Monophasic Variant of Salmonella Typhimurium Infection Affects the Serum Metabolome in Swine.

Salmonella is the most relevant foodborne zoonotic agent found in swine, and its presence in French herds is significant. Its carriage is asymptomatic, which makes it difficult to detect during rearing, thus increasing the risk of its presence on pork meat. Studies have shown that enteric infection in animals could be associated with changes in the serum metabolome composition, through the immune response or changes in the digestive microbiota composition. We hypothesized that these changes in the serum metabolome composition could be used as markers for the detection of asymptomatic animals infected by Salmonella. Using untargeted analysis by liquid chromatography coupled with mass spectrometry, we showed that significant differences in the composition of the serum metabolome could be detected between infected or noninfected animals both 1 and 21 days after experimental infection. This serum metabolome composition significantly changed during the 21 days postinfection in the infected animal groups, suggesting an evolution of the impact of infection with time. Despite this evolution, differences in the serum metabolome composition persisted between infected and noninfected animals 21 days after the initial infection. We also showed a possible difference between high-shedding and low-shedding animals 21 days postinfection. Finally, some of the variations in the metabolome were found to be significantly associated with variations of specific members of the fecal microbiota. Thus, excreting and asymptomatic animals, but also high-shedding animals, could be identified on the basis of their serum metabolome composition.

Effects of Major Families of Modulators on Performances and Gastrointestinal Microbiota of Poultry, Pigs and Ruminants: A Systematic Approach.

Considering the ban on the use of antibiotics as growth stimulators in the livestock industry, the use of microbiota modulators appears to be an alternative solution to improve animal performance. This review aims to describe the effect of different families of modulators on the gastrointestinal microbiota of poultry, pigs and ruminants and their consequences on host physiology. To this end, 65, 32 and 4 controlled trials or systematic reviews were selected from PubMed for poultry, pigs and ruminants, respectively. Microorganisms and their derivatives were the most studied modulator family in poultry, while in pigs, the micronutrient family was the most investigated. With only four controlled trials selected for ruminants, it was difficult to conclude on the modulators of interest for this species. For some modulators, most studies showed a beneficial effect on both the phenotype and the microbiota. This was the case for probiotics and plants in poultry and minerals and probiotics in pigs. These modulators seem to be a good way for improving animal performance.

Inflammatory Responses Induced by the Monophasic Variant of Salmonella Typhimurium in Pigs Play a Role in the High Shedder Phenotype and Fecal Microbiota Composition.

Pigs infected with Salmonella may excrete large amounts of Salmonella, increasing the risk of spread of this pathogen in the food chain. Identifying Salmonella high shedder pigs is therefore required to mitigate this risk. We analyzed immune-associated markers and composition of the gut microbiota in specific-pathogen-free pigs presenting different shedding levels after an oral infection with Salmonella. Immune response was studied through total blood cell counts, production of anti-Salmonella antibodies and cytokines, and gene expression quantification. Total Salmonella shedding for each pig was estimated and hierarchical clustering was used to cluster pigs into high, intermediate, and low shedders. Gut microbiota compositions were assessed using 16S rRNA microbial community profiling. Comparisons were made between control and inoculated pigs, then between high and low shedders pigs. Prior to infection, high shedders had similar immunological profiles compared to low shedders. As soon as 1 day postinoculation (dpi), significant differences on the cytokine production level and on the expression level of several host genes related to a proinflammatory response were observed between high and low shedders. Infection with Salmonella induced an early and profound remodeling of the immune response in all pigs, but the intensity of the response was stronger in high shedders. In contrast, low shedders seroconverted earlier than high shedders. Just after induction of the proinflammatory response (at 2 dpi), some taxa of the fecal microbiota were specific to the shedding phenotypes. This was related to the enrichment of several functional pathways related to anaerobic respiration in high shedders. In conclusion, our data show that the immune response to Salmonella modifies the fecal microbiota and subsequently could be responsible for shedding phenotypes. Influencing the gut microbiota and reducing intestinal inflammation could be a strategy for preventing Salmonella high shedding in livestock. IMPORTANCE Salmonellosis remains the most frequent human foodborne zoonosis after campylobacteriosis and pork meat is considered one of the major sources of human foodborne infections. At the farm, host heterogeneity in pig infection is problematic. High Salmonella shedders contribute more significantly to the spread of this foodborne pathogen in the food chain. The identification of predictive biomarkers for high shedders could help to control Salmonella in pigs. The purpose of the present study was to investigate why some pigs become super shedders and others low shedders. We thus investigated the differences in the fecal microbial composition and the immune response in orally infected pigs presenting different Salmonella shedding patterns. Our data show that the proinflammatory response induced by S. Typhimurium at 1 dpi could be responsible for the modification of the fecal microbiota composition and functions observed mainly at 2 and 3 dpi and to the low and super shedder phenotypes.

Longitudinal study describing time to Salmonella seroconversion in piglets on three farrow-to-finish farms.

BACKGROUND: Pigs are frequently colonised with Salmonella enterica, and this constitutes a major risk for human salmonellosis. The infection can be assessed by the serological response of pigs to S enterica. A longitudinal study was undertaken on-farm to correctly describe this serological response and investigate factors associated with age at Salmonella seroconversion. METHODS: Three pig farms and in each farm three successive batches were considered. Per batch, 40 piglets were selected at random from 10 sows (four piglets per sow). Blood was sampled from sows one week after farrowing and from piglets at weeks 1, 6, 10, 14, 18 and 22 and at the slaughterhouse. Salmonella antibodies were detected in serum using a commercial ELISA test. Factors related to farm characteristics, batch management system, porcine reproductive and respiratory syndrome infection, and sows' Salmonella serological status were recorded to assess their effect on age at seroconversion. RESULTS: At week 1 after farrowing, 96.5 per cent of the sows had antibodies against Salmonella. The serological results of piglets at weeks 1 and 6 only were positively correlated with those of the sows. The average age at Salmonella seroconversion was 137±2.2 days (confidence interval at 95 per cent). The first seroconversions occurred from weeks 10 to 14, but most of the pigs (54.6 per cent) were seropositive at the end of the fattening period, with variations between farms and batches (28.9-75.7 per cent). Herd/farm was significantly associated with age at seroconversion. CONCLUSION: This longitudinal study allowed the authors to follow precisely the evolution of Salmonella seroconversion from maternity to slaughterhouse and confirm the relationship between the seroconversion of sows and serology of their piglets. Moreover, factors related to farm practices and management as a whole are more influential than individual factors (at the pig level) on age at Salmonella seroconversion.

Experimental infection of pigs by Salmonella Derby, S. Typhimurium and monophasic variant of S. Typhimurium: Comparison of colonization and serology.

Salmonella serovars Derby, Typhimurium and the monophasic variant of Salmonella Typhimurium are the most frequently isolated serovars in pigs in France. To compare the excretion patterns, seroconversion to Salmonella and contamination of the organs of pigs inoculated with strains of all three serovars, we conducted an experimental trial with 28 SPF piglets. Four were used as a negative control, while the other 24 were divided equally into three groups. Each group was inoculated at 7 weeks of age with a different strain: S. Derby (SDb), S. Typhimurium (ST), and the monophasic variant of S. Typhimurium (mST). Fecal and blood samples were collected twice a week up until necropsy, on 21 days post-inoculation (DPI) for half of each group and 49 DPI for the remaining piglets. During necropsy, the tonsils, mesenteric lymph nodes and various intestinal contents were collected from each pig. Salmonella bacteria were quantified in CFU/g by a bacteriological method, and levels of Salmonella antibodies were measured using an ELISA Kit. Piglets inoculated with mST continuously excreted Salmonella in their feces throughout the trial. For each of the other serovars, one piglet was Salmonella-negative on one DPI. The quantity of Salmonella excreted was statistically different between the group inoculated with ST and mST (p < 0.05), but no differences were found between the other serovars. The tonsils, cecum and jejunum were the most contaminated organs in all groups. Seroconversion for all the piglets was completed by different DPI: 28 for ST, 31 for mST and 38 for SDb. No major differences were found in terms of excretion and colonization among the studied serovars.

Colonization of Pigs Experimentally Infected with a Monophasic Variant of Salmonella Typhimurium.

The monophasic variant of Salmonella Typhimurium is highly prevalent in human and in pork. However, little is known about colonization dynamics and serology in pigs. We orally inoculated 24 seven-week-old piglets with 109 CFU/pig of a porcine strain of monophasic Salmonella Typhimurium in an experimental trial. Three groups of eight piglets were orally inoculated and monitored for 21, 49, or 84 days post-inoculation until necropsied. From 3 days post-inoculation to necropsy, individual feces were sampled twice weekly and blood once weekly. At necropsy, the tonsils, mesenteric lymph nodes, and the contents of the duodenum, jejunum, ileum, and cecum were collected from each pig. We determined the number of CFU/g in all the samples and measured also Salmonella antibodies in OD% in all blood samples. At different times during the trial, we tested by MLVA (Multilocus Variable Number Tandem Repeat Analysis) the genomic stability of the strain after passing through the intestinal tract. Salmonella was continuously excreted by pigs, ranging from 1.4 to 5.8 log10 CFU/g. At necropsy, Salmonella was present in all samples, but the tonsils were particularly infected. Salmonella antibodies were detected in five pigs 7 days post-inoculation. At 49 days post-inoculation, all the pigs were seropositive. We observed new MLVA types for 3.3% of the isolates tested over the trial. Our study allowed us to show the serovar's ability to persist in pigs after infection up to 84 days post-inoculation. We demonstrated that Salmonella seroconversion appeared earlier than in naturally infected pigs and that the strain's genome can evolve after passing through the digestive tract of pigs.

Population Genetic Structure of Listeria monocytogenes Strains Isolated From the Pig and Pork Production Chain in France.

Listeria monocytogenes is an ubiquitous pathogenic bacterium, transmissible to humans through the consumption of contaminated food. The pork production sector has been hit hard by a series of L. monocytogenes-related food poisoning outbreaks in France. An overview of the diversity of strains circulating at all levels of the pork production chain, from pig farming (PF) to finished food products (FFP), is needed to identify the contamination routes and improve food safety. Until now, no typing data has been available on strains isolated across the entire pig and pork production chain. Here, we analyzed the population genetic structure of 687 L. monocytogenes strains isolated over the last 20 years in virtually all the French départements from three compartments of this production sector: PF, the food processing environment (FPE), and FFP. The genetic structure was described based on Multilocus sequence typing (MLST) clonal complexes (CCs). The CCs were obtained by mapping the PFGE profiles of the strains. The distribution of CCs was compared firstly between the three compartments and then with CCs obtained from 1106 strains isolated from other food production sectors in France. The predominant CCs of pig and pork strains were not equally distributed among the three compartments: the CC37, CC59, and CC77 strains, rarely found in FPE and FFP, were prevalent in PF. The two most prevalent CCs in the FPE and FFP compartments, CC9 and CC121, were rarely or never detected in PF. No CC was exclusively associated with the pork sector. Three CCs (CC5, CC6, and CC2) were considered ubiquitous, because they were observed in comparable proportions in all food production sectors. The two most prevalent CCs in all sectors were CC9 and CC121, but their distribution was disparate. CC9 was associated with meat products and food products combining several food categories, whereas CC121 was not associated with any given sector. Based on these results, CC121 is likely able to colonize a larger diversity of food products than CC9. Both CCs being associated with the food production suggests, that certain processing steps, such as slaughtering or stabilization treatments, favor their settlement and the recontamination of the food produced.

No Clear Differences between Organic or Conventional Pig Farms in the Genetic Diversity or Virulence of Campylobacter coli Isolates.

To evaluate the impact of pig farm management on the genetic diversity and on the virulence of Campylobacter coli, we characterized isolates from 19 organic pig farms (62 isolates) and from 24 conventional pig farms (58 isolates). The 120 C. coli isolates were typed using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and the presence of nine virulence genes was screened using real-time PCR. The capacity of adhesion and invasion of 61 isolates (32 from organic and 29 from conventional farms) were then tested on human intestinal Caco-2 cells. A total of 59 PFGE types and of 50 sequence types (STs) were identified. Twelve PFGE types and nine STs, accounting for 34 and 41.6% of the isolates, respectively, were common between the two production systems with ST854 dominating (18.3% of the isolates). Twenty-nine PFGE types and 25 STs were only found in isolates from organic farms, and 18 PFGE types and 16 STs from conventional farms. No significant differences were found in diversity despite the differences in rearing systems, except at the locus level for the glnA, gltA, and uncA genes. All isolates, regardless of their origin, carried the ceuE, iam, ciaB, and flaA genes and more than 95% of the isolates carried the cadF and cdtABC genes. No significant differences were found in pathogenicity between the two farming systems. The pathogenicity of the C. coli isolates was low compared to C. jejuni control strains tested. The plasmid gene virb11 was detected in only 13 isolates from organic farms; these isolates showed greater invasion capacity than those without this gene. Our study indicates that pig farm management does not significantly affect the diversity and the virulence of Campylobacter coli isolated from pigs. The common genotypes between conventional and organic farms may indicate that some genotypes are adapted to pigs.

Campylobacter coli in Organic and Conventional Pig Production in France and Sweden: Prevalence and Antimicrobial Resistance.

The purpose of the study was to evaluate and compare the prevalence and antimicrobial resistance of Campylobacter coli in conventional and organic pigs from France and Sweden. Fecal or colon samples were collected at farms or at slaughterhouses and cultured for Campylobacter. The minimum inhibitory concentrations of ciprofloxacin, nalidixic acid, streptomycin, tetracycline, erythromycin, and gentamicin were determined by microdilution for a total of 263 French strains from 114 pigs from 50 different farms and 82 Swedish strains from 144 pigs from 54 different farms. Erythromycin resistant isolates were examined for presence of the emerging rRNA methylase erm(B) gene. The study showed that within the colon samples obtained in each country there was no significant difference in prevalence of Campylobacter between pigs in organic and conventional productions [France: conventional: 43/58 (74%); organic: 43/56 (77%) and Sweden: conventional: 24/36 (67%); organic: 20/36 (56%)]. In France, but not in Sweden, significant differences of percentages of resistant isolates were associated with production type (tetracycline, erythromycin) and the number of resistances was significantly higher for isolates from conventional pigs. In Sweden, the number of resistances of fecal isolates was significantly higher compared to colon isolates. The erm(B) gene was not detected in the 87 erythromycin resistant strains tested.

Antibiotic Resistance in Escherichia coli from Pigs in Organic and Conventional Farming in Four European Countries.

Organic pig production differs in many ways from conventional production of pigs, e.g., in antibiotic use, herd structure, feeding regimes, access to outdoor areas and space allowance per pig. This study investigated if these differences result in a lower occurrence of antibiotic resistance in organic slaughter pigs in Denmark, France, Italy and Sweden. Samples were taken from the colon content and/or faeces and minimum inhibitory concentrations (MIC) of ten antibiotics were determined in isolates of Escherichia coli. In addition, the proportion of tetracycline (TET) resistant E. coli in colon content and/or faeces from individual pigs was determined. In all four countries the percentage resistance to ampicillin, streptomycin, sulphonamides or trimethoprim was significantly lower in E. coli from organic pigs. In France and Italy, the percentage of isolates resistant to chloramphenicol, ciprofloxacin, nalidixic acid or gentamicin was also significantly lower in the E. coli from organic pigs. Resistance to cefotaxime, was not found in any country. The percentage of E. coli isolates resistant to TET as well as the proportion of TET-resistant E. coli was significantly lower in organic than in conventional pigs, except in Sweden where TET-resistance was equally low in both production types. There were also differences between countries within production type in the percentage resistance to individual antibiotics as well as the proportion of TET-resistant E. coli with lower median proportions in Sweden and Denmark compared to France and Italy. The study shows that in each of the four countries resistance in intestinal E. coli was less common in organic than in conventional pigs, but that there were also large differences in resistance between countries within each production type, indicating that both country- and production-specific factors influence the occurrence of resistance.

Contribution of Avian Salmonella enterica Isolates to Human Salmonellosis Cases in Constantine (Algeria).

An epidemiological investigation was carried out on one hundred Salmonella isolates from broiler farms, slaughterhouses, and human patients in the Constantine region of Algeria, in order to explore the contribution of avian strains to human salmonellosis cases in this region over the same period of time. The isolates were characterized by phenotypic as well as genotypic methods. A large variety of antimicrobial resistance profiles was found among human isolates, while only seven profiles were found among avian isolates. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), Insertion Sequence 200-PCR (IS200-PCR), and Pulsed Field Gel Electrophoresis (PFGE) resulted in the allocation of the isolates to 16, 20, and 34 different profiles, respectively. The 3 genotyping methods led to complementary results by underlining the clonality of some serovars with the diffusion and persistence of a single clone in the Constantine area as well as stressing the polymorphism present in isolates belonging to other serovars, indicating the diversity of potential reservoirs of nontyphoidal Salmonella. Altogether, our results seem to indicate that nontyphoidal avian Salmonella may play an important role in human salmonellosis in the Constantine region.

First Complete Genome Sequence of a Salmonella enterica subsp. enterica Serovar Derby Strain Associated with Pork in France.

In France, Salmonella enterica subsp. enterica serovar Derby is one of the most often isolated serovars in pigs. Here, we describe the draft genome sequence of a strain isolated from a pig. This strain had the most frequent pulsed-field gel electrophoresis (PFGE) and antimicrobial patterns (S, SSU, T) usually observed in pig production in France. Those patterns have been also highlighted in human isolates.

Characterization of Antibiotic Resistance Gene Abundance and Microbiota Composition in Feces of Organic and Conventional Pigs from Four EU Countries.

One of the recent trends in animal production is the revival of interest in organic farming. The increased consumer interest in organic animal farming is mainly due to concerns about animal welfare and the use of antibiotics in conventional farming. On the other hand, providing animals with a more natural lifestyle implies their increased exposure to environmental sources of different microorganisms including pathogens. To address these concerns, we determined the abundance of antibiotic resistance and diversity within fecal microbiota in pigs kept under conventional and organic farming systems in Sweden, Denmark, France and Italy. The abundance of sul1, sul2, strA, tet(A), tet(B) and cat antibiotic resistance genes was determined in 468 samples by real-time PCR and the fecal microbiota diversity was characterized in 48 selected samples by pyrosequencing of V3/V4 regions of 16S rRNA. Contrary to our expectations, there were no extensive differences between the abundance of tested antibiotic resistance genes in microbiota originating from organic or conventionally housed pigs within individual countries. There were also no differences in the microbiota composition of organic and conventional pigs. The only significant difference was the difference in the abundance of antibiotic resistance genes in the samples from different countries. Fecal microbiota in the samples originating from southern European countries (Italy, France) exhibited significantly higher antibiotic resistance gene abundance than those from northern parts of Europe (Denmark, Sweden). Therefore, the geographical location of the herd influenced the antibiotic resistance in the fecal microbiota more than farm's status as organic or conventional.

Analysis of Listeria monocytogenes strain distribution in a pork slaughter and cutting plant in the province of Quebec.

Following the 2008 Canadian listeriosis outbreak associated with ready-to-eat (RTE) meat products, regulations on the presence of Listeria monocytogenes in RTE food production facilities were modified by Health Canada, confirming the need to control this pathogen, not only in the final product but also in the plant environment. Information on the occurrence of this microorganism during the early steps of production, such as the slaughtering process and in the cutting area, is scarce in Canada. In this study, we sampled different production steps in a slaughtering and cutting plant in the province of Quebec over a 2-year period. The lairage pens, representative areas of the slaughter line, and cutting zones were targeted after their respective cleaning procedures. A total of 874 samples were analyzed for the presence of L. monocytogenes. Characterization was done by first genoserogrouping the isolates using multiplex PCR and then using a pulsed-field gel electrophoresis approach. L. monocytogenes was detected throughout all production stages. The 108 positive samples found were analyzed further, and we established that there were 4 different serogroups, with serogroup IIb being the most prevalent. The results of pulsed-field gel electrophoresis analysis showed a significant decrease in the diversity of strains from the first areas of the plant to the cutting room (10 pulsotypes in 13 positive samples in lairage and 9 in 86 positive samples in cutting) and also showed the overrepresentation of a single predominant strain in the cutting room environment (type 1, representing 96.1% of the isolates). Biofilm formation analysis of the strains cannot exclusively explain the transitions we observed. A strong genotypic similarity between strains isolated in the early production areas and some strains in the cutting room was shown. These results support the need for better surveillance of L. monocytogenes prior to RTE food production in order to design control strategies that are better adapted from a public health perspective.

Experimental study of the impact of antimicrobial treatments on Campylobacter, Enterococcus and PCR-capillary electrophoresis single-strand conformation polymorphism profiles of the gut microbiota of chickens.

An experiment was conducted to compare the impact of antimicrobial treatments on the susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis, and on the diversity of broiler microbiota. Specific-pathogen-free chickens were first orally inoculated with strains of Campylobacter and Enterococcus faecium. Birds were then orally treated with recommended doses of oxytetracycline, sulfadimethoxine/trimethoprim, amoxicillin or enrofloxacin. Faecal samples were collected before, during and after antimicrobial treatment. The susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis strains isolated on supplemented or non-supplemented media was studied and PCR-capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) profiles of the gut microbiota were analysed. Enrofloxacin-resistant Campylobacter were selected in the enrofloxacin-treated group and showed the Thr86Ile mutation in the gyrA gene. Acquisition of the tetO gene in Campylobacter coli isolates was significantly more frequent in birds given oxytetracycline. No impact of amoxicillin treatment on the susceptibility of Campylobacter could be detected. Ampicillin- and sulfadimethoxine/trimethoprim-resistant Enterococcus faecium were selected in amoxicillin-treated broilers, but no selection of the inoculated vancomycin-resistant Enterococcus faecium could be detected, although it was also resistant to tetracycline and sulfadimethoxine/trimethoprim. PCR-CE-SSCP revealed significant variations in a few peaks in treated birds as compared with non-treated chickens. In conclusion, antimicrobial treatments perturbed chicken gut microbiota, and certain antimicrobial treatments selected or co-selected resistant strains of Campylobacter and Enterococcus.

Genetic diversity and antimicrobial resistance profiles of Salmonella enterica serotype derby isolated from pigs, pork, and humans in France.

In France, Salmonella enterica serotypes Typhimurium and Derby are the most often isolated serotypes in pigs. Moreover, serotype Derby usually ranks between third and fourth in prevalence among human isolates in France. The aim of this study was to evaluate the genetic relationships between human and pig Salmonella Derby isolates based on their pulsed-field gel electrophoresis (PFGE) patterns after XbaI, BlnI, and SpeI restriction and on their antimicrobial resistance profiles. The 196 studied isolates were isolated in 2006 and 2007: 73 from fattening pigs, 27 from pork, and 96 from humans. Forty-four PFGE XbaI patterns were identified. A major pattern (SDX01) was identified for 96 isolates (49%). This pattern was common to pig, pork, and human isolates. Among the 146 isolates tested for their antimicrobial resistance, 84.2% (n=123) showed resistance to at least one antibiotic and 69.2% (n=101) were simultaneously resistant to at least streptomycin, sulfonamides, and tetracycline. Most of the isolates that are resistant to these three antibiotics also displayed the major SDX01 pattern. The use of two other restriction enzymes on a part of the panel (155 isolates) brought a significant increase in the discriminatory index, in particular for SDX01 strains. As Salmonella Derby is essentially isolated from pigs, and major resistance and PFGE patterns of isolates from pigs and pork were very similar to human isolates, human salmonellosis due to Salmonella Derby may be related to pigs.

Polyphasic characterization and genetic relatedness of low-virulence and virulent Listeria monocytogenes isolates.

BACKGROUND: Currently, food regulatory authorities consider all Listeria monocytogenes isolates as equally virulent. However, an increasing number of studies demonstrate extensive variations in virulence and pathogenicity of L. monocytogenes strains. Up to now, there is no comprehensive overview of the population genetic structure of L. monocytogenes taking into account virulence level. We have previously demonstrated that different low-virulence strains exhibit the same mutations in virulence genes suggesting that they could have common evolutionary pathways. New low-virulence strains were identified and assigned to phenotypic and genotypic Groups using cluster analysis. Pulsed-field gel electrophoresis, virulence gene sequencing and multi-locus sequence typing analyses were performed to study the genetic relatedness and the population structure between the studied low-virulence isolates and virulent strains. RESULTS: These methods showed that low-virulence strains are widely distributed in the two major lineages, but some are also clustered according to their genetic mutations. These analyses showed that low-virulence strains initially grouped according to their lineage, then to their serotypes and after which, they lost their virulence suggesting a relatively recent emergence. CONCLUSIONS: Loss of virulence in lineage II strains was related to point mutation in a few virulence genes (prfA, inlA, inlB, plcA). These strains thus form a tightly clustered, monophyletic group with limited diversity. In contrast, low-virulence strains of lineage I were more dispersed among the virulence strains and the origin of their loss of virulence has not been identified yet, even if some strains exhibited different mutations in prfA or inlA.

Genotypic and phenotypic characterisation of a collection of Cronobacter (Enterobacter sakazakii) isolates.

Enterobacter sakazakii has been identified as the causative agent of serious neonatal infections, associated with high mortality rate. In many cases, powdered infant formula (PIF) has been identified as the source of infection. Recently, E. sakazakii was proposed to be classified in a new genus, Cronobacter. Since knowledge on this pathogen is still incomplete, there is a need for molecular characterization schemes in order to help with epidemiological investigation and evaluate strain variability. The objectives of this study were to combine genotypic (pulsed-field gel electrophoresis [PFGE], 16S rRNA gene sequencing, and automated ribotyping) methods with traditional phenotypic biochemical methods to characterize a collection of Cronobacter isolates from various origins. In addition, the relative growth dynamics were compared by estimating the growth rates for each isolate in non-selective broth (BHI) at 25 degrees C and 37 degrees C. According to biochemical test profiles the majority of isolates were identified as Cronobacter sakazakii, which seemed to be the most common species distributed in the environment of PIF production plants. Furthermore, the PFGE technique displayed very high discriminatory power as 61 distinct pulsotypes were revealed among the 150 Cronobacter isolates. Combining information on sample origin and pulse type, 64 isolates were deemed as unique strains. Although genetic typing data for the strains clearly delineated them into clusters closely corresponding to biochemical speciation results, it was not without discrepancies as some strains did not group as predicted. Important for quantitative risk assessment is the fact that despite the high genetic heterogeneity observed for this collection, most Cronobacter strains displayed similar growth rates irrespective of species designation.

Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping.

Listeria monocytogenes serotyping is commonly used as the first level of characterisation in the epidemiological surveillance of food and clinical isolates and is therefore widely accepted. The aim of this study was to define a scheme for multiplex molecular serotyping of L. monocytogenes based on a previously described PCR assay and then to evaluate and compare this new procedure with conventional serotyping by agglutination. The study included 1204 Listeria strains collected from food products in France, from March 2005 to October 2006. Two multiplex PCR assays were designed to cluster L. monocytogenes strains into five molecular serogroups: IIa, IIb, IIc, IVa, IVb in agreement with the most commonly encountered serotypes. Amplification of the prfA gene was added to the multiplex PCR to check for L. monocytogenes species; forty-eight (4%) of the isolates tested belonged to the genus Listeria but were not L. monocytogenes. Using this first multiplex PCR, the concordance between conventional and molecular methods was 90.6%, 97.8%, 100% and 100%, for 1/2a, 1/2c, 1/2b and 4b serotypes respectively. False results were observed for some atypical 1/2a, 3a and 1/2c strains. Therefore, this lack of specificity was resolved by using an additional PCR assay based on amplification of the flaA gene, a specific target of 1/2a and 3a strains. When applying the second PCR assay to IIa and IIc molecular serogroup strains, total agreement was obtained between molecular and conventional serotyping methods with a lower level of discrimination for the molecular one. This study proposes to define a strategy for molecular serotyping using both PCR assays: a multiplex and the flaA PCR in order to assign the atypical 1/2a, 3a and 1/2c strains. Moreover, prs gene detection was added for Listeria genus recognition as a positive control in association with flaA detection. Indeed, this molecular serotyping scheme could be considered as a useful and rapid method for first-level characterisation of the most frequently encountered L. monocytogenes serotypes.

Human infections due to Salmonella Napoli: a multicountry, emerging enigma recognized by the Enter-net international surveillance network.

Human infections caused by Salmonella enterica serovar Napoli are relatively uncommon in Europe. Napoli was ranked 22nd in the Enter-net Salmonella database for 2006 with 295 cases (0.28%) of the 105,635 from 29 European countries. For the 18 countries that provided data for all the years 2000-2006, the number of cases rose from 122 out of 116,915 (0.10%) in 2000 to 293 out of 80,318 (0.36%) in 2006-an increase of 140.2%. Over 87% of cases came from three countries, France, Italy, and Switzerland. The epidemiology of the human cases showed an increased frequency in those aged under 5 or over 64, and both sexes were equally represented. Napoli isolates were also reported from nonhuman sources, mainly environmental samples and poultry. Strains compared by pulsed-field gel electrophoresis exhibited high levels of diversity between human, animal, and environmental sources. No single factor has been recognized as causing this rise, hence no public health interventions can be made or advice given to ensure that it does not persist. A 140% rise in 7 years indicates that the public health problem will continue, and further multidisciplinary investigations are needed to solve this enigma.