, 121 isolates from human listeriosis cases, collected 2005-2009, were subtyped. Subtyping consisted of both serotyping (conventional method and PCR) and genotyping using pulsed-field gel electrophoresis (PFGE). Serotype 1/2a dominated in all three groups of isolates (range 73-96%). Eighteen percent of the human isolates (2010) belonged to serotype 4b, but only 1.4% of the food isolates. The food isolates differentiated into 19 pulsotypes (ID=0.843), the human isolates collected 2010 into 31 pulsotypes (ID=0.950) and the processing plant isolates into 22 pulsotypes (ID=0.991). Six of the pulsotypes were shared between the food and human isolates. These pulsotypes comprised 42% of the human isolates and 59% of the food isolates. For some processing plants, there was suggested persistence of one or more specific L. monocytogenes strains, as indicated by repetitive isolation of the same pulsotype from food. This study indicated the presence of L. monocytogenes in the processing plant environment as a likely source of contamination of gravad and cold-smoked fish, and this food category as an important source of human exposure to the pathogen.

Occurrence of pathogens in wild rodents caught on Swedish pig and chicken farms.

A total of 207 wild rodents were caught on nine pig farms, five chicken farms and five non-farm locations in Sweden and surveyed for a selection of bacteria, parasites and viruses. Lawsonia intracellularia and pathogenic Yersinia enterocolitica were only detected in rodents on pig farms (9% and 8% prevalence, respectively) which indicate that these agents are more likely to be transmitted to rodents from pigs or the environment on infected farms. Brachyspira hyodysenteriae (1%), Brachyspira intermedia (2%), Campylobacter jejuni (4%), Campylobacter upsaliensis (2%), leptospires (7%) and encephalomyocarditis virus (9%) were also detected from rodents not in contact with farm animals. Giardia and Cryptosporidium spp. were common, although no zoonotic types were verified, and Salmonella enterica was isolated from 1/11 mice on one farm but not detected by PCR from any of the rodents. Trichinella spp. and Toxoplasma gondii were not detected.

Prevalence and level of Listeria monocytogenes in ready-to-eat foods in Sweden 2010.

An increasing trend in human listeriosis cases over the past five years (2005-2009) in Sweden encouraged the authorities to examine the prevalence and levels of Listeria monocytogenes in ready-to-eat (RTE) foods in 2010. The combined results of two surveys are presented: the Swedish part of an EU-wide survey and a national survey. A total of 1590 samples covering three categories of RTE food able to support growth of L. monocytogenes: (i) soft and semi-soft cheeses (mould- and smear-ripened); (ii) heat-treated meat products; and (iii) smoked and gravad fish, were collected at retail outlets and analysed at the end of shelf life. L. monocytogenes was detected in 0.4% of 525 cheese samples, 1.2% of 507 meat-product samples and 12% of 558 fish samples. In the latter category, L. monocytogenes was found in 14% of both gravad and cold-smoked fish samples and in approximately 2% of hot-smoked fish samples. The percentage of cold-smoked or gravad fish testing positive for L. monocytogenes was significantly lower in samples processed in Sweden (8%) than in samples processed in other countries (45%). Levels of L. monocytogenes exceeding 100 cfu/g were found in one (0.2%) of the cheese samples and in three (0.5%) of the fish samples. The high prevalence of contaminated cold-smoked and gravad fish samples suggests that these products constitute the main problem. This has induced the development of a national strategy plan with the aim to halve the prevalence of L. monocytogenes in cold-smoked and gravad fish at retail in Sweden by the end of year 2015.

Occurrence of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis in small wild rodents.

Rodents are a potential source of pathogenic Yersinia enterocolitica and Y. pseudotuberculosis. In order to study this, 190 rodents were captured and sampled on seven pig farms (n=110), five chicken farms (n=55) and six other locations (n=25) in Sweden. Pigs from three of the pig farms were also sampled (n=60). Pathogenic Y. enterocolitica was detected by TaqMan PCR in about 5% of rodent samples and 18% of pig samples. Only rodents caught on pig farms tested positive for the pathogen. Y. enterocolitica bioserotype 4/O:3 strains isolated from the rodent and pig samples were compared by pulsed-field gel electrophoresis and revealed a high degree of similarity, which was confirmed by random amplified polymorphic DNA. Y. pseudotuberculosis was only detected in one rodent sample. Thus, rodents may be vectors for the transmission of pathogenic Y. enterocolitica to pigs, acting as carriers rather than a reservoir, and should therefore remain an important issue in hygiene control measures on farms.

TaqMan-based real-time PCR method for detection of Yersinia pseudotuberculosis in food.

A sensitive and specific assay for detection of food-borne pathogenic Yersinia pseudotuberculosis was developed. The primer-probe set was designed to target a 157-bp sequence of the chromosomally located gene ail. The complete method, including an internal amplification control, was evaluated for several different food items.

Real-time PCR method for detection of pathogenic Yersinia enterocolitica in food.

The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.

Microbiological baseline study of swine carcasses at Swedish slaughterhouses.

This 13-month survey was conducted to estimate the prevalence and counts of foodborne pathogenic bacteria and indicator bacteria on swine carcasses in Sweden. A total of 541 swine carcasses were sampled by swabbing prechill at the 10 largest slaughterhouses in Sweden. Pathogenic Yersinia enterocolitica was detected by PCR in 16% of the samples. The probability of finding Y. enterocolitica increased with increasing counts of Escherichia coli. No samples were positive for Salmonella. The prevalences of Campylobacter, Listeria monocytogenes, and verocytotoxin-producing E. coli were low (1, 2, and 1%, respectively). None of the verocytotoxin-positive enrichments, as determined by a reverse passive latex agglutination assay, tested positive for the virulence genes eaeA or hlyA by PCR. Coagulase-positive staphylococci, E. coli, and Enterobacteriaceae were recovered from 30, 57, and 87% of the samples, respectively, usually at low levels (95th percentiles, 0.79, 1.09, and 1.30 log CFU/cm2, respectively). The mean log level of Enterobacteriaceae was 0.35 log CFU/cm2 higher than that of E. coli on carcasses positive for both bacteria. The mean log level of aerobic microorganisms was 3.48 log CFU/cm2, and the 95th percentile was 4.51 log CFU/cm2. These data may be useful for risk assessment purposes and can serve as a basis for risk management actions, such as the use of E. coli as an alternative indicator organism for process hygiene control.

Microbiological baseline study of broiler chickens at Swedish slaughterhouses.

This 1-year study was conducted to estimate the prevalence and concentrations of pathogenic and indicator bacteria on Swedish broiler chickens. A total of 636 chilled carcasses were collected from 10 slaughterhouses and sent to the National Food Administration for analyses of carcass rinses. No carcasses were positive for Salmonella. Campylobacter, predominantly Campylobacter jejuni, were detected on 15% (by enrichment) or 14% (by direct plating) of the carcasses. With one exception, all samples from late December through April were Campylobacter negative. The 10th and 90th percentiles of Campylobacter numbers per carcasses were 3.0 and 5.0 log CFU, respectively, and the maximum was 7.1 log CFU. Coagulase-positive staphylococci were detected on 68% of the carcasses, with a maximum of 3.5 log CFU/cm2. The 10th and 90th percentiles were 3.4 and 4.4 log CFU/cm2 for total aerobic microorganisms, 1.8 and 3.3 log CFU/cm2 for Enterobacteriaceae, and 2.0 and 3.6 log CFU/cm2 for Escherichia coli. No correlation was found between numbers of any indicator bacteria and numbers of pathogenic bacteria. Subsets of the samples were analyzed for Listeria monocytogenes, Clostridium perfringens, pathogenic Yersinia enterocolitica, and Enterococcus, resulting in prevalence estimates of 29, 18, 9 (as determined by a PCR assay), and 97%, respectively. L. monocytogenes was most common at slaughterhouses with a low prevalence of coagulase-positive staphylococci, and vice versa. These results will improve the ability of researchers to assess the importance of chicken as a source of foodborne pathogens and can serve as a basis for risk management actions.