Prevalence of extended-spectrum and AmpC ß-lactamase-producing Escherichia coli in Dutch dairy herds.

This study was conducted to assess: (1) a change in between-herd prevalence of extended-spectrum and AmpC ß-lactamase-producing Escherichia coli (ESBL/AmpC-EC) between 2011 and 2013, the period during which the antimicrobial policy in animal husbandry in the Netherlands changed significantly, and (2) the prevalence of ESBL/AmpC-EC in individual calves, young stock, and dairy cows in the Netherlands. In 196 randomly selected conventional dairy herds, faecal samples were collected from calves (maximum n = 15), and randomly selected young stock (n = 5) and dairy cows (n = 15). Additionally, fresh faecal samples were collected from five different places on the floors where the dairy cows were housed. Samples were screened for E. coli with non-wild type susceptibility for cefotaxime and isolates were phenotypically confirmed as ESBL/AmpC-producing by disc diffusion, using cefotaxime and ceftazidime with and without clavulanic acid, and cefoxitin. Samples containing ESBL/AmpC-EC were examined semi-quantitatively. In 59.6% of the dairy herds one or more samples tested positive for ESBL/AmpC-EC. The between-herd prevalence based on floor samples in 2013 (18.0%) was significantly lower than the prevalence in 2011 based on comparable samples (32.7%). The individual animal prevalence of ESBL/AmpC-EC, with a minimum shedding level of 103 cfu/g of faeces, was 19.3% in calves, 0.9% in young stock, and 0.8% in dairy cows. Although ESBL/AmpC-EC was found in the majority of dairy herds, the herd prevalence declined significantly between 2011 and 2013. Calves were found to have both, a much higher individual animal prevalence and a higher level of shedding than young stock and cows.

Agreement between four commercial diagnostic tests and routine bacteriological culture of milk to determine the udder infection status of dairy cows.

Mastitis is usually treated based on clinical signs or somatic cell count information rather than on results of bacteriological culture of milk. In many countries an optimal mastitis treatment is considered important from the perspective of therapy efficacy, prudent antimicrobial use and farm economics. Farmers can optimize their mastitis treatment decisions if they know whether and which mastitis pathogen is involved. Information on the mastitis pathogen involved can be acquired from diagnostic mastitis tests such as culture-based tests. This study aimed to determine the agreement of four commercial culture-based mastitis tests with routine bacteriological culture of milk to determine the intramammary infection status of a quarter or cow. The commercial culture-based tests evaluated in this study were CHROMagar Mastitis (CHROMagar, France), Hardy Diagnostics Mastitis Triplate (Hardy Diagnostics, USA), Minnesota Easy Culture System II Tri-plate (University of Minnesota, USA), and VétoRapid (Vetoquinol, the Netherlands). We used 866 prospectively collected milk samples, routinely submitted to the bacteriological laboratory of GD Animal Health for routine bacteriological culture of milk from April to June 2016. Samples were cultured on routine bacteriological culture of milk and on the commercial culture-based tests. We calculated the agreement beyond chance of each commercial culture-based test result with the result of routine bacteriological culture using 2x2 contingency tables. Furthermore, inter-reader agreement was determined for 597 samples read by two masked readers. The agreement of the four commercial culture-based mastitis tests with routine bacteriological culture of milk for Gram-positive bacteria ranged from 0.14 (95% CI 0.11-0.16) using Hardy Diagnostics Mastitis Triplate to 0.25 (95% CI 0.22-0.28) using Minnesota Easy Culture System II Tri-plate. The agreement for Gram-negative bacteria was approximately 0.70 (95% CI 0.66-0.74) for all four commercial culture-based tests. The agreement for no growth ranged from 0.22 (95% CI 0.19-0.25) using Hardy Diagnostics Mastitis Triplate to 0.34 (95% CI 0.31-0.38) using VétoRapid. This category was affected by prevalence and bias as the prevalence adjusted and bias adjusted kappa ranged from 0.63 (95% CI 0.56-0.69) using CHROMagar Mastitis to 0.68 (95% CI 0.62-0.74) using Hardy Diagnostic Mastitis Triplate. Agreement between readers was almost perfect. Although only for Gram-negative bacteria a good agreement was found between commercial culture-based tests and routine bacteriological culture of milk, and further on-farm evaluations are needed to determine the effect of these findings on udder health, commercial culture-based tests are of added value to support decisions whether and how to treat cows with mastitis.

Genetic features differentiating bovine, food, and human isolates of shiga toxin-producing Escherichia coli O157 in The Netherlands.

The frequency of Escherichia coli O157 genotypes among bovine, food, and human clinical isolates from The Netherlands was studied. Genotyping included the lineage-specific polymorphism assay (LSPA6), the Shiga-toxin-encoding bacteriophage insertion site assay (SBI), and PCR detection and/or subtyping of virulence factors and markers [stx1, stx(2a)/stx(2c), q21/Q933, tir(A255T), and rhsA(C3468G)]. LSPA6 lineage II dominated among bovine isolates (63%), followed by lineage I/II (35.6%) and lineage I (1.4%). In contrast, the majority of the human isolates were typed as lineage I/II (77.6%), followed by lineage I (14.1%) and lineage II (8.2%). Multivariate analysis revealed that the tir(A255T) SNP and the stx(2a)/stx(2c) gene variants were the genetic features most differentiating human from bovine isolates. Bovine and food isolates were dominated by stx(2c) (86.4% and 65.5%, respectively). Among human isolates, the frequency of stx(2c) was 36.5%, while the frequencies of stx(2a) and stx(2a) plus stx(2c) were 41.2% and 22.4%, respectively. Bovine isolates showed equal distribution of tir(255A) (54.8%) and tir(255T) (45.2%), while human isolates were dominated by the tir(255T) genotype (92.9%). LSPA6 lineage I isolates were all genotype stx(2c) and tir(255T), while LSPA6 lineage II was dominated by tir(255A) (86.4%) and stx(2c) (90.9%). LSPA6 lineage I/II isolates were all genotype tir(255T) but showed more variation in stx(2) types. The results support the hypothesis that in The Netherlands, the genotypes primarily associated with human disease form a minor subpopulation in the bovine reservoir. Comparison with published data revealed that the distribution of LSPA6 lineages among bovine and human clinical isolates differs considerably between The Netherlands and North America.

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.

Consumption of fresh fruit juice: how a healthy food practice caused a national outbreak of Salmonella Panama gastroenteritis.

In spring 2008, 15 Salmonella Panama laboratory-confirmed cases were reported within 2 weeks, twice the average annual number of reported cases of this infrequent serotype in The Netherlands. To identify the source responsible for this national outbreak, we carried out an epidemiological, microbiological, and trace-back investigation. In total, 33 cases were reported, and a matched case-control study (23 cases/24 controls) identified consumption of fresh (unpasteurized) fruit juice purchased from a large retailer (X) as the only significant risk factor for illness (matched odds ratio: 7.4, 95% confidence interval: 1.5-37.2). Though the bacterium could not be isolated from fruit juice, the minimal pH value for growth of the causative strain of the outbreak (3.4) was compatible with survival in fruit juice from X. The outbreak strain showed acid resistance and adaptive properties that may explain how it could have caused infection through fresh orange juice. To our knowledge, this is the first documented outbreak related to fresh fruit juice consumption in western Europe since 1922. A growing number of consumers who are seeking healthy food practices are exposed to the infectious risks related to unpasteurized fresh fruit juice. Labeling regulations should be adapted to properly indicate to the consumers that unpasteurized fresh fruit juices remain vulnerable to microbial contamination. Frequent microbiological screening and strict compliance with food safety procedures should reduce the infectious hazards of fresh fruit juices.

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.

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.

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.

Development, validation, and standardization of polymerase chain reaction-based detection of E. coli O157.

A diagnostic polymerase chain reaction assay was developed for the detection of E. coli O157 as the first part of a multicenter validation and standardization project. The assay is based on amplification of sequences of the rfbE O157 gene and includes an internal amplification control. The selectivity of the assay was evaluated against 155 strains, including 32 E. coli O157, 38 E. coli non-O157, and 85 non-E. coli. It was shown to be highly inclusive (100%) and exclusive (100%). The assay has a 100% detection probability of approximately 2 x 10(3) cells per reaction.

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.