Detection of Extended-Spectrum ß-Lactamase (ESBL) E. coli at Different Processing Stages in Three Broiler Abattoirs.

The European Food Safety Authority (EFSA) identified extended-spectrum ß-lactamase/AmpC ß-lactamase (ESBL/AmpC)-producing E. coli as one of the main priority hazards for poultry. Different studies detected ESBL-producing E. coli at broiler fattening farms and in abattoirs, concluding that poultry meat is a potential source of human infection. Broiler breast skin samples taken in three abattoirs with different scalding techniques were examined for ESBL-producing Escherichia (E.) coli and their phylogenetic groups. A total of 307 ESBL-producing E. coli isolates were found, and the abattoir with conventional immersion scalding with thermal treatment of the water had the lowest incidence. Phylogroups D/E and B1 were mostly detected, while phylogroups C, D, and E were not detected. Phylogroup B2 was detected in low proportions. The phylogroups B2 and D are important as they have been associated with urinary tract infections in humans, but were only detected in low proportions at different processing stages in this study. Since the risk for the consumer of being infected via chicken meat with ESBL-producing E. coli and E. coli of highly pathogenic phylogroups cannot be excluded, good kitchen hygiene is of great importance.

Detection and genome characterization of bovine polyomaviruses in beef muscle and ground beef samples from Germany.

Polyomaviruses are small, non-enveloped, circular double-stranded DNA viruses. Some polyomaviruses can induce tumors and cancer under certain circumstances. The bovine polyomaviruses (BPyV) 1-3 have been only scarcely analyzed so far. It was hypothesized that the consumption of beef meat containing polyomaviruses could contribute to the development of cancer in humans. In order to assess the distribution of the BPyV genome in meat from Germany, 101 beef muscle samples and 10 ground beef samples were analyzed here. A specific sample preparation method combined with or without rolling circle amplification (RCA), and BPyV-specific PCRs were developed and applied. BPyV-1 DNA was detected in 1/101 (1%) samples from beef meat and in 2/10 (20%) ground beef samples. BPyV-2 DNA was detected in 3/10 (30%) ground beef samples, whereas BPyV-3 was not detected in the samples. Application of RCA did not increase the detection rate in ground beef samples. Sequence analysis of the PCR products indicated the presence of BPyV-1, BPyV-2a and BPyV-2b. The whole genome of a BPyV-1 strain from ground beef meat showed 97.8% sequence identity to the BPyV-1 reference strain and that of a BPyV-2a strain from ground beef meet showed 99.9% sequence identity to strain 2aS11. It can be concluded that BPyV genomes can be frequently detected in ground beef samples, although higher sample numbers should be investigated in future to confirm this finding. Further studies should focus on the infectivity, tumorigenicity and heat resistance of the contained viruses in order to assess the risk of cancer induction through consumption of BPyVs present in beef products.

Listeria monocytogenes in Different Specimens from Healthy Red Deer and Wild Boars.

In the past, Listeria monocytogenes has been isolated from game feces and meat. However, less information is available on the occurrence of L. monocytogenes in other specimens originating from game animals. Hence, the aim of this study was to get an overview of the occurrence and distribution of L. monocytogenes in game animals by characterization of isolates from different matrices. For that purpose, samples were collected from red deer (Cervus elaphus), wild boars (Sus scrofa), and feed during the hunting season 2011-2012 in three different regions of Germany and Austria. Six samples from each animal were examined: tonsils, content of the rumen or the stomach, liver, intestinal lymph nodes, cecum content, and feces. Nineteen of 45 red deer and 12 of 49 wild boars were found to be positive for L. monocytogenes as well as 4 of 22 pooled feed samples. L. monocytogenes was isolated most frequently from the rumen of red deer (14 of 19) and the tonsils of wild boars (7 of 12). Serotypes 1/2a, 1/2b, 4a, and 4b were detected in samples of game animals and feed, and serotypes 1/2a and 4b were the most prevalent serotypes. The presence of L. monocytogenes serotype 4a had not yet been described in red deer. This might be due to the fact that it was only isolated from the content of rumen and that no other study has yet examined ruminal content. Pulsed-field gel electrophoresis showed a wide variety of strains. Some strains occurred in both species and feed samples, but one strain was dominant in one region. The results show that red deer and wild boars can be carriers of L. monocytogenes in different matrices, although the feces samples can be negative.

Detection of hepatitis E virus RNA in raw sausages and liver sausages from retail in Germany using an optimized method.

Hepatitis E virus (HEV) is a pathogen of increasing importance, which can be zoonotically transmitted from domestic pigs, wild boar, and deer to humans. Foodborne transmission by consumption of raw and undercooked liver, meat, or sausages prepared from infected animals has been documented. The aim of this study was to investigate the distribution of HEV in different types of sausages sold in Germany. As no standardized methods for HEV detection in food exist, several techniques of sample homogenization, virus concentration and nucleic acid extraction followed by real-time RT-PCR were compared using artificially contaminated sausages. A method using TRI Reagent® Solution showed the best efficacy of matrix disruption and a treatment with chloroform followed by a silica-based RNA extraction method resulted in the highest HEV detection rates. The detection limit of the method was 2.9 × 10(3) and 5.3 × 10(4) genome equivalents per 5 g raw sausage and 2 g liver sausage, respectively. Application of the method to raw and liver sausages from retail in Germany resulted in the HEV genome detection in 14 out of 70 (20%) raw sausages and in 11 out of 50 (22%) liver sausages. The detected HEV sequences showed a high diversity and belonged to different subtypes of HEV genotype 3. The results indicate a broad distribution of HEV-RNA in meat products sold in Germany; however, the infectivity of the detected virus remains to be assessed in future.

"Limits of control"--crucial parameters for a reliable quantification of viable campylobacter by real-time PCR.

The unsuitability of the "CFU" parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.

Detection and characterization of potentially zoonotic viruses in faeces of pigs at slaughter in Germany.

Pigs can harbour a variety of viruses in their gastrointestinal tract. Some of them are closely related to human viruses and are therefore suspected to have a zoonotic potential. Only little is known about the presence of those viruses in pigs at slaughter. However, by contamination of meat with zoonotic viruses during the slaughtering process, food-borne transmission to humans may be possible. Here we analyzed 120 faecal samples of pigs at slaughter from 3 different geographical regions of Germany for the presence of astrovirus (AstV), encephalomyocarditis virus (EMCV), hepatitis E virus (HEV), norovirus genogroup II (NoV GII) and group A rotavirus (GARV). Using real-time RT-PCR, the most frequently detected virus was AstV, which was present in 20.8% of the samples, followed by NoV GII with a detection rate of 14.2%. EMCV, HEV and GARV were found only occasionally with detection rates of 4.2%, 2.5% and 0.8%, respectively. Analyses of partial genome sequences of the viruses indicated that the detected AstV and NoV GII mainly represented typical pig virus strains, which have not been detected in humans so far. However, the GARV and HEV strains were more closely related to human strains. The results indicate that enteric viruses, some of them with zoonotic potential, are present in pig faeces at slaughter. Application of good hygiene practice is necessary to minimize the risk of introducing these viruses into the food and to prevent virus transmission to highly exposed persons such as slaughterers and veterinarians.

Serotype distribution of Salmonella isolates from turkey ground meat and meat parts.

The aim of the study was to find out the serotype distribution of 169 Salmonella colonies recovered from 112 Salmonella positive ground turkey (115 colonies) and 52 turkey meat parts (54 colonies). Out of 15 Salmonella serotypes: S. Corvallis, S. Kentucky, S. Bredeney, S. Virchow, S. Saintpaul and S. Agona were identified as the predominant serovars at the rates of 27%, 13%, 12%, 12%, 11%, and 10%, respectively. Other serotypes were below 6% of the total isolates. All S. Kentucky and S. Virchow and most of the S. Corvallis (39/46) and S. Heidelberg (9/9) serotypes were recovered from ground turkey. The results indicate that turkey ground meat and meat parts were contaminated with quite distinct Salmonella serotypes. This is the first study reporting Salmonella serotype distribution in turkey meat and S. Corvallis as predominant serotype in poultry meat in Turkey.

[Surveillance systems for status monitoring of Trichinella-free declared pig farms: concepts and their confidence for freedom from disease]. / Surveillance-Systeme zur Herdenstatus-überwachung: Konzepte und deren Sicherheit für den Trichinella-freien Herdenstatus in Schweinemastbetrieben.

Trichinella surveillance data in Germany show for indoor housed pigs hardly any cases. Nevertheless, obligatory testing is in place for each slaughtered pig. According to EU legislation systematic Trichinella testing can be replaced by a risk-based surveillance system if the risk of Trichinella infection in fattening pigs is negligible. The probability to detect a positive herd (herd sensitivity) was taken as an indicator for the effectiveness of the surveillance. Four different diagnostic methods: a) digestion method, b) E/S-ELISA, c) Western Blot, and d) ELISA sequentially combined with Western Blot, were compared regarding herd sensitivity and specificity for different herd and sample sizes and different levels of prevalence. In a further step three potential surveillance systems were compared with regard to their suitability for herd classification: (i) classical Trichinella examination by artificial digestion method, (ii) ELISA screening followed by classical Trichinella examination and (iii) ELISA screening followed by Western Blot. Results show that: 1) testing by the artificial digestion method (i) provides only low sensitivity of detection for positive herds at present levels of prevalence despite perfect specificity. 2) The ELISA alone provides a high sensitivity of detection even at low sample sizes but at the cost of a very low herd specificity, converging towards zero at increasing sample sizes. In surveillance system (ii), a large number of farms would still need to be tested with the classical digestion method, as they would be misclassified as positive by the ELISA. 3) The Western Blot as well as ELISA screening followed by Western Blot offer a high probability of correct herd classification. The latter diagnostic system appears to be the most suitable for a risk based surveillance (iii) and provides--despite reduced sample sizes--a higher probability for a correct herd classification than the traditional Trichinella examination.

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans. RESULTS: Here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified. CONCLUSIONS: The genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments.

Comparison of two extraction methods for viruses in food and application in a norovirus gastroenteritis outbreak.

Noroviruses are important causes of gastroenteritis; however, due to a lack of sensitive detection methods, the distinct role of contaminated food in norovirus outbreaks remains unclear. Two published virus extraction procedures combined with real-time RT-PCR for the detection of norovirus from food inoculated experimentally were compared. The elution-precipitation method was most efficient in all food matrices tested showing detection limits of 20 RT-PCRU for lettuce and ham, and 200 RT-PCRU for raspberries. The average recovery rates were 23%, 7% and 24% for lettuce, raspberries and ham, respectively. The ultrafiltration method yielded detection limits of 200 RT-PCRU for lettuce and ham, and 2000 RT-PCRU for raspberries; recovery rates were 9%, 7%, 3%, respectively. Subsequently, food items implicated in a norovirus outbreak were examined by the elution-precipitation method. Virus recovery rates determined by using MS2 phage ranged from 1 to 20% depending on the food matrix. However, norovirus could not be detected in the food items examined. This negative result may be explained by a low virus titer and heterogeneous virus distribution, or by random selection of food samples that contained no norovirus. Both, proper sampling and virus extraction from foods may be improved further to identify vehicles of infection.

Food-borne norovirus-outbreak at a military base, Germany, 2009.

BACKGROUND: Norovirus is often transmitted from person-to-person. Transmission may also be food-borne, but only few norovirus outbreak investigations have identified food items as likely vehicles of norovirus transmission through an analytical epidemiological study.During 7-9 January, 2009, 36 persons at a military base in Germany fell ill with acute gastroenteritis. Food from the military base's canteen was suspected as vehicle of infection, norovirus as the pathogen causing the illnesses. An investigation was initiated to describe the outbreak's extent, to verify the pathogen, and to identify modes of transmission and source of infection to prevent further cases. METHODS: For descriptive analysis, ill persons were defined as members of the military base with acute onset of diarrhoea or vomiting between 24 December 2008, and 3 February 2009, without detection of a pathogen other than norovirus in stools. We conducted a retrospective cohort study within the headquarters company. Cases were military base members with onset of diarrhoea or vomiting during 5-9 January. We collected information on demographics, food items eaten at the canteen and contact to ill persons or vomit, using a self-administered questionnaire. We compared attack rates (AR) in exposed and unexposed persons, using bivariable and multivariable logistic regression modelling. Stool specimens of ill persons and canteen employees, canteen food served during 5-7 January and environmental swabs were investigated by laboratory analysis. RESULTS: Overall, 101/815 (AR 12.4%) persons fell ill between 24 December 2008 and 3 February 2009. None were canteen employees. Most persons (n = 49) had disease onset during 7-9 January. Ill persons were a median of 22 years old, 92.9% were male. The response for the cohort study was 178/274 (72.1%). Of 27 cases (AR 15.2%), 25 had eaten at the canteen and 21 had consumed salad. Salad consumption on 6 January (aOR: 8.1; 95%CI: 1.5-45.4) and 7 January (aOR: 15.7; 95%CI: 2.2-74.1) were independently associated with increased risk of disease.Norovirus was detected in 8/28 ill persons' and 4/25 canteen employees' stools, 6/55 environmental swabs and 0/33 food items. Sequences were identical in environmental and stool samples (subtype II.4 2006b), except for those of canteen employees. Control measures comprised cohort isolation of symptomatic persons, exclusion of norovirus-positive canteen employees from work and disinfection of the canteen's kitchen. CONCLUSIONS: Our investigation indicated that consumption of norovirus-contaminated salad caused the peak of the outbreak on 7-9 January. Strict personal hygiene and proper disinfection of environmental surfaces remain crucial to prevent norovirus transmission.

[Tasks and duties of veterinary reference laboratories for food borne zoonoses]. / Aufgaben der veterinärmedizinischen Referenzlaboratorien im Bereich der lebensmittelbedingten Zoonosen.

Reference laboratories are of central importance for consumer protection. Field expertise and high scientific competence are basic requirements for the nomination of a national reference laboratory. To ensure a common approach in the analysis of zoonotic hazards, standards have been developed by the reference laboratories together with national official laboratories on the basis of Art. 33 of Directive (EG) No. 882/2004. Reference laboratories function as arbitrative boards in the case of ambivalent or debatable results. New methods for detection of zoonotic agents are developed and validated to provide tools for analysis, e. g., in legal cases, if results from different parties are disputed. Besides these tasks, national reference laboratories offer capacity building and advanced training courses and control the performance of ring trials to ensure consistency in the quality of analyses in official laboratories. All reference laboratories work according to the ISO standard 17025 which defines the grounds for strict laboratory quality rules and in cooperation with the respective Community Reference Laboratories (CRL). From the group of veterinary reference laboratories for food-borne zoonoses, the national reference laboratories are responsible for Listeria monocytogenes, for Campylobacter, for the surveillance and control of viral and bacterial contamination of bivalve molluscs, for E. coli, for the performance of analysis and tests on zoonoses (Salmonella), and from the group of parasitological zoonotic agents, the national reference laboratory for Trichinella.

Risk based meat hygiene--examples on food chain information and visual meat inspection.

The Regulation (EC) No. 854/2004 refers in particularly to a broad spectrum of official supervision of products of animal origin. The supervision should be based on the most current relevant information which is available. The proposal presented includes forms for implementation of Food Chain Information (FCI) as laid down in the Regulation (EC) No. 853/2004 as well as suggestions for the implementation of the visual inspection and criteria for a practical approach to assist the competent authority and the business operator. These suggestions are summarised in two forms including the FCI and practical information from the farm of origin as well as from the slaughterhouse needed for the competent authority to permit the visual meat inspection of fattening pigs. The requested information from the farm level include i.e. an animal loss rate during the fattening period and diagnostic findings of carcasses and respectively on organs of the animals during meat inspection procedure. The evaluation of findings in liver and pleura at the slaughterhouse level indicate a correlation to the general health status of the fattening conditions at farm level. The criteria developed are in principle suited for a "gold standard" of the health status of fattening pigs and the criteria may serve as practical implementation of the term Good Farming Practice in relation to consumer protection. Only for fattening pigs deriving from farms fulfilling this "gold standard" the visual meat inspection shall be permitted. It is proposed to integrate the two forms for FCI and additional information for authorisation of visual meat inspection for fattening pigs in the working document of the DG SANCO titled "Commission regulation of laying down specific rules on official controls for the inspection of meat" (SANCO/2696/2006).

Expert opinion based modelling of the risk of human infection with H5N1 through the consumption of poultry meat in Germany.

According to current scientific opinion the risk of human infection with H5N1 via preparation and consumption of poultry meat is negligible.This opinion has not yet been challenged by a formal risk assessment, due to the lack of empirical data. We have developed a scenario pathway model as a conceptual framework for a formal assessment of the H5N1 risk to humans through consumption of poultry meat and parameterise the model using information derived from expert opinions. The aim of this study was to investigate whether the notion of an overall negligible risk via the oral infection route is consistent with ad hoc data and expert opinions on the relevant parameters of the model. The model is mainly based on expert opinion. A stochastic Monte-Carlo simulation was conducted which took into consideration (amongst others) the exposure and infection of chicken (broiler and layer), turkeys, ducks and geese, the probabilities of detection prior to slaughter, virus survival and contamination during slaughter, as well as during the cutting and preparation of meat in commercial plants and in private households, respectively. The empirical consumption pattern for poultry meat in Germany was taken into account in the simulation. The results show that the risk for the individual consumer is practically zero whereas up to 23 cases per year in Germany might occur if the upper (more pessimistic) ranges of the expert opinions apply. The finding of a low but non-negligible risk to the population is discussed in relation to the epidemiological information available from recent outbreaks in South East Asia.

Tracing flock-related Campylobacter clones from broiler farms through slaughter to retail products by pulsed-field gel electrophoresis.

A total of 237 Campylobacter isolates from broiler flocks at farm (45 isolates) and slaughter (192 isolates) were typed by pulsed-field gel electrophoresis (PFGE) for epidemiological tracing studies. For PFGE, a modification of the Campynet method was used, which was standardized in a European Union project. The goal of the study was to trace flock-related Campylobacter clones through the whole production chain, from farm through slaughter to retail products, to investigate the introduction of thermophilic Campylobacter spp. on incoming contaminated carcasses during processing to the final products. The results of this study showed that identical clones of this pathogen, which had previously been found within the flocks during primary production, were also detected at individual stages of processing, including final products, which were packed and ready for sale. Most of the detected clones dominated during primary production and at slaughter. This study found PFGE to be suitable for examining epidemiological field data in the same region and time contexts. The discriminatory power of SmaI restriction enzyme digestion was sufficient. Relationships of the isolated Campylobacter strains could be confirmed by use of a second restriction enzyme, KpnI.

Influence of inoculation levels and processing parameters on the survival of Campylobacter jejuni in German style fermented turkey sausages.

This study investigated the influence of inoculum levels and manufacturing methods on the survival of Campylobacter (C.) jejuni in raw fermented turkey sausages. Sausages were prepared and inoculated with C. jejuni. After inoculation, these sausages were processed and ripened for 8 days. Samples were taken throughout the ripening process. The presence of C. jejuni was established bacteriologically. Additionally, lactic acid bacteria were enumerated, pH values and water activity were measured to verify the ripening process. To detect changes in genotype and verify the identity of the recovered clones, AFLP analysis was carried out on the re-isolated strains. Whereas no C. jejuni were detectable when inoculating the sausages with the lowest inoculum (0.08-0.44 log(10) cfu/g sausage emulsion), C. jejuni were detectable for 12-24h by enrichment when inoculated with approximately 2 log(10) cfu/g. After inoculation with 4 and 6 log(10) cfu/g respectively, C. jejuni were detectable without enrichment for 12-48 h and by enrichment for 144 h at the most. The greatest decrease of the C. jejuni population occurred during the first 4 h of ripening. Only a very high inoculum level allowed the survival of the organism during a fermentation process and during ripening to pose a potential risk for consumers. Lower initial Campylobacter inoculums will be eliminated during proper ripening of the sausages, if sufficient decrease in water activity and pH-value is ensured.

[Multiplex PCR in the framework of an enterococci monitoring project]. / Multiplex-PCR im Rahmen eines Enterokokken-Monitoring Projektes.

A multiplex-PCR (MPCR) assay was optimised in the framework of an enterococci monitoring project in order to accelerate the previous time-consuming biochemical testing of enterococcal strains isolated from animal materials and food. The MPCR was optimised by variation of annealing temperature, MgCl2-concentration but more over by primer combinations and primer concentrations. Reference strains were used during the optimization. Afterwards 522 enterococcal field strains were examined by the MPCR. Ambiguous results occurred for 20 strains (17 with false-positive and 3 with false-negative results) which could be explained by the minor specificity ("EM"-primers) and sensitivity (ddlE.faecium-primers) respectively. Differences also existed between the genus-specific primers "E" and "Ent". The first mentioned primers showed weaknesses for the identification of enterococci whereas the use of the "Ent" primers inhibited other primers in the MPCR. However, the majority of the examined strains could be detected unambiguously, so that a differentiation of enterococcal isolates can be recommended by MPCR in contrast to the high time-, material- and work-consumption of conventional biochemical testings.

Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method.

A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole broiler carcass-rinse resulted in a detection limit of less than 5 cells per 25 g meat or 100 ml broiler rinse. A total of 435 samples from four countries, including pig carcass swabs (n = 285), whole broiler carcass-rinse (n = 25), various raw meat (n = 33), and environmental samples (n = 92) were investigated. The interlaboratory diagnostic accuracy, i.e. diagnostic specificity and sensitivity, was shown to be 97.5%. The co-amplification of an internal amplification control indicated possible inhibitory substances derived from the sample. This work can contribute to the quality assurance of PCR-based diagnostic methods and is currently proposed as international standard document.

Species identification and detection of vancomycin resistance genes in enterococci of animal origin by multiplex PCR.

A multiplex PCR assay for the differentiation of species and detection of vancomycin (van) resistance genes in enterococci was established. Three hundred sixty-seven enterococcal strains isolated from food were selected from a sample collection and were examined with regards to the existence of four vancomycin resistance genes (vanA, vanB, vanC1 and vanC2) and to species differentiation (E. faecalis, E. faecium, E. casseliflavus and E. gallinarum) by multiplex PCR. Apart from unambiguous results for the majority of strains, the E. faecium specific primers used here, showed weak points in the specificity and sensitivity, respectively. Despite these rare instances, we succeeded in optimising the multiplex PCR assay by variation of annealing temperature and primer combination. The assay presented here, with its optimised parameters, is therefore suitable for routine use and because of time and material saving, it is an alternative and rapid method in comparison to conventional tests.

Species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in Germany.

Presently, enterococci take the third place of bacterial pathogens associated with nosocomial infections, after staphylococci and Escherichia coli. Especially, the resistances of enterococci to several available antibiotics are threatening. We attempted to determine which species of enterococci could be found in food of animal origin and their significance according to their antibiotic resistances for human beings. From November 2000 to May 2002 we investigated 155 samples of food of animal origin bought in retail outlets in Germany: 27 samples of sausages, 19 of ham, 83 of minced meat, 26 of cheese. From these food samples we isolated 416 enterococcal strains. The most frequent species was Enterococcus faecalis (299 strains); furthermore, we found Enterococcus faecium (54 strains), Enterococcus durans together with Enterococcus hirae (24 strains), Enterococcus casseliflavus (22 strains), Enterococcus avium (9 strains) and Enterococcus gallinarum (8 strains). We focused on the resistance patterns of 118 selected E. faecium and E. faecalis strains to 13 antimicrobial active agents (ampicillin, amoxicillin/clavulanic acid, avilamycin, chloramphenicol, enrofloxacin, erythromycin, flavomycin, gentamicin, penicillin, quinupristin/dalfopristin, teicoplanin, tetracycline and vancomycin). From the clinical point of view, the situation of antibiotic resistance to the examined antimicrobial agents seemed to be favourable. The investigated strains were sensitive to ampicillin and amoxicillin/clavulanic acid. These antibiotics are, in combination with an aminoglycoside, for example gentamicin, agents of choice for the treatment of enterococcal infections in human medicine. Only one E. faecium strain was resistant to penicillin, while all strains were sensitive to the glycopeptide antibiotics, vancomycin and teicoplanin. Resistances found against the antibiotics, tetracycline, quinupristin/dalfopristin and erythromycin, are causes for concern.