Bacteriophages Isolated from Chicken Meat and the Horizontal Transfer of Antimicrobial Resistance Genes.

Antimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested into Escherichia coli ATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P = 0.01) relationship between the presence of phages transducing kanamycin resistance and E. coli isolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance to E. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed in E. coli isolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health.

Shelf-life extension of vacuum-packaged meat from pheasant (Phasianus colchicus) by lactic acid treatment.

We investigated the influence of lactic acid treatment of pheasant meat before vacuum-packaged storage of 3, 7, and 10 d at +6°C on microbiota and pH. Breast muscle samples were collected from carcasses of slaughtered as well as from hunted (shot) wild pheasants. Immersion of meat samples in 3% (wt/wt) lactic acid for 60 s effectuated a significant drop in pH of approximately 0.5 to 0.7 units, which remained during the entire storage period. In parallel, total aerobic counts of such treated and stored samples were on an average 1.5 to 1.7 log units lower than in non-acid-treated samples. Similar results were found for Enterobacteriaceae. A significant decrease in pH was measured at d 7 and 10 in the acid-treated samples in comparison with the untreated ones. In summary, the immersion of pheasant breast meat cuts in dilute lactic acid significantly reduced microbiota during vacuum-packed storage, even at slight temperature abuse conditions.

Treatment of Ready-To-Eat Cooked Meat Products with Cold Atmospheric Plasma to Inactivate Listeria and Escherichia coli.

Ready-to-eat meat products have been identified as a potential vehicle for Listeria monocytogenes. Postprocessing contamination (i.e., handling during portioning and packaging) can occur, and subsequent cold storage together with a demand for products with long shelf life can create a hazardous scenario. Good hygienic practice is augmented by intervention measures in controlling post-processing contamination. Among these interventions, the application of 'cold atmospheric plasma' (CAP) has gained interest. The reactive plasma species exert some antibacterial effect, but can also alter the food matrix. We studied the effect of CAP generated from air in a surface barrier discharge system (power densities 0.48 and 0.67 W/cm2) with an electrode-sample distance of 15 mm on sliced, cured, cooked ham and sausage (two brands each), veal pie, and calf liver pâté. Colour of samples was tested immediately before and after CAP exposure. CAP exposure for 5 min effectuated only minor colour changes (ΔE max. 2.7), due to a decrease in redness (a*), and in some cases, an increase in b*. A second set of samples was contaminated with Listeria (L.) monocytogenes, L. innocua and E. coli and then exposed to CAP for 5 min. In cooked cured meats, CAP was more effective in inactivating E. coli (1 to 3 log cycles) than Listeria (from 0.2 to max. 1.5 log cycles). In (non-cured) veal pie and calf liver pâté that had been stored 24 h after CAP exposure, numbers of E. coli were not significantly reduced. Levels of Listeria were significantly reduced in veal pie that had been stored for 24 h (at a level of ca. 0.5 log cycles), but not in calf liver pâté. Antibacterial activity differed between but also within sample types, which requires further studies.

Toward harmonization of the European food hygiene/veterinary public health curriculum.

Prompted by developments in the agri-food industry and associated recent changes in European legislation, the responsibilities of veterinarians professionally active in veterinary public health (VPH), and particularly in food hygiene (FH), have increasingly shifted from the traditional end-product control toward longitudinally integrated safety assurance. This necessitates the restructuring of university training programs to provide starting competence in this area for veterinary graduates or a sub-population of them. To date, there are substantial differences in Europe in the way in which graduate programs in FH/VPH are structured and in the time allocated to this important curricular group of subjects. Having recognized this, the European Association of Establishments for Veterinary Education (EAEVE) recently instituted a working group to analyze the current situation, with a view to produce standard operating procedures allowing fair and transparent evaluations of universities/faculties constituting its membership and in concurrence with explicit European legislation on the professional qualifications deemed necessary for this veterinary discipline. This article summarizes the main conclusions and recommendations of the working group and seeks to contribute to the international efforts to optimize veterinary training in FH/VPH.

Treatment of Fresh Meat, Fish and Products Thereof with Cold Atmospheric Plasma to Inactivate Microbial Pathogens and Extend Shelf Life.

Assuring the safety of muscle foods and seafood is based on prerequisites and specific measures targeted against defined hazards. This concept is augmented by 'interventions', which are chemical or physical treatments, not genuinely part of the production process, but rather implemented in the framework of a safety assurance system. The present paper focuses on 'Cold Atmospheric pressure Plasma' (CAP) as an emerging non-thermal intervention for microbial decontamination. Over the past decade, a vast number of studies have explored the antimicrobial potential of different CAP systems against a plethora of different foodborne microorganisms. This contribution aims at providing a comprehensive reference and appraisal of the latest literature in the area, with a specific focus on the use of CAP for the treatment of fresh meat, fish and associated products to inactivate microbial pathogens and extend shelf life. Aspects such as changes to organoleptic and nutritional value alongside other matrix effects are considered, so as to provide the reader with a clear insight into the advantages and disadvantages of CAP-based decontamination strategies.

Nitrogen Accumulation in Oyster (Crassostrea gigas) Slurry Exposed to Virucidal Cold Atmospheric Plasma Treatment.

Viral contamination of edible bivalves is a major food safety issue. We studied the virucidal effect of a cold atmospheric plasma (CAP) source on two virologically different surrogate viruses [a double-stranded DNA virus (Equid alphaherpesvirus 1, EHV-1), and a single-stranded RNA virus (Bovine coronavirus, BCoV)] suspended in Dulbecco's Modified Eagle's Medium (DMEM). A 15 min exposure effectuated a statistically significant immediate reduction in intact BCoV viruses by 2.8 (ozone-dominated plasma, "low power") or 2.3 log cycles (nitrate-dominated, "high power") of the initial viral load. The immediate effect of CAP on EHV-1 was less pronounced, with "low power" CAP yielding a 1.4 and "high power" a 1.0 log reduction. We observed a decline in glucose contents in DMEM, which was most probably caused by a Maillard reaction with the amino acids in DMEM. With respect to the application of the virucidal CAP treatment in oyster production, we investigated whether salt water could be sanitized. CAP treatment entailed a significant decline in pH, below the limits acceptable for holding oysters. In oyster slurry (a surrogate for live oysters), CAP exposure resulted in an increase in total nitrogen, and, to a lower extent, in nitrate and nitrite; this was most probably caused by absorption of nitrate from the plasma gas cloud. We could not observe a change in colour, indicative for binding of NOx to haemocyanin, although this would be a reasonable assumption. Further studies are necessary to explore in which form this additional nitrogen is deposited in oyster flesh.

Prevalence of Foodborne Pathogenic Bacteria, Microbial Levels of Hygiene Indicator Bacteria, and Concentrations of Biogenic Amines in Ready-to-Eat Meat Products at Retail in the Republic of Kosovo.

HIGHLIGHTS: RTE meat products from the Republic of Kosovo were tested for contamination. L. monocytogenes was more prevalent in dried or fermented than in cooked-cured meats. E. coli and Enterobacteriaceae were more prevalent in nonpackaged dried or fermented meats. Concentrations of biogenic amines were higher in dried or fermented than in cooked-cured meats.

Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR.

Recently, ethidium monoazide (EMA) has been proposed as a means of reducing the real-time PCR signal originating from free DNA and dead bacterial cells by selectively entering damaged cells and blocking the DNA for PCR amplification via photoactivation. The present study investigated the effect of EMA on viable and dead bacterial cells using real-time PCR, plate count method and microscopy. The foodborne pathogens Campylobacter jejuni and Listeria monocytogenes were used as a Gram-negative and a Gram-positive model organism, respectively. EMA/real-time PCR analysis of heat-treated cultures of C. jejuni and L. monocytogenes containing 2.6x10(5) and 4x10(5) viable and 3x10(6) and 2x10(6) dead cells/ml, respectively, yielded 2x10(3) and 5.2x10(4) bacterial cell equivalents/ml after EMA treatment, thus underestimating the viable cell count in the samples. Similar results were obtained when analyzing late exponential phase cultures of C. jejuni and L. monocytogenes. Inhibition of growth by EMA was observed. It depended on the concentration of the bacterial cells present in the sample and the EMA concentration used (100-1 microg/ml). An EMA concentration at which dead cells would stain brightly and viable cells would not stain at all or would be very pale was not identified, as revealed by comparison with the results of a commercial live/dead stain. The results suggest that EMA influences not only dead but also viable cells of C. jejuni and L. monocytogenes. Thus EMA/real-time PCR is a poor indicator of cell viability.

Preliminary analysis of tetracycline residues in marketed pork in Hanoi, Vietnam.

A cross-sectional survey was designed to investigate the proportion of tetracycline residues in marketed pork in suburb and urban districts in Hanoi. A total of 290 raw muscle samples were randomly collected from open markets in these districts. The samples were qualitatively screened for tetracycline residues using the agar inhibition test, and Bacillus cereus (ATCC 11778) as the reference strain. The inconclusive samples were then analyzed using high-performance liquid chromatography (HPLC). The positive samples from either test were defined as positive results. Overall, 5.5% of all collected samples were positive for tetracycline residues. The proportion of positive samples from shops in suburb districts was significantly (P < 0.05) different from those collected from shops in urban districts. So, the factor of region was identified as a risk factor of tetracycline residue proportion in raw pork with an odds ratio (OR) of 4.03 (95% CI = 1.12, 14.45). For the other factors, such as season, type of shop, type of abattoir, origin of meat, etc., the difference in proportion of positive samples within each factor was substantial but not statistically significant. These factors were identified as nonrisk factors. Such a high proportion may pose a potential hazard to public health, particularly since they might induce drug resistance of pathogenic micro-organisms.

Antimicrobial resistance in commensal Escherichia coli isolated from muscle foods as related to the veterinary use of antimicrobial agents in food-producing animals in Austria.

Controversy exists on veterinary drug application in food animal production and the relevance for human health of antimicrobial resistant commensals isolated from food. The aim of this study was to analyze antimicrobial resistance in Escherichia coli isolated from retail meat of various animal species (including wild roe deer) in Austria. Our results were analyzed taking into consideration the current practices of Austrian veterinarians with regard to their use of antibiotic drugs during pig, poultry, and beef production. Resistant isolates were found most often in pork (76%) followed by poultry (63%) and beef (40%). On wild deer carcasses purchased from Austrian hunters only one isolate was found to be resistant. The latter indicates that antimicrobial resistance is not yet an environmental problem in animals living in the wild. The common use of tetracyclines in veterinary medication in various animal species is clearly reflected in the incidence of antimicrobial resistant isolates in commensal E. coli. The intensive use of fluoroquinolones in poultry could explain the high numbers of nalidixic acid resistant isolates found on poultry meat. Our findings partly explain the impact of veterinary drug application on the resistance development of E. coli isolated from meat.

Comparison of three methods for detecting Campylobacter spp. in chilled or frozen meat.

There is a demand from the meat industry as well as from public health authorities for a simple and rapid detection method for thermophilic Campylobacter spp. from food. Hence, we compared different isolation procedures for their usefulness for this purpose. Bolton enrichment medium without blood, incubated statically in stomacher bags in microaerophilic atmosphere, detected more samples positive for thermophilic Campylobacter spp. than did Preston enrichment broth in bottles with small headspace and tight caps, incubated in aerobic atmosphere. Use of an automated antigen detection system to identify enrichment cultures positive for Campylobacter spp. was as sensitive as selective agars, and reduced the detection time by 24 h. Campylobacter spp. were recovered from 18.4% of the 461 samples tested. The prevalence was highest in refrigerated poultry meat (52% of the 80 samples tested) and poultry offal (41% of the 44 samples tested).

Rapid urease screening of Yersinia on CIN agar plates.

Yersinia enterocolitica is an important foodborne pathogen, but isolation of virulent Yersinia from food sources is still time consuming and requires skills. In this article, we describe a rapid urease screening on cefsulodin-irgasan-novobiocin (CIN) agar plates with an agar overlay assay. This test is simple to perform, all colonies on a plate can be checked simultaneously, it only takes minutes for detection of urease-positive colonies and the colonies survive for transfer, further characterisation, and storage. Additionally, this method is useful to isolate virulent (urease-positive and pYV harbouring) Y. enterocolitica from foodstuffs.

Antimicrobial resistance profile of five major food-borne pathogens isolated from beef, pork and poultry.

This study was performed to evaluate the resistance rate against antimicrobials of food isolates of the five major food-borne pathogens to compare these and to possibly distinguish a pattern. A total of 922 samples of the major meat species (pork, beef and poultry) were analysed for thermophilic Campylobacter, Salmonella, Yersinia enterocolitica, pathogenic Escherichia coli and Listeria monocytogenes. Isolates were subjected to antimicrobial resistance testing by the disc diffusion method. Roughly the same overall rate of resistance was identified for thermophilic Campylobacter, Salmonella and pathogenic E. coli. Resistance to quinolones and tetracycline was determined most frequently. In contrast, food isolates of Y. enterocolitica and L. monocytogenes were rarely tested resistant. The significance of our findings is that resistance rates in enteric bacteria seem to be much higher than in pathogens found in a variety of environments, closely associated to the host environment.

On the efficacy of current biosecurity measures at EU borders to prevent the transfer of zoonotic and livestock diseases by travellers.

Although many animal diseases have been eradicated from the European Union (EU), the animal production sectors in the EU are still under a major threat of disease pathogens introduced by travellers into a country through illegal importation of wildlife or production animals, and/or food products of animal origin. These may carry (exotic) pathogens or toxic metabolites that are hazardous for public health and have a zoonotic potential. According to experts, newly emerging diseases will most probably be zoonotic in nature. The control systems and inspection measures at the borders are, in general, sufficient to control the import of disease pathogens through commercial consignments, as regularly reported by the Food and Veterinary Office (FVO). The Schengen Agreement in the EU has pushed such inspections to the outer borders of the EU in the context of freedom of movement of 'goods' - including live animals and foods of animal origin - people and services within the EU (Treaty of Rome). However, it is questionable whether this policy and the inspection measures taken are effective in reducing public and animal health risks in the EU to an acceptable level. Risk assessment studies point to the potential dangers of illegal imports by travellers. This review article discusses the current status quo and more, in particular, the weaknesses of the current inspection procedures related to biosecurity and suggestions for improvement are made.

Risk management in primary apicultural production. Part 1: bee health and disease prevention and associated best practices.

Prompted by FAO/WHO's and the European Commission's recognition that documents on Good Farming Practices (GFPs) and Good Veterinary Practices (GVPs) in apicultural production are hardly available, part 1 of this contribution provides an update of current apicultural production and associated best practices to ensure animal and public health. Major bee health and disease prevention issues and risk management options at the primary production level are summarised with particular reference to the role of the veterinary practitioner/consultant and the official veterinarian in a control function in the safe production of honey.

Simple media and conditions for inter-laboratory transport of Campylobacter jejuni isolates.

BACKGROUND: Campylobacter jejuni is one of the most important agents of zoonotic disease. Production as well as companion animals can be the infectious source for Campylobacteriosis in humans. Hence, epidemiological research on animal colonization, survival in food of animal origin, and human Campylobacteriosis is of high priority. As such studies involve worldwide co-operations and should include further typing of isolates in reference centers, using a reliable method for transportation is essential. In the case of C. jejuni, a pathogenic and microaerophilic bacterium, special safety precautions as well as particular transport conditions that guarantee survival of isolates are required. OBJECTIVE: The purpose of this study was to test various media and temperatures for the transportation of C. jejuni under aerobic conditions and to identify a cheap, effective and easy method that is appropriate for long distance transportation and can be applied by most veterinary/medical laboratories with a basic infrastructure. MATERIALS AND METHODS: We examined Mueller-Hinton (MH) agar with and w/o 2% horse blood and m-CCDA at room temperature and 2 ± 2 (SD)°C under atmospheric conditions for survival of Campylobacter strains. RESULTS: MH agar with 2% horse blood, suitable transport vials, and an optimum temperature of 2 ± 2°C provided survival of three Campylobacter type strains for at least one month under atmospheric conditions. This was validated by a transport test in which 101 isolates were shipped from Turkey to Austria. All isolates could be recultured and 97% survived more than one month in the transport medium. CONCLUSION: These findings indicate that the described approach is suitable for inter-laboratory transport of C. jejuni isolates.

Microbiological and sensory effects of the combined application of hot-cold organic acid sprays and steam condensation at subatmospheric pressure for decontamination of inoculated pig tissue surfaces.

We studied microbiological and sensory effects of treating pig tissue for 15 s with 55 and 10°C sprays of acetic acid (AA; 0.15 to 0.3 M) and lactic acid (LA; 0.1 to 0.2 M) solutions prior to the tissue being subjected to steam condensation (18 s at 65°C or 10 s at 75°C). LA or AA spraying and then steam treatment resulted in 3- to 4-log average reductions of Pseudomonas fragi and Yersinia enterocolitica inocula (6 to 7 log CFU/cm(2)), regardless of acid temperature or concentration. Buffered LA or 1:1 mixtures of AA:LA and then steam treatment yielded similar reductions. Most of the acid-steam-treated samples had microbial counts below the limit of detection (2 log CFU/cm(2)); thus, the results likely underestimate the potential of this procedure. When the period between inoculation and acid-steam treatment was extended from 0.5 to 24 h, up to a 1-log-higher microbial reduction was observed, due to a 1- to 2-log-greater initial contamination. Increasing the LA contact time to 6 min increased the microbial reduction by 0.8 log. Acid-steam treatment effected lower L* values (darker color) on pigskin, but higher L* values on muscle and fat tissue (paler color). Many muscle samples exhibited lower a* values and off-color brown hues. Off-odors were observed immediately after treatment, but with the exception of fat tissue and AA-treated samples, they largely disappeared during further storage. Off-flavors were only detected in AA-treated muscle samples.

Risk management in primary apicultural production. Part 2: a Hazard Analysis Critical Control Point approach to assuring the safety of unprocessed honey.

In managing risks associated with the human consumption of honey, all sectors of the production chain must be considered, including the primary production phase. Although the introduction of the Hazard Analysis Critical Control Point (HACCP) system has not been made compulsory for purposes of quality and safety control in farming operations, European legislation makes many references to the key role of primary production in food safety management and the HACCP system has been indicated as the preferred tool to ensure that consumers are provided with safe foods. This article describes a systematic HACCP-based approach to identifying, preventing and controlling food safety hazards occurring in primary apicultural production. This approach serves as a useful tool for beekeepers, food business operators, veterinary advisors, and for Food and Veterinary Official Control Bodies in their planning and conducting of audits and for establishing priorities for the evaluation of training programmes in the apicultural sector.

Food-borne zoonoses, the EU zoonosis legislation and the prospects for food safety and consumer protection during primary animal production.

Zoonoses are diseases that are transmitted naturally between animals and humans. The control of food-borne zoonoses within the European Union is a prerequisite for assuring a functional internal market and consequently represents an important item on the political agenda. Unfortunately, until recently, gaining a clear view of the current incidence of food-borne zoonoses and the prevalence of its causative agents has been frustrated by the absence of reliable monitoring and reporting systems. Similarly, it has become clear that, Europe wide, one has witnessed only limited success with regard to the control of important food-borne agents such as Salmonella spp. The European Union has adopted legislation to remedy this situation and to control food-borne zoonoses in primary production. This contribution discusses the incentives for introducing EU Directive 2003/99/EC and EU Regulation No. 2160/2003, summarises their essentials and discusses major ramifications of both pieces of legislation for the prevention of food-borne zoonoses. It is concluded that there is reason for cautious optimism concerning human salmonellosis, while for other food-borne zoonoses there should be a call for action.

Real-time PCR method with statistical analysis to compare the potential of DNA isolation methods to remove PCR inhibitors from samples for diagnostic PCR.

A real-time PCR method for fast comparison of different DNA isolation methods to remove PCR inhibitors from samples is presented. A fixed amount of target-200 copies of a 79-bp region of the COCH gene of the zebrafish (Danio rerio)-was added to each PCR reaction together with isolated DNA from different types of samples including chicken feces. Four commercial DNA isolation kits and a chelex-based technique were compared using this method. The copy numbers calculated and the endpoint fluorescence were statistically compared to the values of 22 control samples containing the control target and water instead of isolated DNA, processed together in the same PCR run. The level of the endpoint fluorescence was more often negatively influenced by inhibitors than the copy number calculated, suggesting a more pronounced effect on the plateau phase of the reaction by limiting one or more compounds in the PCR reaction.