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.

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.

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.

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.

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).

Tracing the sources of human salmonellosis: a multi-model comparison of phenotyping and genotyping methods.

Salmonella source attribution is usually performed using frequency-matched models, such as the (modified) Dutch and Hald models, based on phenotyping data, i.e. serotyping, phage typing, and antimicrobial resistance profiling. However, for practical and economic reasons, genotyping methods such as Multi-locus Variable Number of Tandem Repeats Analysis (MLVA) are gradually replacing traditional phenotyping of salmonellas beyond the serovar level. As MLVA-based source attribution of human salmonellosis using frequency-matched models is problematic due to the high variability of the genetic targets investigated, other models need to be explored. Using a comprehensive data set from the Netherlands in 2005-2013, this study aimed at attributing sporadic and domestic cases of Salmonella Typhimurium/4,[5],12:i:- and Salmonella Enteritidis to four putative food-producing animal sources (pigs, cattle, broilers, and layers/eggs) using the modified Dutch and Hald models (based on sero/phage typing data) in comparison with a widely applied population genetics model - the asymmetric island model (AIM) - supplied with MLVA data. This allowed us to compare model outcomes and to corroborate whether MLVA-based Salmonella source attribution using the AIM is able to provide sound, comparable results. All three models provided very similar results, confirming once more that most S. Typhimurium/4,[5],12:i:- and S. Enteritidis cases are attributable to pigs and layers/eggs, respectively. We concluded that MLVA-based source attribution using the AIM is a feasible option, at least for S. Typhimurium/4,[5],12:i:- and S. Enteritidis. Enough information seems to be contained in the MLVA profiles to trace the sources of human salmonellosis even in presence of imperfect temporal overlap between human and source isolates. Besides Salmonella, the AIM might also be applicable to other pathogens that do not always comply to clonal models. This would add further value to current surveillance activities by performing source attribution using genotyping data that are being collected in a standardized fashion internationally.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae in hospital food: a risk assessment.

OBJECTIVE: Determine the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE) contamination of food and colonization of food handlers in a hospital kitchen and compare retrieved ESBL-PE strains with patient isolates. DESIGN: Cross-sectional study. SETTING: A 2,200-bed tertiary care university hospital in Switzerland. PARTICIPANTS: Food handlers. METHODS: Raw and prepared food samples were obtained from the hospital kitchen, with a comparator group from local supermarkets. Fecal samples collected from food handlers and selectively pre-enriched homogenized food samples were inoculated onto selective chromogenic media. Phenotypic confirmation of ESBL production was performed using the double disk method. Representative ESBL-PE were characterized using polymerase chain reaction (PCR) and sequencing for blaCTX-M, blaSHV, and blaTEM genes, and Escherichia coli strains were typed using phylotyping, repetitive element palindromic PCR, and multilocus sequence typing. Meat samples were screened for antibiotic residues using liquid chromatography time-of-flight mass spectrometry. RESULTS: Sixty (92%) of the raw chicken samples were ESBL-PE positive, including 30 (86%) of the hospital samples and all supermarket samples. No egg, beef, rabbit, or cooked chicken samples were ESBL-PE positive. No antibiotic residues were detected. Six (6.5%) of 93 food handlers were ESBL-PE carriers. ESBL-PE strains from chicken meat more commonly possessed blaCTX-M-1 and blaCTX-M-2, whereas blaCTX-M-14 and blaCTX-M-15 were predominant among strains of human origin. There was partial overlap in the sequence type of E. coli strains of chicken and human origin. No E. coli ST131 strains or blaCTX-M-15 genes were isolated from meat. CONCLUSIONS: Although there is significant ESBL-PE contamination of delivered chicken meat, current preventive strategies minimize risks to food handlers, hospital staff, and patients.

Risk factors for human salmonellosis originating from pigs, cattle, broiler chickens and egg laying hens: a combined case-control and source attribution analysis.

Several case-control studies have investigated risk factors for human salmonellosis while others have used Salmonella subtyping to attribute human infections to different food and animal reservoirs. This study combined case-control and source attribution data into a single analysis to explore risk factors at the point of exposure for human salmonellosis originating from four putative food-producing animal reservoirs (pigs, cattle, broilers and layers/eggs) in the Netherlands. We confirmed that most human cases (∼ 90%) were attributable to layers/eggs and pigs. Layers/eggs and broilers were the most likely reservoirs of salmonellosis in adults, in urban areas, and in spring/summer, whereas pigs and cattle were the most likely reservoirs of salmonellosis in children, in rural areas, and in autumn/winter. Several reservoir-specific risk factors were identified. Not using a chopping board for raw meat only and consuming raw/undercooked meat were risk factors for infection with salmonellas originating from pigs, cattle and broilers. Consuming raw/undercooked eggs and by-products were risk factors for layer/egg-associated salmonellosis. Using antibiotics was a risk factor for pig- and cattle-associated salmonellosis and using proton-pump inhibitors for salmonellosis attributable to any reservoir. Pig- and cattle-associated infections were also linked to direct contact with animals and environmental exposure (e.g. playing in sandboxes). Eating fish, meat in pastry, and several non-meat foods (fruit, vegetables and pasteurized dairy products) were protective factors. Consuming pork and occupational exposure to animals and/or raw meats were protective against layer/egg-associated salmonellosis. We concluded that individuals acquiring salmonellosis from different reservoirs have different associated risk factors, suggesting that salmonellas may infect humans through various transmission pathways depending on their original reservoirs. The outcome of classical case-control studies can be enhanced by incorporating source attribution data and vice versa.

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.

Multiple-locus variable number tandem repeat analysis of Staphylococcus aureus: comparison with pulsed-field gel electrophoresis and spa-typing.

BACKGROUND: Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution. METHODOLOGY/PRINCIPAL FINDINGS: A new MLVA scheme for S. aureus was validated using 1681 S. aureus isolates collected from Dutch patients and 100 isolates from pigs. MLVA using 8 tandem repeat loci was performed in 2 multiplex PCRs and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. The assessed number of repeats was used to create MLVA profiles consisting of strings of 8 integers that were used for categorical clustering. MLVA yielded 511 types that clustered into 11 distinct MLVA complexes which appeared to coincide with MLST clonal complexes. MLVA was at least as discriminatory as PFGE and twice as discriminatory as spa-sequence typing. There was considerable congruence between MLVA, spa-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. CONCLUSIONS: The MLVA described in this study is a high throughput, relatively low cost genotyping method for S. aureus that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology.

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.