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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Characterization of Cefotaxime- and Ciprofloxacin-Resistant Commensal Escherichia coli Originating from Belgian Farm Animals Indicates High Antibiotic Resistance Transfer Rates.

Food-producing animals represent one of the sources of antibiotic resistant commensal bacteria. There is an increasing awareness that these bacteria might have the potential to transfer their resistance genes to other (pathogenic) bacteria. In this study, 50 commensal Escherichia coli strains originating from food-producing animals and resistant to the "highest priority, critically important antibiotics" cefotaxime and/or ciprofloxacin, were selected for further characterization. For each strain (i) an antibiogram, (ii) the phylogenetic group, (iii) plasmid replicon type, (iv) presence and identification of integrons, and (v) antibiotic resistance transfer ratios were determined. Forty-five of these strains were resistant to 5 or more antibiotics, and 6 strains were resistant to 10 or more antibiotics. Resistance was most common to ampicillin (100%), sulfamethoxazole, ciprofloxacin (82%), trimethoprim, tetracycline (74%), cefotaxime, (70%) and ceftazidime (62%). Phylogenetic groups A (62%) and B1 (26%) were most common, followed by C (8%) and E (4%). In 43 strains, more than 1 replicon type was detected, with FII (88%), FIB (70%), and I1 (48%) being the most encountered types. Forty strains, positive for integrons, all harbored a class I integron and seven of them contained an additional class II integron. No class III integrons were detected. The antibiotic resistance transfer was assessed by liquid mating experiments. The transfer ratio, expressed as the number of transconjugants per recipient, was between 10-5 and 100 for cefotaxime resistance and between 10-7 and 10-1 for ciprofloxacin resistance. The results of the current study prove that commensal E. coli in food-production animals can be a source of multiple resistance genes and that these bacteria can easily spread their ciprofloxacin and cefotaxime resistance.

The Biodiversity of the Microbiota Producing Heat-Resistant Enzymes Responsible for Spoilage in Processed Bovine Milk and Dairy Products.

Raw bovine milk is highly nutritious as well as pH-neutral, providing the ideal conditions for microbial growth. The microbiota of raw milk is diverse and originates from several sources of contamination including the external udder surface, milking equipment, air, water, feed, grass, feces, and soil. Many bacterial and fungal species can be found in raw milk. The autochthonous microbiota of raw milk immediately after milking generally comprises lactic acid bacteria such as Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc species, which are technologically important for the dairy industry, although they do occasionally cause spoilage of dairy products. Differences in milking practices and storage conditions on each continent, country and region result in variable microbial population structures in raw milk. Raw milk is usually stored at cold temperatures, e.g., about 4°C before processing to reduce the growth of most bacteria. However, psychrotrophic bacteria can proliferate and contribute to spoilage of ultra-high temperature (UHT) treated and sterilized milk and other dairy products with a long shelf life due to their ability to produce extracellular heat resistant enzymes such as peptidases and lipases. Worldwide, species of Pseudomonas, with the ability to produce these spoilage enzymes, are the most common contaminants isolated from cold raw milk although other genera such as Serratia are also reported as important milk spoilers, while for others more research is needed on the heat resistance of the spoilage enzymes produced. The residual activity of extracellular enzymes after high heat treatment may lead to technological problems (off flavors, physico-chemical instability) during the shelf life of milk and dairy products. This review covers the contamination patterns of cold raw milk in several parts of the world, the growth potential of psychrotrophic bacteria, their ability to produce extracellular heat-resistant enzymes and the consequences for dairy products with a long shelf life. This problem is of increasing importance because of the large worldwide trade in fluid milk and milk powder.

Antibiotic use and resistance in animals: Belgian initiatives.

The widespread use of antibiotics in animals is causing concerns about the growing risk for development and the spread of antibiotic-resistant bacteria. Antibiotic consumption is higher in animals than in humans as reported in a joint publication of EFSA (European Food Safety Agency), ECDC (European Centre for Disease Prevention and Control), and EMA (European Medicines Agency) using data from 2011 and 2012. Both in humans and animals, positive associations between the consumption of antibiotics and resistant bacteria are observed. Responsible use of antibiotics in humans and animals should therefore be promoted. In this paper some general aspects of antibiotic resistance such as microbiological versus clinical resistance, intrinsic versus acquired resistance, resistance mechanisms, and transfer of resistance are briefly introduced. In 2012, the Belgian Center of Expertise on Antimicrobial Consumption and Resistance in Animals (AMCRA) was founded. Its mission is to collect and analyze all data related to antibiotic use and resistance in animals in Belgium and to communicate these findings in a neutral and objective manner. One of AMCRA's 10 objectives is a 50% reduction in antibiotic consumption in veterinary medicine in Belgium by 2020. The aim of this paper is to report on the achievements of this national project. The Institute for Agricultural and Fisheries Research (ILVO, Merelbeke-Melle), in collaboration with Ghent University, is currently working on three nationally funded projects on antibiotic resistance in animal husbandry. In the first project, an in vitro model is used to study the influence of low antibiotic concentrations due to carry-over after production and usage of medicated feed on the development of resistance in the pig gut. Part of that project is to develop a quantitative risk assessment model. A second project focuses on tracking excreted antibiotics used in pig rearing and their influence on the development of antibiotic resistance in pig manure and the environment. In the last project, the relation between the use of biocides in animal husbandry and antibiotic resistance development are being studied. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.

Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

Identification and characterization of a heat-resistant protease from Serratia liquefaciens isolated from Brazilian cold raw milk.

The cold storage of raw milk before heat treatment in dairy industry promotes the growth of psychrotrophic microorganisms, which are known for their ability to produce heat-resistant proteolytic enzymes. Although Pseudomonas is described as the main causative genus for high proteolytic spoilage potential in dairy products, Serratia liquefaciens secretes proteases and may be found in raw milk samples as well. However, at the present there is no information about the proteolytic spoilage potential of S. liquefaciens in milk after heat-treatment. The main aim of this research was to assess the proteolytic spoilage potential of S. liquefaciens isolated from Brazilian raw milk and to characterize the involved protease. S. liquefaciens was shown to secrete one heat-resistant spoilage metalloprotease of, approximately, 52 kDa encoded by the ser2 gene. The heat-resistance of Ser2 was similar to the aprX encoded metalloprotease produced by Pseudomonas. Although the ser2 gene was detected in all S. liquefaciens isolates tested in this study, the proteolytic activity of the isolates in milk was highly heterogeneous. Since nucleotide and deduced amino acid sequences of ser2 of all tested isolates are identical, this heterogeneity may be attributed to differences in enzyme expression levels or post-translational modifications.

Low Temperature and Modified Atmosphere: Hurdles for Antibiotic Resistance Transfer?

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 sakei subsp. sakei to Listeria monocytogenes was 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(-4) to 10(-6). With low-density inocula, transfer was only detected under the 100% N2 atmosphere 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 sakei subsp. sakei to Listeria monocytogenes.

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.

Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants.

Since phenotypic methods to identify coagulase negative staphylococci (CNS) from the milk of ruminants often yield unreliable results, methods for molecular identification based on gene sequencing or fingerprinting techniques have been developed. In addition to culture-based detection of isolates, culture-independent methods may be of interest. On the basis of molecular studies, the five CNS species commonly causing intramammary infections (IMI) are Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus simulans and Staphylococcus xylosus. Current knowledge suggests that S. chromogenes is a bovine-adapted species, with most cases of IMI due to this bacterium being opportunistic. S. haemolyticus also appears to be an opportunistic pathogen, but this bacterium occupies a variety of habitats, the importance of which as a source of IMI remains to be elucidated. S. xylosus appears to be a versatile species, but little is known of its epidemiology. S. epidermidis is considered to be a human-adapted species and most cases of IMI appear to arise from human sources, but the organism is capable of residing in other habitats. S. simulans typically causes contagious IMI, but opportunistic cases also occur and the ecology of this bacterium requires further study. Further studies of the ecology and epidemiology of CNS as a cause of IMI in cattle are required, along with careful attention to classification of these bacteria and the diseases they cause.

A quantitative exposure model simulating human norovirus transmission during preparation of deli sandwiches.

Human noroviruses (HuNoVs) are a major cause of food borne gastroenteritis worldwide. They are often transmitted via infected and shedding food handlers manipulating foods such as deli sandwiches. The presented study aimed to simulate HuNoV transmission during the preparation of deli sandwiches in a sandwich bar. A quantitative exposure model was developed by combining the GoldSim® and @Risk® software packages. Input data were collected from scientific literature and from a two week observational study performed at two sandwich bars. The model included three food handlers working during a three hour shift on a shared working surface where deli sandwiches are prepared. The model consisted of three components. The first component simulated the preparation of the deli sandwiches and contained the HuNoV reservoirs, locations within the model allowing the accumulation of NoV and the working of intervention measures. The second component covered the contamination sources being (1) the initial HuNoV contaminated lettuce used on the sandwiches and (2) HuNoV originating from a shedding food handler. The third component included four possible intervention measures to reduce HuNoV transmission: hand and surface disinfection during preparation of the sandwiches, hand gloving and hand washing after a restroom visit. A single HuNoV shedding food handler could cause mean levels of 43±18, 81±37 and 18±7 HuNoV particles present on the deli sandwiches, hands and working surfaces, respectively. Introduction of contaminated lettuce as the only source of HuNoV resulted in the presence of 6.4±0.8 and 4.3±0.4 HuNoV on the food and hand reservoirs. The inclusion of hand and surface disinfection and hand gloving as a single intervention measure was not effective in the model as only marginal reductions of HuNoV levels were noticeable in the different reservoirs. High compliance of hand washing after a restroom visit did reduce HuNoV presence substantially on all reservoirs. The model showed that good handling practices such as washing hands after a restroom visit, hand gloving, hand disinfection and surface disinfection in deli sandwich bars were an effective way to prevent HuNoV contamination of the prepared foods, but it also demonstrated that further research is needed to ensure a better assessment of the risk of HuNoV transmission during preparation of foods.