Validation by an interlaboratory collaborative trial of EN ISO 21528 - microbiology of the food chain - horizontal methods for the detection and enumeration of Enterobacteriaceae.

The methods for the detection and enumeration of Enterobacteriaceae, described in EN ISO 21528, parts 1 and 2, were validated by order of the European Commission under the mandate M/381. Fourteen laboratories from seven European countries participated in the collaborative trials, organized by The Netherlands Food and Consumer Product Safety Authority (Wageningen/Utrecht, The Netherlands). Five different matrices from different food categories were selected to be tested in the collaborative trials, in order to validate the method horizontally, according to ISO 16140. The matrices included meat, tiramisu, infant formula, liquid egg, smoked salmon (detection method only) and animal feed (enumeration method only). The raw meat and liquid egg were naturally contaminated and the other matrices were artificially contaminated with a cocktail of four different Enterobacteriaceae strains. The samples used in the trial were tested for homogeneity and stability before distribution. The method for detection of Enterobacteriaceae showed a specificity and sensitivity above 95% for all matrices. The method for the enumeration had a repeatability limit r of 0.37 (expressed as a difference between log10-transformed test results) and a reproducibility limit R of 0.87 (expressed as a difference between log10-transformed test results). The validation data were incorporated in the newly published ISO standards EN ISO 21528:2017- Microbiology of the food chain - Horizontal methods for the detection and enumeration of Enterobacteriaceae - Part 1: Detection method, and Part 2: Colony-count technique.

Validation by interlaboratory trials of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique.

The validation in an interlaboratory study of the International Standards Organization standard method for the enumeration of Campylobacter in foods (ISO 10272-2) was performed after preparation of the revised Standard based on scientifically sound and validated methods of analysis. The matrices selected for testing in the collaborative trial were frozen spinach, minced meat, raw milk, chicken skin, and broiler caecal material. Each matrix was artificially inoculated with a different Campylobacter strain. Fifteen laboratories participated in the interlaboratory study. As a general indication of repeatability limit (r), the following overall values can be used when testing chicken skin samples: As a general indication of reproducibility limit (R), the following overall values can be used when testing chicken skin samples: The validation data for all matrices were incorporated in the newly published ISO standard EN ISO 10272-2:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 2: colony-count technique.

Validation by interlaboratory trials of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method.

During the last decade Campylobacter has been the most commonly reported gastrointestinal bacterial infection in humans in the European Union. The use of a sensitive detection method based on enrichment of Campylobacter spp. is often needed when examining foods. However, as background flora developed resistance to third generation ß-lactams used in selective culture media, the ISO method was adapted. It now consists of three different procedures (A, B, and C) depending on the expected concentration and condition of Campylobacter and the background microflora. As the diagnostic sensitivity of the detection test varies between laboratories, this justifies the validation of the method in an interlaboratory study. The matrices selected for testing in the collaborative trials were frozen spinach (procedure A, Bolton enrichment broth), minced meat (procedure A, Bolton enrichment broth), raw milk (procedure B, Preston enrichment broth), chicken skin (procedure B, Preston enrichment broth), and broiler caecal material (procedure C, direct plating on mCCD agar). Each matrix was artificially inoculated with a different Campylobacter strain at a low and high contamination level, and with sterile diluent for 'blanks'. Seventeen laboratories participated in the interlaboratory study. The sensitivity and specificity of the methods for the five selected matrices were determined, as well as the level of detection (LOD50). Calculated LOD50 values ranged from 0.84 cfu/test portion in frozen spinach and 2.2 cfu/test portion in minced meat to 14 cfu/test portion in chicken skin and 57 cfu/test portion in raw milk, all based on test portions of 10 g. The test portion size for broiler caecal material was a 10 µl-loop, yielding a LOD50 of 6.1 cfu/test portion. The validation data were incorporated in the newly published ISO standard EN ISO 10272-1:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 1: Detection method.

Evidence for Human Adaptation and Foodborne Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

Prevalence of Clostridium difficile in retailed meat in the Netherlands.

Recent reports indicate that a large proportion of community-acquired Clostridium difficile infections (CA-CDI) are not linked to recent antibiotic therapy, older age, significant comorbidity or previous hospitalization. Possible community sources for CA-CDI include animals and food, and therefore a surveillance study on the prevalence of C. difficile in meat was performed. Samples of different meat species were collected from the retail trade and analyzed for the presence of C. difficile using a method that included selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping. Of 500 samples tested, 8 (1.6%) were positive for the presence of C. difficile: 1 from lamb (6.3%) and 7 from chicken meat (2.7%). The isolated strains belonged to PCR ribotypes different from those that are currently most frequently found in patients with CDI in the Netherlands, except for C. difficile PCR ribotype 001 which was found in one chicken meat sample. This observation suggests that other matrices than meat may serve as a source for CA-CDI.

Occurrence and characterization of Shiga toxin-producing Escherichia coli in raw meat, raw milk, and street vended juices in Bangladesh.

The major objective of this study was to investigate the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in different types of food samples and to compare their genetic relatedness with STEC strains previously isolated from animal sources in Bangladesh. We investigated a total of 213 food samples, including 90 raw meat samples collected from retail butcher shops, 20 raw milk samples from domestic cattle, and 103 fresh juice samples from street vendors in Dhaka city. We found that more than 68% (n = 62) of the raw meat samples were positive for the stx gene(s); 34% (n = 21) of buffalo meats and 66% (n = 41) of beef. Approximately 10% (n = 2) of the raw milk and 8% (n = 8) of the fresh juice samples were positive for stx. We isolated STEC O157 from seven meat samples (7.8%), of which two were from buffalo meats and five from beef; and no other STEC serotypes could be isolated. We could not isolate STEC from any of the stx-positive raw milk and juice samples. The STEC O157 isolates from raw meats were positive for the stx(2), eae, katP, etpD, and enterohemorrhagic E. coli hly virulence genes, and they belonged to three different phage types: 8 (14.3%), 31 (42.8%), and 32 (42.8%). Pulsed-field gel electrophoresis (PFGE) typing revealed six distinct patterns among seven isolates of STEC O157, suggesting a heterogeneous clonal diversity. Of the six PFGE patterns, one was identical and the other two were ≥90% related to PFGE patterns of STEC O157 strains previously isolated from animal feces, indicating that raw meats are readily contaminated with fecal materials. This study represents the first survey of STEC in the food chain in Bangladesh.

Two outbreaks of campylobacteriosis associated with the consumption of raw cows' milk.

The present paper summarises the investigation of two different outbreaks of milk-associated Campylobacter enteritis in the Netherlands. In 2005, after a school trip to a dairy farm, 22 out of a group of 34 children developed diarrhoeal illness and Campylobacterjejuni was cultured from the stool samples of 11 of the cases. The illness was found to be epidemiologically associated with drinking raw milk during the farm visit; 86% of the cases could be explained by drinking raw milk. C.jejuni was also isolated from three of 10 faecal samples from dairy cattle collected at the farm. The human isolates and C.jejuni isolates from one of these three samples of cattle faeces revealed identical restriction patterns by both pulsed-field gel electrophoresis (PFGE) and flagellin (fla) typing by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Both epidemiological and bacteriological evidence implicated contaminated raw milk as the vehicle of transmission, though C.jejuni was not isolated from the bulk tank milk or the milk filter collected during the farm investigation. In 2007, an outbreak of enteritis was notified among people who had attended a lunch at a dairy farm where bulk tank milk was served. Of the 19 persons who had consumed raw milk, 16 (84%) had become ill. Of the persons who did not drink the raw milk, none became ill. A significant association was found between tasting the raw milk and being ill (risk difference=0.84, p=0.0011). C.jejuni was cultured from four of seven cases who had submitted a stool specimen. C. jejuni was also isolated from a sample of bulk tank milk and the isolate had an identical flaA PCR-RFLP genotype to isolates obtained from patients. Also in this outbreak both the epidemiological and bacteriological findings support raw milk as the vehicle for the enteritis. These two outbreaks highlight the health risks associated with the consumption of raw milk. As long as legislation allows the sale and distribution of untreated milk these risks will continue. Therefore, consumers need to be continuously informed about the dangers inherent in consuming unpasteurised milk or products made from raw milk. Farmers need to be strongly discouraged from serving raw milk to their visitors.

Prevalence and genetic characterization of shiga toxin-producing Escherichia coli isolates from slaughtered animals in Bangladesh.

To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx(1) and/or stx(2), respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx(2), eae, katP, etpD, and enterohemorrhagic E. coli hly (hly(EHEC)) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx(1). Only 7.0% (n = 5) of the isolates were positive for hly(EHEC), and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf(O113), saa, lpfA(O157/01-141), and lpfA(O157/OI-154) genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.

An efficient and rapid method for recovery of norovirus from food associated with outbreaks of gastroenteritis.

Noroviruses have emerged as the most common cause of foodborne outbreaks of acute nonbacterial gastroenteritis. In this study, two methods for the extraction of viruses from deli ham were compared. Using both methods, as little as 1 to 10 reverse transcription (RT)-PCR units of inoculated norovirus and enterovirus could be detected by nested RT-PCR assays. The fastest and most efficient extraction method based on TRIzol LS Reagent was chosen to identify viruses in food items associated with three different outbreaks. Norovirus was detected using nested (real time) RT-PCR assays that target the genome region routinely used for diagnosis of human cases, thereby facilitating the comparison of sequences detected in food and clinical specimens. For one outbreak, a norovirus sequence (163/163 nucleotides) identical to those detected in clinical samples was found on salami sliced by a food handler with a recent history of gastroenteritis. For the other two outbreaks, norovirus was detected on leftovers of spareribs and ham, but fecal samples from affected persons were not available. The methods used in this study may be useful in future outbreak investigations because the extraction method is easy to perform and suitable for this particular type of food and the detection method facilitates direct comparison of patient and food data.

Evaluation of immunomagnetic separation and PCR for the detection of Escherichia coli O157 in animal feces and meats.

Series of animal feces and meat samples artificially contaminated with strains of Escherichia coli O157 isolated from different sources were tested by both an immunomagnetic separation (IMS)-based method and a PCR method using primers specific for a portion of the rfbE gene of E. coli O157. IMS is laborious and time consuming but ends up with the isolation of the pathogen. PCR is fast and less laborious, but it can only be used for screening purposes, so a further culture step is required to isolate the organism. For both fecal and meat samples, the IMS method was found to be more sensitive than the PCR. Furthermore, the detection efficiency of the PCR was influenced by the origin of the fecal sample and the type of meat. For sheep feces, the efficiency of the PCR appeared to be systematically lower than for cattle feces. And the efficiency of the PCR in detecting E. coli O157 in spiked samples of raw minced beef and dry-fermented sausages was systematically lower than in samples of filet americain. Based on this study, it can be concluded that both for animal feces and meat, IMS can be used more successfully to detect E. coli O157 than PCR, because IMS showed to be more sensitive and the outcome was not influenced by the type of animal feces or meat.

Detection of noroviruses in shellfish in the Netherlands.

Shellfish from oyster farms in the Netherlands and imported from other European countries were examined for viral contamination. A method that allows sequence matching between noroviruses from human cases and shellfish was used. The samples of shellfish (n = 42) were analyzed using a semi-nested RT-PCR that had been optimized for detection of norovirus in shellfish (SR primer sets). In addition, a different genome region was targeted using a second primer set which is routinely used for diagnosis of norovirus infection in humans (JV12Y/JV13I). To improve the detection limit for this RT-PCR a semi-nested test format was developed (NV primer sets). One of 21 oyster samples (4.8%) from Dutch farms was norovirus positive, whereas norovirus was detected in 1 out of 8 oyster samples (12.5%) and 5 out of 13 mussel samples (38.5%) collected directly after importation in the Netherlands. RNA from samples associated with an outbreak of gastro-enteritis in the Netherlands in 2001 was re-analyzed using the NV primer sets. At least one identical sequence (142/142 nt) was found in three fecal and in two oyster samples related to this outbreak. Further surveillance of norovirus by detection and typing of viruses from patients with gastroenteritis and shellfish is warranted to clarify the causes of future outbreaks.

Immunoconcentration of Shiga toxin-producing Escherichia coli O157 from animal faeces and raw meats by using Dynabeads anti-E. coli O157 and the VIDAS system.

To identify the reservoirs and routes of transmission of Shiga toxin-producing Escherichia coli (STEC) O157, sensitive detection and isolation methods are necessary. The sensitivity of traditional culture methods can be improved significantly by the inclusion of an immunoconcentration step, resulting in less false-negatives. In this report, we evaluated the results of two commercially available test systems: Dynabeads anti-E. coli O157 and the Vitek Immunodiagnostic Assay System (VIDAS) Immuno-Concentration E. coli O157 (ICE) kit. Additionally, we compared two selective isolation media for STEC O157. Statistical analysis of the results obtained for animal faecal samples (n=637) examined by both immunoconcentration methods showed that by the manual Dynabeads anti-E. coli O157 procedure systematically more samples were identified as positive than by the VIDAS ICE. In case of meat samples (n=360), no difference between the results of the two methods was found. In addition to being accurate, the Dynabeads anti-E. coli O157 method is a less expensive method than the VIDAS ICE. But, the Dynabeads method is laborious and there is a risk of cross-contamination. The VIDAS ICE procedure on the other hand is fully automated with a standardised performance; fast and safe for the user. Irrespective of the type of sample (faeces or meat) and the immunoconcentration technique applied (Dynabeads anti-E. coli O157 or VIDAS ICE) more samples were found positive after plating onto CHROMagar O157 with cefixime (0.025 mg l(-1)) and tellurite (1.25 mg l(-1)) than after plating onto sorbitol-MacConkey agar with cefixime (0.05 mg l(-1)) and tellurite (2.5 mg l(-1)). However, only in case of meat samples examined by the VIDAS ICE the difference between the isolation media was not statistically significant.

Determination of streptomycin and dihydrostreptomycin in milk and honey by liquid chromatography with tandem mass spectrometry.

Two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed for the determination of streptomycin (STR) and its derivative dihydrostreptomycin (DHSTR) in milk and honey. These aminoglycoside antibiotics are used as veterinary drugs. In the EU, the presence of dihydro- and streptomycin residues in honey is forbidden, the maximum residue level (MRL) in milk is 200 microg/kg. The methods were optimised with regard to sensitivity and chromatographic efficiency, and validated by a procedure consistent with EU directive 2002/657. Average recoveries and accompanying standard deviations were satisfactory. The limit of quantification of STR was 2 microg/kg in honey and 10 microg/kg in milk, of DHSTR it was a factor two lower. The precision of the milk analysis was improved by using STR as the internal standard for DHSTR and vice versa. In a survey of 186 honeys available on the Dutch market, 26% of the honeys of foreign origin were positive for (DH)STR. This occurence rate was consistent with previous surveys, but lower concentrations were found.

Evaluation of Media and Test Kits for the Detection and Isolation of Escherichia coli O157 from Minced Beef.

This study has evaluated the efficacy of selective enrichment and plating media used for the isolation of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157, by examining pure bacterial cultures. In addition, the performance of a variety of commercial test kits for the detection of E. coli O157 strains inoculated into minced beef was compared, using the Ampcor E. coli O157:H7 Kit, 3M Petrifilm™ Test Kit-HEC, Dynabeads anti- E coli O157, EHEC-TEK™, and the Tecra E. coli O157 visual immunoassay. The commercial Verotox F test for the determination of the VT type of VTEC isolates was compared with a polymerase chain reaction (PCR) assay for VT-coding genes. Modified E. coli broth containing novobiocin (mEC + n) and sorbitol MacConkey agar supplemented with cefixime and tellurite (CT-SMAC) were the most efficacious media for selective enrichment and isolation, respectively. After enrichment of the inoculated samples, all kits tested could detect less than one O157 VTEC cell per g of minced beef. While the results of the immunoassays need to be confirmed by isolating the organisms, the use of the immunomagnetic separation technique directly yields isolates. The results of the Verotox F test were consistent with PCR results. A sensitive and cost-effective method for the isolation of O157 VTEC from minced beef in food industry and epidemiological studies involving large numbers of samples is the following: enrichment in mEC + n at 37°C for 6 to 8 h with shaking at 100 rpm, followed by immunomagnetic separation using Dynabeads anti- E. coli O157 and spread plating of the concentrated target cells onto CT-SMAC. The Verotox F test can be used to determine whether the isolates produce VTl and/or VT2.

Occurrence of Escherichia coli O157 and Other Verocytotoxin-Producing E. coli in Retail Raw Meats in the Netherlands.

Raw meats obtained from retail outlets in the Netherlands were examined for the presence of Escherichia coli of serogroup O157 and other verocytotoxin (VT)-producing E. coli (VTEC), in three different surveys. In the first survey O157 VTEC were detected and isolated by selective plating onto sorbitol MacConkey agar following selective enrichment in modified tryptone soy broth with acriflavin. The organisms were isolated from 2 (0.3%) of 770 samples of minced mixed beef and pork, but not detected in samples of raw minced beef (n = 1,000), minced pork (n = 260), or poultry products (n = 300). In the second survey an additional 360 raw meats were examined with the 3M Petrifilm™ Test Kit-HEC, after selective enrichment in modified E. coli broth containing novobiocin. VT-negative E. coli O157 strains were isolated from 22 (6.1%) samples. In the third survey 180 enrichment cultures of the first survey were screened for the presence of VT1 and VT2 genes with a polymerase chain reaction (PCR). Twenty-nine (16.1%) of the 180 enrichment cultures showed a positive PCR: one for the VT1 gene only, 17 for the VT2 gene only, and 11 for both the VT1 and VT2 gene. A total of 46 VTEC strains were isolated from 10 randomly selected PCR-positive samples. Serotyping revealed that 41 of the 46 VTEC isolates belonged to nine different O serogroups; the remaining five were unidentifiable. A number of the serogroups recovered have been associated with human disease.

Cross-contamination with Campylobacter jejuni and Salmonella spp. from Raw Chicken Products During Food Preparation.

Campylobacter jejuni was isolated from 170 (61%) of 279 samples of chicken products and Salmonella from 44 (54%) of 81 samples. Cross-contamination experiments showed that C. jejuni and to a lesser extent Salmonella were easily transferred from raw chicken products to cutting-boards, plates, and hands. These organisms were also isolated from raw vegetables and cooked chicken products, which were in contact with plates on which raw chicken products had been placed. Measures for the prevention of infection by cross-contamination through handling of raw chicken are mentioned.