Outbreak of Salmonella Bovismorbificans associated with the consumption of uncooked ham products, the Netherlands, 2016 to 2017.

In January 2017, an increase in reported Salmonellaenterica serotype Bovismorbificans cases in the Netherlands was observed since October 2016. We implemented a case-control study to identify the source, including all cases after December 2016. Adjusted odds ratios were calculated using logistic regression analysis. We traced back the distribution chain of suspected food items and sampled them for microbiological analysis. Human and food isolates were sequenced using whole genome sequencing (WGS). From October 2016 to March 2017, 54 S. Bovismorbificans cases were identified. Sequencing indicated that all were infected with identical strains. Twenty-four cases and 37 controls participated in the study. Cases were more likely to have consumed ham products than controls (aOR = 13; 95% CI: 2.0-77) and to have shopped at a supermarket chain (aOR = 7; 95% CI: 1.3-38). Trace-back investigations led to a Belgian meat processor: one retail ham sample originating from this processor tested positive for S. Bovismorbificans and matched the outbreak strain by WGS. All ham products related to the same batch were removed from the market to prevent further cases. This investigation illustrates the importance of laboratory surveillance for all Salmonella serotypes and the usefulness of WGS in an outbreak investigation.

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015.

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data.

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.

Increase in reptile-associated human salmonellosis and shift toward adulthood in the age groups at risk, the Netherlands, 1985 to 2014.

While the contribution of the main food-related sources to human salmonellosis is well documented, knowledge on the contribution of reptiles is limited. We quantified and examined trends in reptile-associated salmonellosis in the Netherlands during a 30-year period, from 1985 to 2014. Using source attribution analysis, we estimated that 2% (95% confidence interval: 1.3-2.8) of all sporadic/domestic human salmonellosis cases reported in the Netherlands during the study period (n = 63,718) originated from reptiles. The estimated annual fraction of reptile-associated salmonellosis cases ranged from a minimum of 0.3% (corresponding to 11 cases) in 1988 to a maximum of 9.3% (93 cases) in 2013. There was a significant increasing trend in reptile-associated salmonellosis cases (+ 19% annually) and a shift towards adulthood in the age groups at highest risk, while the proportion of reptile-associated salmonellosis cases among those up to four years-old decreased by 4% annually and the proportion of cases aged 45 to 74 years increased by 20% annually. We hypothesise that these findings may be the effect of the increased number and variety of reptiles that are kept as pets, calling for further attention to the issue of safe reptile-human interaction and for reinforced hygiene recommendations for reptile owners.

Assessing the public health risk of Shiga toxin-producing Escherichia coli by use of a rapid diagnostic screening algorithm.

Shiga toxin-producing Escherichia coli (STEC) is an enteropathogen of public health concern because of its ability to cause serious illness and outbreaks. In this prospective study, a diagnostic screening algorithm to categorize STEC infections into risk groups was evaluated. The algorithm consists of prescreening stool specimens with real-time PCR (qPCR) for the presence of stx genes. The qPCR-positive stool samples were cultured in enrichment broth and again screened for stx genes and additional virulence factors (escV, aggR, aat, bfpA) and O serogroups (O26, O103, O104, O111, O121, O145, O157). Also, PCR-guided culture was performed with sorbitol MacConkey agar (SMAC) and CHROMagar STEC medium. The presence of virulence factors and O serogroups was used for presumptive pathotype (PT) categorization in four PT groups. The potential risk for severe disease was categorized from high risk for PT group I to low risk for PT group III, whereas PT group IV consists of unconfirmed stx qPCR-positive samples. In total, 5,022 stool samples of patients with gastrointestinal symptoms were included. The qPCR detected stx genes in 1.8% of samples. Extensive screening for virulence factors and O serogroups was performed on 73 samples. After enrichment, the presence of stx genes was confirmed in 65 samples (89%). By culture on selective media, STEC was isolated in 36% (26/73 samples). Threshold cycle (CT) values for stx genes were significantly lower after enrichment compared to direct qPCR (P < 0.001). In total, 11 (15%), 19 (26%), 35 (48%), and 8 (11%) samples were categorized into PT groups I, II, III, and IV, respectively. Several virulence factors (stx2, stx2a, stx2f, toxB, eae, efa1, cif, espA, tccP, espP, nleA and/or nleB, tir cluster) were associated with PT groups I and II, while others (stx1, eaaA, mch cluster, ireA) were associated with PT group III. Furthermore, the number of virulence factors differed between PT groups (analysis of variance, P < 0.0001). In conclusion, a diagnostic algorithm enables fast discrimination of STEC infections associated with a high to moderate risk for severe disease (PT groups I and II) from less-virulent STEC (PT group III).

Extended-spectrum ß-lactamase-producing Escherichia coli from retail chicken meat and humans: comparison of strains, plasmids, resistance genes, and virulence factors.

BACKGROUND: The worldwide prevalence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is increasing rapidly both in hospitals and in the community. A connection between ESBL-producing bacteria in food animals, retail meat, and humans has been suggested. We previously reported on the genetic composition of a collection of ESBL-producing Escherichia coli (ESBL-EC) from chicken meat and humans from a restricted geographic area. Now, we have extended the analysis with plasmid replicons, virulence factors, and highly discriminatory genomic profiling methods. METHODS: One hundred forty-five ESBL-EC isolates from retail chicken meat, human rectal carriers, and blood cultures were analyzed using multilocus sequence typing, phylotyping, ESBL genes, plasmid replicons, virulence genes, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE). RESULTS: Three source groups overlapped substantially when their genetic composition was compared. A combined analysis using all variables yielded the highest resolution (Wilks lambda [Λ]: 0.08). Still, a prediction model based on the combined data classified 40% of the human isolates as chicken meat isolates. AFLP and PFGE showed that the isolates from humans and chicken meat could not be segregated and identified 1 perfect match between humans and chicken meat. CONCLUSIONS: We found significant genetic similarities among ESBL-EC isolates from chicken meat and humans according to mobile resistance elements, virulence genes, and genomic backbone. Therefore, chicken meat is a likely contributor to the recent emergence of ESBL-EC in human infections in the study region. This raises serious food safety questions regarding the abundant presence of ESBL-EC in chicken meat.

Livestock density as risk factor for livestock-associated methicillin-resistant Staphylococcus aureus, the Netherlands.

To determine whether persons living in areas of high animal density are at increased risk for carrying livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), we used an existing dataset of persons in the Netherlands with LA-MRSA carriage and controls who carried other types of MRSA. Results of running univariate and multivariate logistic regression models indicated that living in livestock-dense areas increases the odds of nasal carriage of LA-MRSA. We found that doubling pig, cattle, and veal calf densities per municipality increased the odds of LA-MRSA carriage over carriage of other types of MRSA by 24.7% (95% CI 0.9%-54.2%), 76.9% (95% CI 11.3%-81.3%), and 24.1% (95% CI 5.5%-45.9%), respectively, after adjusting for direct animal contact, living in a rural area, and the probable source of MRSA carriage. Controlling the spread of LA-MRSA thus requires giving attention to community members in animal-dense regions who are unaffiliated with livestock farming.

A regional Salmonella enterica serovar Typhimurium outbreak associated with raw beef products, The Netherlands, 2010.

Between April and May 2010, several medical microbiological laboratories in the Netherlands notified a total of 90 cases of Salmonella enterica serovar Typhimurium with the same antibiogram type (resistant for ampicillin, tetracycline, and co-trimoxazol) and the same multiple locus variable number tandem repeats analysis pattern (03-16-09-NA-311) or single locus variants. Date of illness onset ranged from end of March to mid-May with a peak in the second week of April. Almost half of the cases were hospitalized. Cases completed a questionnaire about food items and other risk factors in the 7 days before illness onset. A matched case-control study was performed. Consumption of "ossenworst" (matched odds ratio 48.2 [95% confidence interval (CI): 3.9-595.9]) and filet américain (8.5 [95% CI: 1.0-73.6]) were found to be significant risk factors for illness. Eighty percent of the cases had eaten at least one or both raw meat products. The producer of the ground beef that was used to produce the "ossenworst" was identified, but no microbiological evidence was found. Consumers should be made more aware of the presence of raw meat in ready-to-eat products and of the potential risk in eating these products. Vulnerable persons such as young children, elderly, and persons with poor health should be discouraged from eating these products. Detection of this outbreak was mainly based on the antibiogram pattern that had identified possible cases 10 days before detailed typing results from the reference laboratory became available, thus facilitating early case findings.

Extended-spectrum ß-lactamase genes of Escherichia coli in chicken meat and humans, The Netherlands.

We determined the prevalence and characteristics of extended-spectrum ß-lactamase (ESBL) genes of Enterobacteriaceae in retail chicken meat and humans in the Netherlands. Raw meat samples were obtained, and simultaneous cross-sectional surveys of fecal carriage were performed in 4 hospitals in the same area. Human blood cultures from these hospitals that contained ESBL genes were included. A high prevalence of ESBL genes was found in chicken meat (79.8%). Genetic analysis showed that the predominant ESBL genes in chicken meat and human rectal swab specimens were identical. These genes were also frequently found in human blood culture isolates. Typing results of Escherichia coli strains showed a high degree of similarity with strains from meat and humans. These findings suggest that the abundant presence of ESBL genes in the food chain may have a profound effect on future treatment options for a wide range of infections caused by gram-negative bacteria.

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.

Selenite enrichment broth to improve the sensitivity in molecular diagnostics of Salmonella.

Selenite enrichment broth (SEB) is used to optimize the recovery of Salmonella enterica subspecies enterica from stool samples. Compared to a direct culture approach, it enhances culture yield by reducing growth of faecal coliforms and faecal streptococci. Over the course of seven years from 2000 to 2017, 47,235 faecal samples were tested with a Salmonella PCR. We investigated the added value of using SEB in combination with faeces for DNA extraction, in order to improve the sensitivity of molecular diagnostics for detection of Salmonella. A Salmonella enterica subspecies enterica strain was tested for growth characteristics, with and without incubation in SEB, to determine the impact of Selenite enrichment in the Salmonella PCR. Retrospectively, a total of 102 Salmonella enterica subspecies enterica PCR positive faecal samples were re-analysed. DNA extraction was performed with the EasyMag® and MagNaPure96® system using three different input volumes of faeces and SEB. Prospectively, 114 Salmonella PCR positive faecal samples were retested within 2 days using five different input volumes for DNA extraction. Retrospectively, PCR that used SEB as part of input in the DNA extraction, 7/102 (7%) Salmonella PCR positive samples were additionally detected compared to no use of SEB. Of these, Salmonella enterica subspecies enterica serovariation Thompson, Enteritidis, 9,12:l.v and Senftenberg have been outbreak related in the past. Prospectively results were combined in collaboration with another microbiology laboratory, 15/114 (13.2%) additional specimens were detected with the Salmonella PCR, including processing Selenite enrichment broth. In conclusion, of the total 47,235 feacal samples, with SEB the prevalence of a positive PCR for Salmonella is 2.2%. Of these 2.2% positive Salmonella PCRs, 0.4% was not detected in culture. By using SEB an improved detection of Salmonella diagnostics could be realized and a substantial part of 13,2% additional Salmonella cases could be detected.

Evaluation of the Premi Test Salmonella, a commercial low-density DNA microarray system intended for routine identification and typing of Salmonella enterica.

A new commercial system based on genetic profiling and aimed at identifying Salmonella enterica serovars was evaluated by comparing its performance with classical serotyping on 443 strains. Within 62 serovars represented, 60 gave unique genetic profiles while 2 were undistinguishable. Results were obtained within 8 h, were reproducible and clear-cut. The system allowed single-tube processing of the samples and required no peculiar technical skill. It showed interesting potential for routine laboratory testing.

Tracing Back the Source of an Outbreak of Salmonella Typhimurium; National Outbreak Linked to the Consumption of Raw and Undercooked Beef Products, the Netherlands, October to December 2015.

INTRODUCTION: On 23 October 2015, six related cases with gastroenteritis called the Netherlands Food and Consumer Product Safety Authority. They suspected filet américain, a raw beef spread, to be the source of infection. Leftovers and stool samples tested positive for Salmonella Typhimurium. Multiple locus variable-number of tandem repeat analysis (MLVA) revealed a MLVA pattern (02-23-08-08-212), which had not been detected in the Netherlands before. Concomitantly, an increase of this MLVA type was observed in the national Salmonella surveillance, amounting to 46 cases between 26 October and 9 December. METHODS: To investigate whether filet américain or an alternative (related) source could  be linked to surveillance-reported cases, cases (n=38) were invited to complete a questionnaire and upstream source tracing to map the food supply chain was initiated. RESULTS: Rapid interdisciplinary action resulted in identification of a contaminated 46-ton batch of beef distributed via a Dutch deboning plant as the likely source of infection. In total, 24/29 respondents (83%) could be linked to the incriminated batch of beef products (predominantly filet américain and minced beef). DISCUSSION: Repeated identification of raw meat products as a source of infection emphasizes the importance of awareness of the risk of infection when handling or consuming these products. Improved measures and procedures on product labelling, pre-treatment or product testing should be considered.

Distribution of Salmonella enterica serovars from humans, livestock and meat in Vietnam and the dominance of Salmonella Typhimurium phage type 90.

Epidemiologically unrelated non-typhoid Salmonella isolates from humans (n = 56) and animal origin (n = 241, from faeces, carcasses and meat) in Vietnam were investigated. Salmonella Typhimurium, S. Anatum, S. Weltevreden, S. Emek, and S. Rissen were the most prevalent serovars. S. Typhimurium phage type 90 was predominant among S. Typhimurium isolates. The serotype and phage type distribution of the Salmonella isolates was different from that in Europe and America. Many sero- and phage types found in humans were also found in cattle, pigs, and poultry suggesting that food producing animals are an important source of human non-typhoid Salmonella infection in Vietnam.

Class 1 integrons in Dutch Salmonella enterica serovar Dublin isolates from clinical cases of bovine salmonellosis.

Fifty-nine Salmonella enterica serovar Dublin (Salmonella Dublin) isolates from clinical cases of bovine salmonellosis between 1993 and 2004 were tested for their susceptibility to 15 antimicrobial agents and the presence of class 1 integrons. Integrons were further analyzed by conserved segment PCR-RFLP. DNA sequencing was used to identify the inserted gene cassette. Twelve (20.3%) isolates were multidrug-resistant. A combination of resistance against chloramphenicol, streptomycin and sulphonamides was the most common phenotype observed. Multidrug-resistance (MDR) was found to be strongly associated with the presence of integrons, since a class 1 integron with the aadA1 gene cassette encoding resistance to streptomycin and spectinomycin was found in all 12 multidrug-resistant isolates. The presence of the aadA1 gene in Salmonella Dublin has not been reported before. None of the integron carrying Salmonella Dublin isolates could transfer its antimicrobial resistance to E. coli K12 by conjugation. Analysis of plasmid profiles and pulsed field gel electrophoresis (PFGE) patterns showed at least some clonality among the Salmonella Dublin isolates, but 11 different types could be distinguished based on both XbaI and BlnI-PFGE patterns. Thus, the Dutch Salmonella Dublin strains were closely related but not clonal.

Community-acquired MRSA and pig-farming.

BACKGROUND: Sporadic cases of CA-MRSA in persons without risk-factors for MRSA carriage are increasing. CASE PRESENTATION: We report a MRSA cluster among family members of a pig-farmer, his co-workers and his pigs. Initially a young mother was seen with mastitis due to MRSA. Six months later her baby daughter was admitted to the hospital with pneumococcal otitis. After staying five days in hospital, the baby was found to be MRSA positive. At that point it was decided to look for a possible source, such as other family members and house-hold animals, including pigs on the farm, since those were reported as a possible source of MRSA earlier. Swabs were taken from the throat and nares of family members and co-workers. A veterinarian obtained swabs from the nares, throat and perineum of 10 pigs. Swabs were cultured following a national protocol to detect MRSA that included the use of an enrichment broth. Animal and human strains were characterized by PFGE, spa-typing, MLST analysis, SSCmec, AGR typing, and the detection for PVL, LukM, and TSST toxin genes. Three family members, three co-workers, and 8 of the 10 pigs were MRSA positive. With the exception of the initial case (the mother) all persons were solely colonized, with no signs of clinical infections. After digestion with SmaI, none of the strains showed any bands using PFGE. All isolates belonged to spa type t108 and ST398. CONCLUSION: 1. This report clearly shows clonal spread and transmission between humans and pigs in the Netherlands. 2. MLST sequence type 398 might be of international importance as pig-MRSA, since this type was shown earlier to be present in epidemiologically unrelated French pigs and pig-farmers. 3. Research is needed to evaluate whether this is a local problem or a new source of MRSA, that puts the until now successful Search and Destroy policy of the Netherlands at risk.

Whole-Genome Sequencing for Routine Pathogen Surveillance in Public Health: a Population Snapshot of Invasive Staphylococcus aureus in Europe.

UNLABELLED: The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http://www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) community-oriented database infrastructure and analysis tools. IMPORTANCE: The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine whole-genome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets.

Multiple-locus variable number tandem repeat analysis is superior to spa typing and sufficient to characterize MRSA for surveillance purposes.

AIM: Assess the best approach to type methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcal protein A (spa) typing, multiple-locus variable number tandem repeat analysis (MLVA) or both. MATERIALS & METHODS: Discriminatory power of spa typing and MLVA was determined using 20,771 MRSA isolates. RESULTS: There were twice as many MLVA types (MTs) as spa types present in the collection. Among the top 70% of the isolates, 37 spa types and 139 MTs were found. MLVA diversity among the top-10 spa types was high (diversity index 0.96), while spa diversity among the top-10 MTs was much lower (diversity index 0.83). The probability that two MRSA isolates with the same spa type also had the same MT was low (Wallace's coefficient 0.27). By contrast, most MRSA isolates yielding the same MT also had the same spa type (Wallace's coefficient 0.90). CONCLUSION: MLVA is superior to spa typing and will suffice to characterize MRSA isolates for surveillance.

Intercenter reproducibility of binary typing for Staphylococcus aureus.

The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al. [J. Clin. Microbiol. 2001 39 (1) 328]. The BT assay, including a standardized DNA extraction protocol was performed in duplicate in eleven medical microbiology laboratories. Two different hybridization detection procedures were applied and a prelabeled DNA sample as process control was included. Only three laboratories accurately identified all strains. Divergence in technical procedures resulted in misinterpretation due to an increasing number of faint or absent hybridization signals in combination with high background staining. The binary type of the process control was determined correctly by all participating laboratories. The feasibility of the BT protocol was related directly to the skill of the laboratory personnel. On the basis of the national study, we concluded that the DNA extraction protocol needed modification to improve DNA yield and purity. Subsequently, seven European laboratories participated in an international study to determine the reproducibility of the modified BT protocol. Each center was asked to analyze 10 DNA samples previously extracted from 10 MRSA strains (phase 1) and, additionally, to analyze 10 MRSA strains, using the standardized or their in-house DNA isolation protocol (phase 2). A prelabeled DNA process control sample was included again. The binary types of all DNA samples were identified correctly by all but one laboratories. This latter laboratory diverged from the protocol by adding an excess of labeled DNA to the hybridization mixture, resulting in a high background and, therefore, noninterpretable BT results. All centers produced identical BT results for the process control. Five of the seven centers correctly identified the binary types of all 10 MRSA strains in phase 2 of the international study. Three of these centers used their in-house DNA extraction protocol. Divergence from the standard BT protocol in the remaining two centers resulted in no interpretable BT data for the 10 MRSA strains. The study demonstrated that each center that followed the BT protocol to the letter could generate reproducible results, irrespective whether or not an in-house DNA isolation protocol was used. The current BT protocol thus represents a simple method generating robust, reproducible genotype data for S. aureus strains.