Determination of the growth potential of Listeria monocytogenes in various types of Belgian artisanal cheeses by challenge tests.

Cheese potentially allowing the growth of Listeria monocytogenes must be free of the pathogen in 25 g before being put on the market, while 100 cfu/g is tolerated when the pathogen is unable to grow. Challenge tests were performed in order to assess the growth potential of L. monocytogenes in at least one batch of 32 Belgian cheese varieties from 32 factories. All varieties were grouped in four categories: unripened acid-curd cheeses, mold-ripened soft cheeses, smear-ripened soft cheeses and ripened semi-hard cheeses. Associated microflora and cheese physicochemical characteristics were also studied. A cocktail of three strains was used to inoculate cheese on the first day of shelf-life, and samples were stored until the end of shelf-life at 7-9 °C. Growth potential was considered as the difference (a) between median contamination at the end and at the beginning of the test or (b) between the highest value at the end of the test and the lowest value at its beginning. L. monocytogenes always decreased in unripened acid-curd cheeses but showed extended growth in 21 out of 25 batches of ripened soft cheese. Contrasting results were obtained for semi-hard cheeses, as important intra- and inter-batch variability was observed. For the latter, the recommended method based on medians to calculate the growth potential led to erroneous food safety considerations, and it should always be advised to focus on absolute levels.

Limited association between disinfectant use and either antibiotic or disinfectant susceptibility of Escherichia coli in both poultry and pig husbandry.

BACKGROUND: Farm disinfectants are widely used in primary production, but questions have been raised if their use can select for antimicrobial resistance. The present study examined the use of disinfectants in poultry and pig husbandry and its contribution to the antibiotic and disinfectant susceptibility of Escherichia coli (E. coli) strains obtained after cleaning and disinfection. On those field isolates antibiotic susceptibility was monitored and susceptibility to commonly used active components of farm disinfectants (i.e. glutaraldehyde, benzalkoniumchloride, formaldehyde, and a formulation of peracetic acid and hydrogen peroxide) was tested. RESULTS: This study showed a high resistance prevalence (> 50%) for ampicillin, sulfamethoxazole, trimethoprim and tetracycline for both production animal categories, while for ciprofloxacin only a high resistance prevalence was found in broiler houses. Disinfectant susceptibility results were homogenously distributed within a very small concentration range. Furthermore, all E. coli strains were susceptible to in-use concentrations of formaldehyde, benzalkoniumchloride and a formulation of peracetic acid and hydrogen peroxide, indicating that the practical use of disinfectants did not select for disinfectant resistance. Moreover, the results showed no indications for the selection of antibiotic resistant bacteria through the use of disinfectants in agricultural environments. CONCLUSION: Our study suggests that the proper use of disinfectants in agricultural environments does not promote antibiotic resistance nor reduce E. coli disinfectant susceptibility.

Presence and fate of antibiotic residues, antibiotic resistance genes and zoonotic bacteria during biological swine manure treatment.

The presence and dissemination of antibiotic residues, antibiotic resistance genes and zoonotic bacteria in the environment is of growing concern worldwide. Manure management practices, such as biological removal of nitrogen from swine manure, may help to decrease levels of antibiotic residues, antibiotic resistance genes and zoonotic bacteria present in manure before fertilization, thereby reducing environmental contamination. Therefore, the aim of this study was to monitor the presence and fate of seven antibiotic residues (colistin, sulfadiazine, trimethoprim, doxycycline, oxytetracycline, ceftiofur and tylosin A), nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) and two zoonotic bacteria (Salmonella Typhimurium and Campylobacter coli) during biological nitrogen removal from swine manure over time. Samples from the raw manure, the solid fraction, the liquid fraction and the storage lagoon were analyzed on two farms at six time points with an interval of two weeks. Only the antibiotics which were used during the three months preceding the first sampling could be detected before and after biological nitrogen removal from swine manure. Of all the antibiotics studied, doxycycline was recovered in all of the samples and sulfadiazine was recovered in most samples on both farms. For both antibiotics, there appears to be a reduction of the amount of residues present in the storage lagoon compared to the liquid fraction, however, this reduction was not statistically significant. A significant reduction of the relative abundances of most of the antibiotic resistance genes studied was observed when comparing the liquid fraction and the storage lagoon. For tet(L), no differences were observed between the fractions sampled and for sul2 and erm(F), a significant increase in relative abundances was observed on the second farm sampled. For the zoonotic bacteria, a reduction of at least 1 log was observed after biological nitrogen removal from swine manure. The results indicate that the concentration of certain antibiotic residues and several antibiotic resistance genes and the amount of zoonotic bacteria present in the manure may be reduced in the end product of the biological nitrogen removal from swine manure.

Detection and identification of xerophilic fungi in Belgian chocolate confectionery factories.

Chocolate confectionery fillings are generally regarded as microbiologically stable. The stability of these fillings is largely due to the general practice of adding either alcohol or preservatives. Consumer demands are now stimulating producers to move away from adding alcohol or other preservatives to their confectionery fillings and instead to search for innovative formulations. Such changes in composition can influence the shelf life of the product and may lead to spoilage by xerophilic fungi. The aim of this study was to test whether the production environment of Belgian chocolate confectionery factories and common ingredients of chocolate confectioneries could be potential sources of contamination with xerophilic fungal species. In the factory environment, the general and strictly xerophilic fungal spore load was determined using an RCS Air Sampler device in combination with DG18 and MY50G medium, respectively. Four basic ingredients of chocolate confectionery fillings were also examined for fungal spore levels using a direct plating technique. Detected fungi were identified to species level by a combination of morphological characterization and sequence analysis. Results indicated a general fungal spore load in the range of 50-250 colony forming units per cubic meter of air (CFU/m(3) air) and a more strict xerophilic spore load below 50 CFU/m(3) air. These results indicate rather low levels of fungal spores present in the factory environment. The most prevalent fungi in the factory environment were identified as Penicillium spp., particularly Penicillium brevicompactum. Examination of the basic ingredients of confectionery fillings revealed nuts to be the most likely potential source of direct contamination. In nuts, the most prevalent fungal species identified were Eurotium, particularly Eurotium repens.

Short communication: Subtyping of Staphylococcus haemolyticus isolates from milk and corresponding teat apices to verify the potential teat-skin origin of intramammary infections in dairy cows.

Coagulase-negative staphylococci (CNS) are a major cause of intramammary infections (IMI) in dairy cows and they colonize the teat skin. Staphylococcus haemolyticus, one of the more common CNS, has been identified as a highly versatile opportunistic species. The aim of the present study was to gain better insight into the adaptation of S. haemolyticus subtypes to the udder ecosystem with respect to IMI development. During a longitudinal observational study conducted over 13 mo on 6 Flemish dairy herds, S. haemolyticus isolates were recovered from milk and teat apices. A total of 44 S. haemolyticus isolates originating from milk (24 isolates) and teat apices (20 isolates) of 6 selected udder quarters were singled out and analyzed using a combined methodology of (GTG)5-PCR and amplified fragment length polymorphism (AFLP) fingerprinting to determine intraspecies differences. Combining both fingerprinting methods, 4 S. haemolyticus subtypes were obtained (I to IV). Subtypes I, II, and IV were recovered from both milk and teat apex samples and were found to be associated with persisting IMI. Subtype III, not apparently related to IMI, was isolated solely from teat apices and not from milk. In general, S. haemolyticus subtypes found in milk from infected quarters could be recovered from the corresponding teat apices, although the latter could be colonized with up to 3 different subtypes. Comparing subtypes from milk and teat apices indicates that the IMI-causing agent likely originates from the teat skin.

The need for harmonization in detection of human noroviruses in food.

Noroviruses (NoV) have been recognized worldwide as a leading cause of foodborne gastroenteritis over the last decade. A broad range of foods- shellfish, fresh produce, and ready-to-eat/catered foods-has been implicated in NoV foodborne outbreaks. The recognition of NoV as an important food pathogen has been aided by the development of sensitive molecular methods for detection of the NoV genome. However, despite advances, NoV detection is still hampered by several limitations. First, NoV detection can often only be implemented by expert laboratories due to the complexity of the virus extraction step, which in most protocols is cumbersome and labor-intensive. Moreover, a very wide selection of automated methods for virus extraction from foods is available, so selection of an adequate method is not straightforward. On the other hand, automated systems have been made available or the RNA purification and real-time RT-PCR (RT-qPCR) is considered the gold standard for detection of NoV. Second, correct interpretation of real-time PCR results is often difficult. From a technical point of view, the interpretation of the often nonsigmoidal amplification curves remains difficult, even for experts. From a food safety perspective, interpretation of very high Cq (or Ct) values-and thus, of low viral genomic copy numbers-is not straightforward, as RT-(q)PCR merely detects the presence of viral genomic material that is not necessarily linked to the presence of infectious viral particles. Despite efforts, both limitations have not completely resolved thus far. Harmonization may be a first step to comprehend and deal with these limitations. The current review provides an overview of a number of validated methods that have been published by food safety and other authorities.

Cultured meat and challenges ahead: A review on nutritional, technofunctional and sensorial properties, safety and legislation.

Cultured meat is introduced as a valuable traditional meat equivalent. However, before marketable end products are available, several hurdles need to be overcome. Among others, these issues comprise obtaining an optimal nutritional profile and approaching the texture, the colour and the unique flavour and taste of conventional meat. Furthermore, the impact of processing on these matters is also still subject of future research. Moreover, more profound knowledge on food-safety aspects, like microbial contamination, prions, possible genetically engineered starting material, etc., and ways to reduce such risks will determine the future success of cultured meat products. Undoubtedly, correct terminology and adequate definitions also require further attention, as these form the starting point of legislative/regulatory aspects. This review provides a state-of-the-art overview on nutritional, technofunctional and sensorial properties, and food-safety and legislative/regulatory aspects on cultured meat production. Additionally, the various challenges and future steps of these aspects of cultured meat are highlighted.

The impact of antibiotic residues on resistance patterns in leek at harvest.

When crops are cultivated on fields fertilized with animal manure, the risk exists that plants may take up antibiotic residues and may be exposed to antibiotic resistance genes and antibiotic resistant bacteria. During cultivation in a greenhouse pot experiment, leek (Allium porrum) was fertilized with either pig slurry or mineral fertilizer and exposed to either no antibiotics, doxycycline (10,000 µg/kg manure), sulfadiazine (1000 µg/kg manure), or lincomycin (1000 µg/kg manure). At harvest, 4.5 months later, lincomycin, sulfadiazine or doxycycline were not detected in any of the leek samples nor in their corresponding soil samples. Further, antimicrobial susceptibility testing was performed on 181 Bacillus cereus group isolates and 52 Pseudomonas aeruginosa isolates from the grown leek. For the B. cereus group isolates, only a small shift in MIC50 for lincomycin was observed among isolates from the lincomycin and control treatment. For P. aeruginosa, only in the setup with doxycycline treatment a higher MIC50 for doxycycline was observed compared to the control, specifically the isolates selected from growth media supplemented with 8 mg/L doxycycline. Nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) were investigated at harvest in the leek and soil samples. In the leek samples, none of the antibiotic resistance genes were detected. In the soil samples fertilized with pig slurry, the genes erm(B), erm(F), tet(M), sul2, tet(W) and tet(O) were detected in significantly higher copy numbers in the lincomycin treatment as compared to the other antibiotic treatments. This could be due to a shift in soil microbiota induced by the addition of lincomycin. The results of this study indicate that consumption of leek carries a low risk of exposure to antibiotic residues or antibiotic resistance to doxycycline, sulfadiazine or lincomycin.

Effect of grape seed extract on human norovirus GII.4 and murine norovirus 1 in viral suspensions, on stainless steel discs, and in lettuce wash water.

The anti-norovirus (anti-NoV) effect of grape seed extract (GSE) was examined by plaque assay for murine norovirus 1 (MNV-1), cell-binding reverse transcription-PCR for human NoV GII.4, and saliva-binding enzyme-linked immunosorbent assay for human NoV GII.4 P particles, with or without the presence of interfering substances (dried milk and lettuce extract). GSE at 0.2 and 2 mg/ml was shown to reduce the infectivity of MNV-1 (>3-log PFU/ml) and the specific binding ability of NoV GII.4 to Caco-2 cells (>1-log genomic copies/ml), as well as of its P particles to salivary human histo-blood group antigen receptors (optical density at 450 nm of >0.8). These effects were decreased as increasing concentrations of dried milk (0.02 and 0.2%) or lettuce extract were added. Under an electron microscope, human NoV GII.4 virus-like particles showed inflation and deformation after treatment with GSE. Under conditions that simulated applications in the food industry, the anti-NoV effect of GSE using MNV-1 as a target organism was shown to be limited in surface disinfection (<1-log PFU/ml, analyzed in accordance with EN 13697:2001). However, a 1.5- to 2-log PFU/ml reduction in MNV-1 infectivity was noted when 2 mg of GSE/ml was used to sanitize water in the washing bath of fresh-cut lettuce, and this occurred regardless of the chemical oxygen demand (0 to 1,500 mg/ml) of the processing water.

Strain-specific transfer of antibiotic resistance from an environmental plasmid to foodborne pathogens.

Pathogens resistant to multiple antibiotics are rapidly emerging, entailing important consequences for human health. This study investigated if the broad-host-range multiresistance plasmid pB10, isolated from a wastewater treatment plant, harbouring amoxicillin, streptomycin, sulfonamide, and tetracycline resistance genes, was transferable to the foodborne pathogens Salmonella spp. or E. coli O157:H7 and how this transfer alters the phenotype of the recipients. The transfer ratio was determined by both plating and flow cytometry. Antibiotic resistance profiles were determined for both recipients and transconjugants using the disk diffusion method. For 14 of the 15 recipient strains, transconjugants were detected. Based on plating, transfer ratios were between 6.8 × 10⁻9 and 3.0 × 10⁻² while using flow cytometry, transfer ratios were between <1.0 × 10⁻5 and 1.9 × 10⁻². With a few exceptions, the transconjugants showed phenotypically increased resistance, indicating that most of the transferred resistance genes were expressed. In summary, we showed that an environmental plasmid can be transferred into foodborne pathogenic bacteria at high transfer ratios. However, the transfer ratio seemed to be recipient strain dependent. Moreover, the newly acquired resistance genes could turn antibiotic susceptible strains into resistant ones, paving the way to compromise human health.

Staphylococcus agnetis sp. nov., a coagulase-variable species from bovine subclinical and mild clinical mastitis.

Thirteen Gram-positive-staining coagulase-variable staphylococci were isolated from subclinical and mild clinical mastitic bovine milk (n=12) and a teat apex (n=1). The results of sequence analysis of the 16S rRNA gene and two housekeeping genes, rpoB and tuf, and DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis showed that the isolates formed a separate branch within the genus Staphylococcus. The phylogenetically most closely related species were Staphylococcus hyicus and Staphylococcus chromogenes. DNA-DNA hybridization with S. hyicus DSM 20459(T) and S. chromogenes DSM 20674(T) confirmed that the isolates belonged to a separate species. The predominant fatty acids were i-C(15:0), ai-C(15:0), i-C(17:0) and C(20:0) and the peptidoglycan type was A3α L-Lys-Gly(5). Based on the results of genotypic and phenotypic analyses, it is proposed that the thirteen isolates represent a novel species, for which the name Staphylococcus agnetis sp. nov. is proposed. Strain 6-4(T) (=DSM 23656(T)=CCUG 59809(T)) is the type strain.

Impact of fertilization with pig or calf slurry on antibiotic residues and resistance genes in the soil.

Antibiotic residues and antibiotic resistance genes can enter the environment via fertilization with calf and pig manure. In a longitudinal study, nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) and 56 antibiotic residues were investigated in 288 soil samples and 8 corresponding slurry samples from 6 pig farms and 2 veal farms using qPCR and LC-MS/MS, respectively. A significant increase in gene copy number of tet(M), erm(B), erm(F) and sul2 was observed in all the soil layers between sampling times prior to (T1) and 2-3 weeks after fertilization (T3). Tet(B), tet(Q) and tet(L) were least abundant in the soil among the genes tested. From 7 classes of antibiotics, 20 residues were detected in soil and slurry using an optimized and validated extraction method. Flumequine was detected in all soil samples in concentrations below 100 µg/kg despite being detected in only half of the corresponding slurry samples. Doxycycline, oxytetracycline, lincomycin and sulfadiazine were also frequently detected in concentrations ranging from 0.1 µg/kg to 500 µg/kg and from 2 µg/kg and 9480 µg/kg in soil and slurry, respectively. Furthermore a positive association between the presence of antibiotic residues (total antibiotic load) and antibiotic resistance genes in soil was found. One possible explanation for this is a simultaneous introduction of antibiotic residues and resistance genes upon application of animal slurry.

Inactivation of murine norovirus 1, coliphage phiX174, and Bacteroides [corrected] fragilis phage B40-8 on surfaces and fresh-cut iceberg lettuce by hydrogen peroxide and UV light.

In this study, the inactivating properties of liquid hydrogen peroxide (L-H(2)O(2)), vaporized hydrogen peroxide (V-H(2)O(2)), UV light, and a combination of V-H(2)O(2) and UV light were tested on murine norovirus 1 (MNV-1) and bacteriophages (φX174 and B40-8) as models for human noroviruses. Disinfection of surfaces was examined on stainless steel discs based on European Standard EN 13697 (2001). For fresh-produce decontamination, a mixture of the viruses was inoculated onto shredded iceberg lettuce and treated after overnight incubation at 2°C. According to our results, L-H(2)O(2) (2.1%) was able to inactivate MNV-1 and φX174 on stainless steel discs by approximately 4 log(10) units within 10 min of exposure, whereas for B40-8, 15% of L-H(2)O(2) was needed to obtain a similar reduction in 10 min. Only a marginal reduction (≤1 log(10) unit after 5 min of exposure) by V-H(2)O(2) (2.52%) was achieved for the tested model viruses, although in combination with UV light, a 4-log(10)-unit decrease within 5 min of treatment was observed on stainless steel discs. Similar trends were observed for the decontamination of shredded iceberg lettuce, but the viral decline was reduced. These results demonstrated that both L-H(2)O(2) and a combination of V-H(2)O(2) and UV light can be used for norovirus inactivation on surfaces; V-H(2)O(2) (2.52%) in combination with UV light is promising for decontamination of fresh produce with much less consumption of water and disinfectant.

Evaluation of a norovirus detection methodology for soft red fruits.

In the present study, a proposed methodology for detection of GI and GII noroviruses (NoV) in soft red fruits was evaluated. The murine norovirus-1 (MNV-1), a recently described cultivable NoV surrogate was integrated in the detection methodology as full process control, reverse transcription control and real-time PCR internal amplification control. Both the performance and robustness of the proposed methodology were analyzed. Firstly, the performance of the method was examined by analysis of the recovery of MNV-1, GI and/or GII NoV inoculated on frozen raspberry crum samples. Results showed that the recovery of MNV-1 was not significantly influenced by the inoculum incubation time (30 min or overnight incubation) or the inoculum level (10(6) or 10(8) genomic MNV-1 copies/10 g of frozen raspberry crum sample). In contrast, a significant influence of the GI and GII NoV inoculum level (10(4) or 10(6) genomic MNV-1 copies/10 g of frozen raspberry crum sample) was noticed on the recovery of respectively GI and GII NoV from frozen raspberry crum samples. Secondly, the robustness of the methodology was evaluated by subjecting three types of artificially MNV-1, GI and/or GII NoV contaminated soft red fruit products (deepfrozen forest fruit mix, fresh raspberries and fresh strawberry puree) to the method. Results showed a significant influence of the soft red fruit product type on the recovery efficiency of GI NoV and MNV-1, while no significant differences could be shown for GII NoV. In general, the recovery of GI and GII NoV in strawberry puree was more efficient from the strawberry puree compared to the two other soft red fruit types. In conclusion, results show that this methodology can be used for detection of NoV in different soft red fruits, although NoV recovery efficiencies can be influenced by (1) the NoV concentration on the soft red fruit type and (2) the tested soft red fruit type.

Parkinson's disease patients' short chain fatty acids production capacity after in vitro fecal fiber fermentation.

Animal models indicate that butyrate might reduce motor symptoms in Parkinson's disease. Some dietary fibers are butyrogenic, but in Parkinson's disease patients their butyrate stimulating capacity is unknown. Therefore, we investigated different fiber supplements' effects on short-chain fatty acid production, along with potential underlying mechanisms, in Parkinson's patients and age-matched healthy controls. Finally, it was investigated if this butyrate production could be confirmed by using fiber-rich vegetables. Different fibers (n = 40) were evaluated by in vitro fermentation experiments with fecal samples of Parkinson's patients (n = 24) and age-matched healthy volunteers (n = 39). Short-chain fatty acid production was analyzed by headspace solid-phase micro-extraction gas chromatography-mass spectrometry. Clostridium coccoides and C. leptum were quantified through 16S-rRNA gene-targeted group-specific qPCR. Factors influencing short-chain fatty acid production were investigated using linear mixed models. After fiber fermentation, butyrate concentration varied between 25.6 ± 16.5 µmol/g and 203.8 ± 91.9 µmol/g for Parkinson's patients and between 52.7 ± 13.0 µmol/g and 229.5 ± 42.8 µmol/g for controls. Inulin had the largest effect, while xanthan gum had the lowest production. Similar to fiber supplements, inulin-rich vegetables, but also fungal ß-glucans, stimulated butyrate production most of all vegetable fibers. Parkinson's disease diagnosis limited short-chain fatty acid production and was negatively associated with butyrate producers. Butyrate kinetics during 48 h fermentation demonstrated a time lag effect in Parkinson's patients, especially in fructo-oligosaccharide fermentation. Butyrate production can be stimulated in Parkinson's patients, however, remains reduced compared to healthy controls. This is a first step in investigating dietary fiber's potential to increase short-chain fatty acids in Parkinson's disease.

Longitudinal screening of antibiotic residues, antibiotic resistance genes and zoonotic bacteria in soils fertilized with pig manure.

Fertilization with animal manure is one of the main routes responsible for the introduction of antibiotic residues, antibiotic resistance genes, and zoonotic bacteria into the environment. The aim of this study was to assess the effect of the use of pig (swine) manure as a fertilizer on the presence and fate of six antibiotic residues, nine antibiotic resistance genes, and bacteria (zoonotic bacteria Salmonella spp. and Campylobacter spp. and E. coli as indicator for Gram-negative bacterial species of the microbiota of livestock) on five fields. To the best of our knowledge, the present study is the first to assess a multitude of antibiotic residues and resistance to several classes of antibiotics in pig manure and in fertilized soil over time in a region with an intensive pig industry (Flanders, Belgium). The fields were sampled at five consecutive time points, starting before fertilization up to harvest. Low concentrations of antibiotic residues could be observed in the soils until harvest. The antibiotic resistance genes studied were already present at background levels in the soil environment prior to fertilization, but after fertilization with pig manure, an increase in relative abundance was observed for most of them, followed by a decline back to background levels by harvest-time on all of the fields studied. No apparent differences regarding the presence of antibiotic resistance genes in soils were observed between those fertilized with manure that either contained antibiotic residues or not. With regard to dissemination of resistance, the results presented in this study confirm that fertilization with animal manure directly adds resistance genes to the soil. In addition, it shows that this direct mechanism may be more important than possible selective pressure in soil-dwelling bacteria exerted by antibiotic residues present in the manure. These results also indicate that zoonotic bacteria detected in the manure could be detected in the soil environment directly after fertilization, but not after 1 month. In conclusion, although some antibiotic residues may be present in both manure and soil at concentrations to exert selective pressure, it seems that antibiotic resistance is mostly introduced directly to soil through fertilization with animal manure.

Efficacy of sodium hypochlorite and peroxyacetic acid to reduce murine norovirus 1, B40-8, Listeria monocytogenes, and Escherichia coli O157:H7 on shredded iceberg lettuce and in residual wash water.

The efficiency of sodium hypochlorite (NaOCl) and peroxyacetic acid (PAA) to reduce murine norovirus 1 (MNV-1), a surrogate for human norovirus, and Bacteroides fragilis HSP40-infecting phage B40-8 on shredded iceberg lettuce was investigated. The levels of removal of viruses MNV-1 and B40-8 were compared with the reductions observed for bacterial pathogens Listeria monocytogenes and Escherichia coli O157:H7. Two inoculation levels, one with a high organic load and the other containing a 10-fold lower number of pathogens and organic matter, showed that the effectiveness of NaOCl was greatly influenced by the presence of organic material, which was not observed for PAA. Moreover, the present study showed that 200 mg/liter NaOCl or 250 mg/liter PAA is needed to obtain an additional reduction of 1 log (compared with tap water) of MNV-1 on shredded iceberg lettuce, whereas only 250 mg/liter PAA achieved this for bacterial pathogens. None of the treatments resulted in a supplementary 1-log PFU/g reduction of B40-8 compared with tap water. B40-8 could therefore be useful as an indicator of decontamination processes of shredded iceberg lettuce based on NaOCl or PAA. Neither MNV-1, B40-8, nor bacterial pathogens could be detected in residual wash water after shredded iceberg lettuce was treated with NaOCl and PAA, whereas considerable numbers of all these microorganisms were found in residual wash water consisting solely of tap water. This study illustrates the usefulness of PAA and NaOCl in preventing cross-contamination during the washing process rather than in causing a reduction of the number of pathogens present on lettuce.

Commensal E. coli rapidly transfer antibiotic resistance genes to human intestinal microbiota in the Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME).

Food-producing animals are indicated as a reservoir of antibiotic resistance genes and a potential vector for transmission of plasmid-encoded antibiotic resistance genes by conjugation to the human intestinal microbiota. In this study, transfer of an antibiotic resistance plasmid from a commensal E. coli originating from a broiler chicken towards the human intestinal microbiota was assessed by using a Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME). This in vitro model mimics the human intestinal ecosystem and received a single dose of 109E. coli MB6212, which harbors a plasmid known to confer resistance towards several antibiotics including tetracycline, sulfamethoxazole and cefotaxime. Since the degree of stress imposed by stomach pH and bile acids vary with the consumed meal size, the effect of meal size on E. coli donor survival and on plasmid transfer towards lumen and mucosal coliforms and anaerobes was determined. The administered commensal E. coli strain survived stomach acid and bile salt stress and was able to grow in the colon environment during the timeframe of the experiment (72 h). Transfer of antibiotic resistance was observed rapidly since cultivable transconjugant coliforms and anaerobes were already detected in the lumen and mucosa after 2 h in the simulated proximal colon. The presence of the resistance plasmid in the transconjugants was confirmed by PCR. Differences in meal size and adapted digestion had neither a detectable impact on antibiotic resistance transfer, nor on the survival of the E. coli donor strain, nor on short chain fatty acid profiles. The median number of resistant indigenous coliforms in the lumen of the inoculated colon vessels was 5.00 × 105 cfu/ml [min - max: 3.47 × 104-3.70 × 108 cfu/ml], and on the mucosa 1.44 × 107 cfu/g [min-max: 4.00 × 103-4.00 × 108 cfu/g]. Exact quantification of the anaerobic transconjugants was difficult, as (intrinsic) resistant anaerobic background microbiota were present. QPCR data supported the observation of plasmid transfer in the simulated colon. Moreover, inoculation of E. coli MB6212 had no significant impact on the microbial diversity in the lumen as determined by 16 S ribosomal gene based next generation sequencing on lumen samples. This study demonstrates that a commensal, antibiotic resistant E. coli strain present in food can transfer its antibiotic resistance plasmid relatively quickly to intestinal microbiota in the M-SHIME. The spread and persistence of antibiotic resistance genes and resistant bacteria in our intestinal system is an alarming scenario which might present clinical challenges, since it implies a potential reservoir for dissemination to pathogenic bacteria.

Effects of ascorbic acid on patulin in aqueous solution and in cloudy apple juice.

Degradation of the mycotoxin patulin (PAT) and the generation of (less toxic) breakdown products, such as (E/Z)-ascladiol (ASC-E/Z) and desoxypatulinic acid (D-PAT), can occur due to chemical, physical and biological treatments. Our study focused on the chemical degradation of PAT in the presence of ascorbic acid (AA) both for pure PAT standard in acidified aqueous solution and for PAT-contaminated cloudy apple juice (CAJ) (obtained via addition of apple mash produced from apples inoculated with Penicillium expansum). Within this framework, different concentrations of AA were evaluated, as well as the presence/absence of oxygen and different storage temperatures. In order to do so, an in-house methodology allowing a good separation of PAT from its reaction and breakdown products was optimized first. The highest PAT reduction (60%) in CAJ with an initial PAT concentration of 100 µg/kg and 0.25% (w/v) AA was achieved after 6 days of incubation at 22 °C in the presence of oxygen. It was also found that the treatment by AA resulted in the generation of degradation products less toxic than PAT (such as (E/Z)-ASC). In conclusion, AA used to improve numerous product quality aspects (e.g. colour (less browning), nutritional value, etc.) and considered as a safe food additive (Food and Drug Administration (FDA) (1999)) has an effect on PAT degradation. It was shown that such degradation generated less toxic compounds in the presence of oxygen. In view of consumers' safety, fortification of apple juice (and possibly apple-based products) with AA could be helpful within an integrated system to ensure products with low levels of patulin. The optimum conditions for such an approach within a legal and practical point of view need to be further explored.

Impact of Cross-Contamination Concentrations of Doxycycline Hyclate on the Microbial Ecosystem in an Ex Vivo Model of the Pig's Cecum.

AIMS: Cross-contamination of feed with antibiotics causes pigs to become unintentionally exposed to low concentrations of antibiotics. This study investigates the effect of residues of doxycycline hyclate (DOX) in an ex vivo model of the intestinal tract of pigs, focusing on the microbial community, microbial activity, and the enrichment of resistant bacteria and resistance genes. RESULTS: The effect of three concentrations DOX were tested; 1 and 4 mg/L correspond to the intestinal concentrations when pigs are fed a compound feed containing 3% of a therapeutic dose, and a reference concentration of 16 mg/L. These were continuously administered to a chemostat, simulating the microbial ecosystem of the pig cecum and inoculated with cecal content of organically grown pigs. The administration of even the lowest DOX concentration caused a significant decrease in bacterial activity, while the microbial community profile appeared to remain unaffected by any of the concentrations. A concentration of 1 mg/L DOX caused minor selection pressure for tetracycline-resistant Escherichia coli but no other groups enumerated with plate cultivation, while 4 mg/L induced major enrichment of tetracycline-resistant E. coli, Enterobacteriaceae and total anaerobes. High abundances of tet(Q), tet(M), tet(W), tet(O), and tet(B) were detected in the inoculum and also before antibiotic administration in the chemostat and did not significantly increase during administration of 1 and 4 mg/L DOX. Only 16 mg/L DOX caused minor enrichments. CONCLUSIONS: Cross-contamination concentrations of doxycycline, as a result of cross-contamination, cause a selection pressure for resistant bacteria and negatively affect microbial activity.