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Since the first description of livestock-associated methicillin-resistant Staphylococcus aureus(LA-MRSA), a high prevalence was observed in pigs. At present, questions remain about the transmission of LA-MRSA to the general human population through pork. The objectives of the present study were to determine the prevalence of LA-MRSA in Belgian pork and to determine the role of the pork production chain and butcheries in transmission of LA-MRSA to the human population. Pig meat samples (chops, bacon, minced pork, ribs, forelimbs, and ears; n=137) originating from four butcheries (A through D) were spread plated on ChromID MRSA plates both before and after overnight enrichment culture. Suspect colonies were confirmed using a MRSA-specific triplex PCR assay and a CC398-specific PCR assay. The isolates (n=147) were further characterized by SCCmectyping, multiple-locus variable number tandem repeat analysis, and antimicrobial susceptibility testing, a selection of isolates were subjected to pulsed-field gel electrophoresis and spatyping. Direct plating revealed a MRSA prevalence of 8%. After enrichment, MRSA was isolated from 98 (72%) of 137 samples of which the majority were from rib, ear, and forelimb. The majority (97%) of obtained isolates belonged to CC398, the main LA-MRSA type. A high level of genetic diversity was noted among the isolates from one butchery. Thirty antimicrobial susceptibility profiles were found; 13 and 9% of the isolates had Cip-Tet-Tri and Gen-Kan-Tet-Tob-Tri profiles, respectively. These results indicate the importance of enrichment for MRSA detection of pork. The observed genetic diversity of the isolates indicated that the pork production chain can be considered a source of multiple MRSA types that could be transmitted to the human population through cross-contaminated meat.

Food is an important dissemination route for antibiotic-resistant bacteria. Factors used during food production and preservation may contribute to the transfer of antibiotic resistance genes, but research on this subject is scarce. In this study, the effect of temperature (7 to 37°C) and modified atmosphere packaging (air, 50% CO2-50% N2, and 100% N2) on antibiotic resistance transfer from Lactobacillus sakeisubsp. sakeito Listeria monocytogeneswas evaluated. Filter mating was performed on nonselective agar plates with high-density inocula. A more realistic setup was created by performing modified atmosphere experiments on cooked ham using high-density and low-density inocula. Plasmid transfer was observed between 10 and 37°C, with plasmid transfer also observed at 7°C during a prolonged incubation period. When high-density inocula were used, transconjugants were detected, both on agar plates and cooked ham, under the three atmospheres (air, 50% CO2-50% N2, and 100% N2) at 7°C. This yielded a median transfer ratio (number of transconjugants/number of recipients) with an order of magnitude of 10-4to 10-6. With low-density inocula, transfer was only detected under the 100% N2atmosphere after 10-day incubation at 7°C, yielding a transfer ratio of 10-5. Under this condition, the highest bacterial density was obtained. The results indicate that low temperature and modified atmosphere packaging, two important hurdles in the food industry, do not necessarily prevent plasmid transfer from Lactobacillus sakeisubsp. sakeito Listeria monocytogenes.

AbstractSalmonellaEnteritidis (SE) is a genetically homogenous serovar, which makes optimal subtype discrimination crucial for epidemiological research. This study describes the development and evaluation of an optimized multiple-locus variable number tandem-repeat assay (MLVA) for characterization of SE. The typeability and discriminatory power of this MLVA was determined on a selected collection of 60 SE isolates and compared with pulsed-field gel electrophoresis (PFGE) using restriction enzymes XbaI, NotI, or SfiI. In addition, the estimated Wallace coefficient (W) was calculated to assess the congruence of the typing methods. Selection of epidemiologically unrelated isolates and more related isolates (originating from layer farms) was also based on the given phage type (PT). When targeting six loci, MLVA generated 16 profiles, while PFGE produced 10, 9, and 16 pulsotypes using XbaI, NotI, and SfiI, respectively, for the entire strain collection. For the epidemiologically unrelated isolates, MLVA had the highest discriminatory power and showed good discrimination between isolates from different layer farms and among isolates from the same layer farm. MLVA performed together with PT showed higher discriminatory power compared to PFGE using one restriction enzyme together with PT. Results showed that combining PT with the optimized MLVA presented here provides a rapid typing tool with good discriminatory power for characterizing SE isolates of various origins and isolates originating from the same layer farm.

AbstractAn isolation method described by Possé et al. (FEMS Microbiol Lett2008;282:124–131) was satisfactorily validated in an international ring-trial using artificially contaminated minced beef samples. Until now, no validated method existed for the simultaneous isolation of Shiga toxin–producing Escherichia coliserogroups O26, O103, O111, and O145 in food. Twelve laboratories from five European countries participated and received 16 inoculated beef samples contaminated with cold-stressed cells of the four serogroups O26, O103, O111, and O145 in two levels (approximately 30 and 300 CFU 25 g−1) in duplicate. In addition, they received four non-inoculated samples. The isolation protocol comprised a selective enrichment step, a selective isolation step on a non-O157 agar plate differentiating the serogroups by color, followed by confirmation by plating on confirmation agar media and agglutination. All laboratories were able to isolate the inoculated serogroups from the samples, both for the high and the low inoculation level. Results did not differ whether in-house-prepared or ready-to-use non-O157 agar plates were used, demonstrating that by following the instructions laboratories managed to perform the complete protocol with success.

AbstractObjectives:To prioritize an extended list of food- and water-borne zoonoses to allow food safety authorities to focus on the most relevant hazards in the food chain.Methods:An evidence-based semiquantitative methodology was developed. Scores were given by 35 scientific experts in the field of animal and public health, food, and clinical microbiology and epidemiology to 51 zoonotic agents according to five criteria related to public health (severity and occurrence in humans), animal health (severity of disease coupled with economic consequences and occurrence in animals), and food (occurrence in food). The scoring procedure was standardized and evidence-based as experts were provided, for each zoonotic agent, a same set of up-to-date help information data related to the five criteria. Independently, the relative importance of the five criteria was weighted by seven food chain risk managers. The zoonotic agents were ranked based on overall weighted scores and were grouped in four statistically different levels of importance.Results:The following foodborne zoonotic pathogens were classified as “most important”: Salmonellaspp., Campylobacterspp., Listeria monocytogenes, and verocytotoxigenic Escherichia coli. A second group of “significant importance” included Toxoplasma gondii, the agent of bovine spongiform encephalopathy, Clostridium botulinum, Staphylococcus aureus, Cryptosporidium parvum, Mycobacterium bovis, Echinococcus granulosus, Streptococcusspp., Echinococcus multilocularis, Yersinia enterocolitica, Mycobacterium avium, Fasciola hepatica, Giardia intestinalis, and Rotavirus.Conclusions:This methodology allowed to rank 51 zoonotic agents with objectivity and taking account of a combined input from risk assessors and risk managers.Applications:These results support food safety policy makers to establish the multiannual monitoring program of foodborne zoonoses. They also enable to identify knowledge gaps on specific zoonotic agents and to formulate key research questions. Principally, this method of prioritization is of general interest as it can be applied for any other ranking exercise and in any country.

ABSTRACTA total of 295 ceftiofur-resistant Escherichia coliisolates were obtained from 489 cloacal samples collected at five different Belgian broiler farms with the aim to evaluate the diversity of this resistance at the farm level. Strains were examined for resistance against β-lactam antibiotics and other antimicrobial agents by using disk diffusion tests. Three different β-lactam resistance phenotypes suggested the presence of an extended-spectrum β-lactamase (ESBL), a class C β-lactamase, or the combination of an ESBL with a class C β-lactamase. Seventy-six percent of these isolates also showed acquired resistance to other antimicrobial agents. After genotyping by repetitive extragenic palindromic-PCR, 51 unrelated E. colistrains were selected for further analyses. Isoelectric focusing and sequencing of the amplicons obtained in PCRs for the detection of genes encoding broad-spectrum β-lactamase enzymes revealed the following ESBLs: TEM-52 (13.2%), TEM-106 (2%), CTX-M-1 (27.4%), CTX-M-2 (7.8%), CTX-M-14 (5.9%), and CTX-M-15 (2%). The only plasmidic AmpC β-lactamase found in this study was the CMY-2 enzyme (49%). Mutations in the promoter and attenuator regions of the chromosomal ampCgene were found only in association with blaCMY-2genes and ESBL genes. The combination of an ESBL (CTX-M-1) with a plasmidic AmpC β-lactamase (CMY-2) was found in 7.8% of the isolates. These data show that ceftiofur-resistant E. colistrains are often present in cloacal samples of broilers at the farm level in Belgium. The diversity of broad-spectrum β-lactamases among these isolates is high, and they may act as a reservoir of ESBL and ampCgenes.

Egg weight, shell thickness, number of pores, cuticle deposition, eggshell strength (dynamic stiffness and damping ratio), and the ability of Salmonella entericaserovar Enteritidis (SE) to penetrate the eggshell were determined. Penetration was assessed by filling the eggs with a selective medium that allowed viewing of Salmonellagrowth on the inside of the shell and membrane complex. After inoculation of each shell with on average 2.71 log CFU, the eggs were stored for up to 14 days at 20°C and 60% relative humidity. Commercially available eggs were used. At 14 days of storage, only 6.0% of the eggs from free-range hens and 16.0% of the generic (i.e., eggs from hens in conventional battery cages that were given standard feed) white eggs were penetrated. The generic brown, organic, and omega-3–enriched eggs were penetrated at a frequency of 30 to 34%. In a second experiment it was shown that the layer strains of the hen (ISA-Brown Warren versus Bovans Goldline), which were kept in furnished cages, did not affect eggshell penetration by SE. For Bovans Goldline hens, the housing system (furnished cage versus aviary) did not affect penetration, while a trend was visible toward a higher fraction of penetrated eggshells when hens were fed corncob mix rather than standard feed. Eggshell penetration was observed more frequently in the absence of cuticle spots and for eggs having lower dynamic stiffness values. Shell contamination at the end of storage was highly correlated with SE penetration.

Shells of agar-filled and whole eggs were inoculated with 103 to 104 CFU of Salmonella entericaserovar Enteritidis per eggshell. The agar-filled eggs were used to study bacterial eggshell penetration, and the whole egg results were used to characterize contamination of the egg contents. In each group, half of the eggs were stored for 21 days at 20°C and 60% relative humidity (RH), and the other half was stored for 24 h at 6°C and then for 20 days at 20°C. The latter conditions resulted in condensation on the eggshell for 30 min from the moment the eggs were placed in the 20°C chamber. Taking into account the ages at which hens were studied (39, 53, and 67 weeks), an average of 62% of the eggshells with condensate were penetrated compared with 43% for the control group; this difference was significant (P< 0.01). No significant difference in whole egg contamination was found; 18% of the control eggs were contaminated compared with 22% of the condensate eggs. Whole egg contamination was significantly higher for eggs from the hens at an older age (67 weeks). This difference probably was not due to a higher penetration potential because differences were not observed for the corresponding agar-filled eggs. Condensation on the eggshell seemed to encourage bacterial penetration of the eggshell but had a smaller impact on whole egg contamination.

At present there is no rapid microbiological method for the detection of viable Mycobacterium paratuberculosis in milk. By combining an extensive milk sample pretreatment with solid phase cytometry as the detection technique we were able to demonstrate viable mycobacterial cells in 50 ml of artificially contaminated pasteurized milk in less than one working day.

Listeria monocytogenesis a major concern to producers of ready-to-eat foods because of the high mortality rate associated with listeriosis and the widespread nature of the organism. To investigate the prevalence of this pathogen in different ready-to-eat food products on the Belgian market, a variety of 252 ready-to-eat food products, mainly fish and meat products, were analyzed. Overall, L. monocytogeneswas detected in 23.4% of the samples. The highest prevalence of L. monocytogeneswas found in prepared minced meat (42.1%) and smoked halibut (33.3%). Contamination levels were in most cases low (<10 CFU/g); however, levels higher than 100 CFU/g were detected in some samples of smoked salmon, smoked halibut, and prepared minced meat. A high prevalence of Listeria innocua(15.8%) and Listeria welshimeri(36.8%) was detected in prepared minced meat. L. monocytogenesstrains isolated from different contaminated products were subjected to repetitive element sequence-based PCR (REP-PCR) typing to determine possible associations with product type, producer, or market. REP-PCR patterns were analyzed using BioNumerics software, and seven different groups with at least 90% similarity were identified. The cluster analysis indicates that cross-contamination occurred at the producer and retail level. Serotype identification of the strains by PCR revealed that most belonged to the 1/2a(3a) serotype group.

A survey assessed the handling of shell eggs and processed eggs from purchase until serving to potentially vulnerable people in care homes, acute hospitals, handicapped institutions, and crèches (i.e. nurseries). In 94% of the institutions shell eggs are consumed at a weekly average of 1.1 ± 0.3 eggs for each person. In 52% of the institutions egg products are used. The shell eggs are held on average at 6.4C for 1–2 weeks. The questionnaire listed a series of egg dishes, which were classified afterwards in three risk categories. Of the high‐risk dishes, shell eggs are particularly used for addition to mashed potatoes, for preparing soft‐boiled eggs and eggs sunny‐side‐up and for inclusion in hot sauces. Of the medium‐risk dishes, shell eggs are used for preparing scrambled eggs and omelets. When egg products are bought next to shell eggs, relatively less high‐risk dishes and more low‐risk dishes on the total dishes were made from shell eggs. Thus, egg products are purchased on a rational base. The distribution of storage temperature, storage time and preparation frequency of high‐risk meals were combined using a Monte Carlo resampling technique, which indicated that the majority of the samples (98.5%) fell into a low‐hazard category.

Specific PCR, amplifying a fragment of the mitochondrially encoded cytochrome b gene, are developed for discrimination of chicken, turkey, pig, cow and sheep. These PCR tests could be applied for detection and discrimination of animal food ingredients. For origin identification of milk, the bovine PCR is applied on DNA extracted with the DNeasy™ Tissue kit. A PCR of 30 cycles reached a sensitivity of 720 somatic cells in the PCR reaction corresponding to 2·9 × 104 cells per ml milk. A PCR of 40 cycles could detect 0·72 to 0·144 somatic cells in the PCR reaction corresponding to 29 to 2·9 cells per ml milk.

A survey of parents of higher education college students assessed the purchasing behavior, storage conditions and consumption of hen eggs in the household, to quantify microbiological unsafe handling. Analysis of the questionnaires indicates an average consumption of 6.4 eggs per month, which are kept at an average temperature of 9.7C for 2 weeks. An additional spot check showed that the interviewees probably underestimate the keeping temperature. The questionnaire also listed a series of egg dishes, which were afterwards categorized according to risk. The distributions of storage temperature, keeping time and type of dish were combined using a Monte Carlo resampling technique, which resulted in a hazard distribution. This distribution showed that the majority of the samples (98.2%) fell into a low hazard category.

We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.

Enzymatic amplification results showed thatListeriaspecies have at least two 16S–23S rRNA spacer regions of different lengths. These spacer regions ofL. monocytogenes,L. ivanoviiandL. seeligeriwere cloned after enzymatic amplification. Sequence analysis of the inserts revealed two spacers of 245–246 bp and 496–498 bp, respectively, of which the latter included tRNA(Ala) and tRNA(Ile) genes. OneListeriaspp.-specific probe, LIS-ICG4, was deduced from the 245-bp spacer and aL. monocytogenes-specific probe, LMO-ICG5, was inferred from the 496-bp spacer. The specificity of both probes was tested in a reverse hybridization assay (Line Probe Assay, LiPA). Both LIS-ICG4 and LMO-ICG5 proved to be highly specific when hybridized to a large collection ofListeriastrains and strains from other relevant taxa. The LiPA test herein described for the simultaneous detection ofListeriaspp. andL. monocytogenescan be expanded to detect other foodborne pathogens.