Roland Lindqvist

The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment

The recent finding that the formation of staphylococcal enterotoxins in food is very different from that in cultures of pure Staphylococcus aureus sheds new light on, and brings into question, traditional microbial risk assessment methods based on planktonic liquid cultures. In fact, most bacteria in food appear to be associated with surfaces or tissues in various ways, and interaction with other bacteria through molecular signaling is prevalent. Nowadays it is well established that there are significant differences in the behavior of bacteria in the planktonic state and immobilized bacteria found in multicellular communities. Thus, in order to improve the production of high-quality, microbiologically safe food for human consumption, in situ data on enterotoxin formation in food environments are required to complement existing knowledge on the growth and survivability of S. aureus. This review focuses on enterotoxigenic S. aureus and describes recent findings related to enterotoxin formation in food environments, and ways in which risk assessment can take into account virulence behavior. An improved understanding of how environmental factors affect the expression of enterotoxins in foods will enable us to formulate new strategies for improved food safety.

Inactivation of Escherichia coli, Listeria monocytogenes and Yersinia enterocolitica in fermented sausages during maturation/storage

The purpose of this study was to evaluate maturation and storage conditions as a way to increase the safety of non-heat treated fermented sausages. The specific objectives were to investigate the effects of storage time and temperature on the levels of Escherichia coli, Listeria monocytogenes and Yersinia enterocolitica in fermented sausages and in broth, and to validate how well the broth experiments and some published models can predict inactivation in sausage. One strain each of E. coli, L. monocytogenes and Y. enterocolitica with induced acid tolerance was inoculated into sausage batters representing a typical Swedish recipe for cold-smoked sausages. The sausages were fermented at 27 degrees C for 39 or 48 h and then stored at different temperatures (8, 15, or 20-22 degrees C) for up to 44 days. The levels of the experimental strains, lactic acid bacteria, and pH, a(w), and lactic acid was measured during the maturation/storage period. Inactivation in BHI broths adjusted to pH 4.4 or 4.6, water activity of 0.93, and with 1, 1.3 or 2% lactic acid added was also studied. For all strains inactivation rates increased with temperature in both broths and sausages. At 8 degrees C the storage time required for a one-log reduction in sausage ranged from 21 days for E. coli, >16 days for L. monocytogenes, to 18 days for Y. enterocolitica. At temperatures of 20 degrees C or more, the storage time needed for a one log reduction was shorter: between 7 to 11 days for E. coli, 4 to 7 days for L. monocytogenes, and 1 to 4 days for Y. enterocolitica. A published model based on temperature only yielded a good prediction of E. coli inactivation in sausage. A linear model based on the rate estimated in broth yielded a fair prediction of L. monocytogenes inactivation. The performance of other inactivation models validated was unsatisfactory. Significant E. coli growth which occurred in batters without salt during initial phases of fermentation resulted in a subsequent increased inactivation rate, possibly due to increased susceptibility to stress of exponential phase bacteria. The results indicate that the practice of utilising a short maturation period and storage at refrigeration temperatures may result in unsatisfactory reductions of pathogens if present. Thus, inclusion of a maturation period above refrigeration temperatures before distribution may increase the safety of these products.

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.

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.

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.

A summary of reported foodborne disease incidents in Sweden, 1992 to 1997.

Reports of foodborne disease incidents in Sweden from 1992 to 1997 are summarized. The results are based on reports from the municipal environmental and public health authorities to the National Food Administration and from medical authorities to the Swedish Institute for Infectious Diseases Control. A total of 555 incidents, of which 84% were outbreaks, were reported, involving 11,076 ill people. In 66% of the incidents, no disease agent was determined. Bacterial agents were implicated in 25% and viruses in 8% of the incidents. Calicivirus was the most reported agent both in terms of incidents and cases. Mixed dishes was the food category most often implicated in outbreaks, and smorgasbord and casserole or stews were the subcategories that caused the most cases. The place of consumption was unknown in 8% of the incidents. In about 60% of the incidents, the implicated food was consumed in commercial food establishments; in approximately 20% of incidents, it was consumed at home. The average annual incidence of reported foodborne disease in Sweden was estimated to be 21 cases per 100,000. The average annual incidence of reported foodborne salmonellosis and campylobacteriosis was estimated to be 2.0 and 0.6 cases per 100,000, respectively. The awareness and motivation to report foodborne diseases need to be improved, but additional sources of information are needed to counteract some of the limitations of reporting discussed in this work.

Quantitative Microbial Risk Assessment for Escherichia coli O157 on Lettuce, Based on Survival Data from Controlled Studies in a Climate Chamber

The aims of the study were to determine the survival of Escherichia coli O157 on lettuce as a function of temperature and light intensity, and to use that information in a screening-level quantitative microbial risk assessment (QMRA) in order to evaluate risk-reducing strategies including irrigation water quality guidelines, rinsing, and holding time between last irrigation and harvest. Iceberg lettuce was grown in a climate chamber and inoculated with E. coli O157. Bacterial numbers were determined with the standard plate count method after inoculation and 1, 2, 4, and 7 day(s) postinoculation. The experiments were carried out at 11, 18, and 25°C in light intensities of 0, 400, and 600 mmol (m(2))(-1) s(-1). There was a significant effect of temperature and light intensity on survival, with less bacteria isolated from lettuce incubated at 25 and 18°C compared with 11°C (P < 0.0001), and in light intensities of 400 and 600 mmol (m(2))(-1) s(-1) compared with 0 mmol (m(2))(-1) s(-1) (P < 0.001). The average log reductions after 1, 2, 4, and 7 day(s) were 1.14, 1.71, 2.04, and 3.0, respectively. The QMRA compared the relative risk with lettuce consumption from 20 scenarios. A stricter water quality guideline gave a mean fivefold risk reduction. Holding times of 1, 2, 4, and 7 day(s) reduced the risk 3, 8, 8, and 18 times, respectively, compared with harvest the same day as the last irrigation. Finally, rinsing lettuce for 15 s in cold tap water prior to consumption gave a sixfold risk reduction compared with eating unrinsed lettuce. Sensitivity analyses indicated that variation in bacterial inactivation had the most significant effect on the risk outcome. A QMRA determining the relative risks between scenarios reduces uncertainty and can provide risk managers with decision support.

Public health burden due to infections by verocytotoxin-producing Escherichia coli (VTEC) and Campylobacter spp. as estimated by cost of illness and different approaches to model disability-adjusted life years

Aims: To estimate disability-adjusted life years (DALY) and cost of illness (COI) associated with the gastrointestinal bacterial pathogens Campylobacter and verocytotoxin-producing Escherichia coli (VTEC) in Sweden and to investigate the impact of variability in health outcomes, data availability, and different assumptions about underreporting on DALY. Methods: Data from the Swedish notification system, public databases, and the literature were used to estimate COI and DALY. DALY was modelled using a deterministic and a stochastic approach, the latter describing variation in health outcomes between individuals. Effects of different assumptions about underreporting of gastroenteritis were evaluated in separate scenarios. Results: COI and DALY were greater for Campylobacter than for VTEC. Years of life lost due to haemolytic uraemic syndrome and years lived with gastroenteritis constituted most of DALY for VTEC and Campylobacter, respectively. Productivity losses due to gastroenteritis constituted the main cost associated with both pathogens. Degree of underreporting had a greater impact on DALY for Campylobacter, due to higher estimated incidence of gastroenteritis associated with campylobacteriosis. Conclusions: Pathogen-specific health outcomes and data quality may influence the preferred modelling approach. There was a fair agreement between modelling approaches, but the stochastic model reflected the contribution of some rare health outcomes not captured in the deterministic model. Health outcomes excluded due to lack of data lead to an underestimation of the total burden associated with the pathogens. Increased knowledge, especially on the degree of underreporting and the contribution of the pathogens to sequelae, is needed to further improve public health burden estimates for these pathogens in Sweden.

The effect of undissociated lactic acid on Staphylococcus aureus growth and enterotoxin A production.

The potential of Staphylococcus aureus cheese isolates to grow and produce staphylococcal enterotoxin A under conditions typical for cheese making was investigated in three broth experiments. The effect of the concentration of undissociated lactic acid (HLac) in conjunction with specific pH values was studied by adjusting pH at a single concentration of lactic acid. First, the time-to-growth of S. aureus was modelled by using survival analysis and absorbance data obtained from an automated turbidity reader. The fitted model describes the time to growth and indicates the growth ⁄ no growth boundary of S. aureus as a function of HLac concentration, temperature and water activity. Second, growth rates and lag times of S. aureus were estimated after two different pre-treatments in skim milk at three HLac concentrations and two temperatures based on optical detection times of serial dilutions of bacterial solutions. Growth rates differed between strains, and increased with increasing temperature and decreasing HLac concentration. Preliminary results indicate that lag times were dependent on pre-treatment suggesting that the growth potential of S. aureus in cheese curd may be greater if milk is used immediately after milking compared to holding at 4°C after milking. Third, growth, inactivation, and enterotoxin A production of S. aureus strains were investigated at twelve combinations of HLac concentration and temperature. Concentrations of enterotoxin A increased linearly during the first four days, with a production rate increasing with increasing temperature and decreasing HLac concentration. Significant amounts of enterotoxin A were produced during extended incubation, up to 14days, but then initial pH had changed. This highlights a potential limitation of modelling based on the initial environmental conditions in batch experiments. In summary, ranges of time-to-growth, growth rates, lag times and enterotoxin A production rates of S. aureus in the presence of HLac were estimated. The results can be used together with process data to indicate the range and magnitude of growth and enterotoxin A production during initial stages of cheese production.

Time to growth and inactivation of three STEC outbreak strains under conditions relevant for fermented sausages

Published models predicting growth and survival capabilities of shiga-toxin-producing Escherichia coli (STEC) under a(w) and lactic acid stress were validated by performing experiments with fermented sausage associated outbreak strains. Strain variation in inactivation and time to growth (TTG) were investigated for strains representing three serotypes (O103, O111, and O157). The TTG and growth boundaries of each strain were compared with predictions of a model for generic acid adapted E. coli and survival with predictions of two inactivation models. In addition, the influence of strain variation on the performance of the inactivation models, in terms of bias and accuracy factors, was illustrated. Strains with induced acid tolerance were used in broths containing 50 or 110 mM total lactic acid. The concentration of undissociated lactic acid (HLac) was adjusted by setting the pH-value, and water activity (0.900 to 0.995 depending on experiment) was adjusted by adding NaCl. The survival capabilities of the outbreak strains were good compared to the model predictions. The average bias factors of inactivation model predictions were within a factor of 2.2 depending on the strain used to validate the model indicating that inactivation rates of outbreak strains were slower than predicted. However, the observed rates were similar to the rates of a previously studied acid tolerant generic E. coli strain. Similarly, the time to growth of two of the strains (O103 and O157) was comparable with model predictions, whereas the growth capability of the third strain (O111) was lower than predicted. These results suggest that the properties of the most tolerant sausage outbreak strains are comparable to tolerant generic E. coli strains, which imply that suitable non-pathogenic E. coli strains are valid surrogates for fermented sausage outbreak strains. The relative sensitivity of strains depended on the environmental parameters and the response evaluated. The strain with the smallest log reduction at 20 °C was O157, whereas it was strain O103 at 8 °C. Under conditions unfavorable for growth, the time to growth was much shorter for strains O103 and O157 than for strain O111, whereas differences between strains were negligible under conditions favorable for growth. Depending on the response variable and the specific application the limitation of not addressing strain variation may lead to biased, fail-dangerous, predictions. Thus, solutions on how to best address strain variation in the development and validation of predictive models are needed.