Phenotypic and genotypic characterization of H2 S-positive and H2 S-negative strains of Shewanella baltica isolated from spoiled whiting (Merlangius merlangus).

UNLABELLED: Four strains were isolated from a spoiled whiting (Merlangius merlangus). All of them were able to grow aerobically from 4 to 30°C and also able to develop anaerobically in the presence of trimethylamine N-Oxide (TMAO) at 25°C. Biochemical characterization did not allow identification of the strains species but showed that one of the four strains was unable to produce H2 S. Two strains synthetized an ornithine decarboxylase being potential putrescine producers. Results of carbon source use highlighted that the four strains were able to use citrate and d-sucrose and one strain was not able to use l-arabinose. Genotypic characterization of the strains thanks to 16S rRNA and gyrB partial gene sequencing led to their identification as members of Shewanella baltica species. These observations suggest that H2 S production may not be the most appropriate screening parameter for Shewanella species and further to monitor the development of spoilage flora. SIGNIFICANCE AND IMPACT OF THE STUDY: Shewanella is a complex genus composed of numerous and heterogeneous species. One of them Shewanella baltica has previously been described as one of the most important H2 S-producing bacterial species in iced stored fish and may act as spoilage organism through the reduction of trimethylamine N-Oxide (TMAO). Four strains of S. baltica were isolated from spoiled whiting (Merlangius merlangus), and description of three H2 S-positive strains and one H2 S-negative strain of S. baltica is highlighted in this short paper. Consequently, H2 S production might not be the most appropriate screening parameter to assess the development of spoilage organisms.

The Bayesian microbial subtyping attribution model: robustness to prior information and a proposition.

Attributing foodborne illnesses to food sources is essential to conceive, prioritize, and assess the impact of public health policy measures. The Bayesian microbial subtyping attribution model by Hald et al. is one of the most advanced approaches to attribute sporadic cases; it namely allows taking into account the level of exposure to the sources and the differences between bacterial types and between sources. This step forward requires introducing type and source-dependent parameters, and generates overparameterization, which was addressed in Hald's paper by setting some parameters to constant values. We question the impact of the choices made for the parameterization (parameters set and values used) on model robustness and propose an alternative parameterization for the Hald model. We illustrate this analysis with the 2005 French data set of non-typhi Salmonella. Mullner's modified Hald model and a simple deterministic model were used to compare the results and assess the accuracy of the estimates. Setting the parameters for bacterial types specific to a unique source instead of the most frequent one and using data-based values instead of arbitrary values enhanced the convergence and adequacy of the estimates and led to attribution estimates consistent with the other models' results. The type and source parameters estimates were also coherent with Mullner's model estimates. The model appeared to be highly sensitive to parameterization. The proposed solution based on specific types and data-based values improved the robustness of estimates and enabled the use of this highly valuable tool successfully with the French data set.

Molecular identification in monophasic and nonmotile variants of Salmonella enterica serovar Typhimurium.

Variant strains of Salmonella enterica serovar Typhimurium, lacking one or both flagellar phases have been widely reported. The monophasic S.1,4,[5],12:i:- variant has emerged worldwide in the past few years and has become one of the most frequently encountered in many countries. In contrast, monophasic S.1,4,[5],12:-:1,2 and nonmotile S.1,4,[5],12:-:- strains are rarely described. This study investigated seven molecular markers to identify and delineate monophasic S.1,4,[5],12:i:- (n = 90), S.1,4,[5],12:-:1,2 (n = 25), nonmotile S.1,4,[5],12:-:- (n = 17) strains, and some serovar Typhimurium strains (n = 124) collected through the French Salmonella network between 2001 and 2010. Three markers were commonly detected in serovar Typhimurium and in all variant strains: STM2757, mdh and fliA-B. Monophasic S.1,4,[5],12:i:- were genotypically confirmed by the absence of the fljB, fljA, and hin genes. Nevertheless, 13 (14.5%) of them were positive for these last three genes, revealing monophasic strains named "inconsistent" as previously described. All nonmotile 1,4,[5],12:-:- strains had the fliC, fljA, fljB, and hin genes and the fliC gene was detected in 88% of monophasic S.1,4,[5],12:-:1,2 strains. The combination of the seven markers detection enables to recognize eight different genotypes within the S.1,4,[5],12:i:- collection, among which the Spanish and the U.S. clones previously described could be distinguished and assigned to a genotype. Based on this molecular approach, 71% of the French S.1,4,[5],12:i:- collection belonged to the Spanish clone, whereas only 2% were assigned to the U.S. clone. This study highlights the usefulness of these molecular markers and genotypes for identifying lineages, especially among the epidemiologically important monophasic S.1,4,[5],12:i:- variant.

Epidemiological and microbiological investigation of a large outbreak of monophasic Salmonella Typhimurium 4,5,12:i:- in schools associated with imported beef in Poitiers, France, October 2010.

An outbreak due to the emerging monophasic Salmonella Typhimurium 4,5,12:i:- occurred in four schools in Poitiers in October 2010. Food trace-back investigation led to the identification of beef burgers as the cause of the outbreak and their subsequent withdrawal. The Institute for Public Health Surveillance conducted a retrospective epidemiological investigation to assess the extent of the outbreak and describe cases. Self-administered questionnaires were completed by students and personnel attending each of the four schools affected. Clinical cases were defined as anyone having eaten at the school when the beef burgers were served and reporting diarrhoea or fever with at least one digestive symptom (nausea, vomiting or abdominal pain), within five days after the incriminated school meal or with unknown date of onset within a 15-day period after the incriminated school meal. Of 1,559 persons exposed, 554 clinical cases were identified corresponding to an overall attack rate of 35.5%. Of 554 clinical cases, a total of 286 (53%) sought medical care and 31 (6%) were hospitalised for more than 24 hours. This multi-school outbreak is one of the biggest food-borne outbreaks of monophasic Salmonella Typhimurium 4,5,12:i:- described in France. Prompt notification of cases and rapid identification and withdrawal of the incriminated batch of beef burgers was crucial to limit the extension of this outbreak.

Methicillin-resistant Staphylococcus aureus ST398 contamination and transmission in pigs after a low dose inoculation.

AIMS: Methicillin-resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. METHODS AND RESULTS: Twelve specific pathogen-free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 10(4) colony-forming units per animal, and three naïve pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naïve contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. CONCLUSION: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. SIGNIFICANCE AND IMPACT OF THE STUDY: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 10(4) colony-forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.

Nationwide outbreak of Salmonella enterica serotype 4,[5],12:i:- infection associated with consumption of dried pork sausage, France, November to December 2011.

An outbreak of the monophasic variant of Salmonella enterica serotype 4,[5],12:i:- occurred in November and December 2011 in France. Epidemiological investigation and food investigation with the help of supermarket loyalty cards suggested dried pork sausage from one producer as the most likely source of the outbreak. Despite the absence of positive food samples, control measures including withdrawal and recall were implemented.

Automated early warning system for the surveillance of Salmonella isolated in the agro-food chain in France.

Non-typhic Salmonella is one of the major bacterial pathogens that cause foodborne infections as well as economic losses for the food production industry. There is therefore a need to improve early detection to prevent the emergence and spread of Salmonella within the agro-food chain. The passive laboratory-based surveillance system of the Salmonella network has been integrated into the French Food Safety Agency's working plan. The objective of this study was to evaluate the ability of this network to detect unusual Salmonella contamination as early as possible in the agro-food chain. Three statistical methods were used to detect unusual events from the time-series of counts. After an experimental period of more than 1 year, this approach detected several unusual events linked to contamination in the agro-food chain that were confirmed in a timely manner at national or regional levels. This evaluation also reinforced the position of the Salmonella network as an integral part of the national public health surveillance system.

Characterization of isolates of Listeria monocytogenes from sludge using pulsed-field gel electrophoresis and virulence assays.

AIMS: To study the diversity and virulence of Listeria monocytogenes isolated from sludge. METHODS AND RESULTS: A total of 60 isolates of L. monocytogenes from sludge were characterized by serotyping, PFGE typing and using in vitro and in vivo virulence assays. The PFGE patterns were compared with those of food and human isolates to determine whether specific group clones are associated with environmental samples. The 60 isolates gave 44 different combined ApaI/AscI PFGE patterns. The PFGE patterns of most isolates were similar or very similar to those of epidemic isolates. The majority (93%) of isolates were found to be virulent by plaque-forming assay and by mouse virulence assay. CONCLUSIONS: Our findings suggest that L. monocytogenes strains found in non-sanitized sludge are virulent and represent a potential health hazard. Although no case of listeriosis related to sludge spread onto agricultural land has been reported, particular attention to this pathogen is needed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study dealing with the characterization of L. monocytogenes isolates from non-sanitized sludge samples by molecular typing methods and in vitro and in vivo virulence assays. Our findings provide relevant information for evaluating the health risks associated with spreading sludge onto agricultural land.

A Listeria monocytogenes strain is still virulent despite nonfunctional major virulence genes.

The low-virulence Listeria monocytogenes strains have been previously assigned to 4 phenotypic groups. This study aimed to characterize the A23 strain, which exhibits a pulsed-field gel electrophoresis profile specific to low-virulence strains. This strain has the same causal mutations as the group III strains and a supplementary mutation in the mpl gene, leading to the absence of internalin A expression and the presence of inactive internalin B, phosphatidyl-inositol phospholipase C, and phosphatidylcholine phospholipase C. Despite these mutations in major virulence genes, the A23 strain formed plaques in cell monolayers and contaminated 100% of inoculated mice, suggesting that it evolved from group III strains by acquiring new virulence genes.

Outbreak of Salmonella enterica serotype Muenster infections associated with goat's cheese, France, March 2008.

Salmonella enterica serotype Muenster (hereafter referred to as S. Muenster) is rare in France and in Europe. In France, a nationwide outbreak of gastrointestinal illness due to S. Muenster occurred during March and April 2008. Twenty-five laboratory-confirmed cases of S. Muenster were documented by telephone using a trawling questionnaire. Four patients were admitted to hospital and no death was recorded. Among the 21 interviewed cases, 16 reported consumption of goat's cheese in the days prior to symptoms. The investigation incriminated goat's cheese from producer X as being the most likely source of the outbreak. S. Muenster was isolated from both cases and the incriminated goat's cheese. The pulsed-field gel electrophoresis profiles of the food isolates of producer X and the isolates from cases were indistinguishable. Following the withdrawal of the contaminated batch of cheese, the number of cases decreased to its usual level. To our knowledge, this is the first published outbreak of S. Muenster associated with food consumption in Europe.

Prevalence of low-virulence Listeria monocytogenes strains from different foods and environments.

Various studies have demonstrated variations in the levels of virulence of different L. monocytogenes strains. In our laboratory, a plaque-forming assay followed by subcutaneous footpad inoculation of mice enabled us to estimate the prevalence of the low-virulence strains. This value fell from 16.3% to 1.7% with bacteria collected before 1994 and after 1997 respectively. This could be related to the modification in 1997 of the reference method EN ISO 11 290-1 of Listeria detection which recommended the use of polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) medium. The aim of this study was to determine whether the percentage of low-virulence strains detected has changed due to the modification of the detection method recommending the use of the ALOA medium. After analyzing 380 L. monocytogenes strains, no increase in the percentage of low-virulence strains could be detected. The prevalence reached only 2.6% (ten of the 380 strains tested). The low virulence of L. monocytogenes strains was not related to rare serotypes and was also observed in serotypes usually involved in human disease. Low-virulence strains were found in dairy, meat, ready-to-eat products and also in the environment, highlighting the absence of one specific source. These results are discussed in terms of detection methods and the definition of low virulence.

Characteristics of Salmonella contamination of broilers and slaughterhouses in the region of Constantine (Algeria).

The present study provides the first data about the prevalence of Salmonella contamination of broilers and slaughterhouses in the region of Constantine, Algeria. The serotypes and anti-microbial resistance phenotypes of the isolates were determined, and risk factors contributing to the contamination were evaluated. A total of 2490 samples, 1800 originating from 30 broiler farms and 690 from 15 slaughterhouses, were taken during two periods: March 2005-June 2006 and September 2006-March 2007. Salmonella contamination concerned 37% of the broiler farms and 53% of the slaughterhouses. Among the 55 isolates recovered, 10 different serotypes were identified. The most frequently recovered serotypes in both slaughterhouses and breeder farms were S. Hadar (36%, n = 20), S. Virchow (16%, n = 9), S. Infantis (10.9%, n = 6), S. Albany (11%, n = 6) and S. Carnac (7%, n = 4). Isolates belonging to S. Heidelberg (2%, n = 1) and S. Rissen (2%, n = 1) were found only in farms, while those belonging to S. Typhimurium (9%, n = 5), S. Enteritidis (4%, n = 2) and S. Montevideo (2%, n = 1) were recovered only from slaughterhouses. Thirty-nine isolates (80%) were resistant to at least one anti-microbial and 51% were multi-resistant, i.e. resistant to two or more anti-microbial molecules. About 58% (n = 32) were resistant to streptomycin, 36% (n = 20) to tetracyclines, 27% (n = 15) to nalidixic acid, 13% (n = 7) to ofloxacin and one isolate to enrofloxacin. Finally, seven distinct anti-microbial resistance profiles were identified. In parallel, four risk factors were found to be significantly associated with Salmonella contamination. Together with the huge spread of Salmonella in the broiler production chain in Constantine, Algeria, these risk factors highlight the hazards of the broiler channels, particularly linked to poor technical and hygiene practices.

Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France.

Enterotoxins produced by Staphylococcus aureus are responsible for staphylococcal food-poisoning outbreaks (SFPO). In France, SFPO are the second cause of food-borne diseases after Salmonella. However, very little is known about the strains involved. The objective of this study was to characterize the staphylococcal strains related to these SFPO through phenotypic and genotypic analyses. A total of 178 coagulase-positive staphylococcal isolates recovered from 31 SFPO (1981-2002) were screened through biotyping. Thirty-three strains representative of the different biotypes in each SFPO were further examined for SmaI macrorestriction-type, phage-type, resistance to various antimicrobial drugs, presence of staphylococcal enterotoxin (se) genes sea to sei, and production of enterotoxins SEA to SED. All these 33 strains were identified as S. aureus species: 27 were of human biotypes and six ovine or non-host-specific biotypes. Most (74.1%) strains reacted with group III phages. Eleven strains were resistant to at least two classes of antibiotics and among them, two were resistant to methicillin. Twenty-nine strains carried one or several of the eight se genes tested; the gene sea was most common (n=23), and often linked to sed (n=12) or seh (n=5). The novel se genes seg-i were in all cases associated with se genes sea to sed except for one strain which carried only seg and sei. Pulsed-Field Gel Electrophoresis (PFGE) of SmaI macrorestriction digests of the 33 strains discriminated 32 PFGE patterns grouped into nine biotype-specific clusters. All five strains carrying sea and seh were grouped together into the same sub-cluster. Three of the four se-gene-negative strains were in one PFGE cluster: all four should be tested for se genes not included in this study and, if negative, be further investigated for the presence of unidentified SEs.

Trends in antimicrobial resistance phenotypes in non-typhoid Salmonellae from human and poultry origins in France.

A total of 1873 strains from human origin and 4283 strains from non-human origin of Salmonella enterica serotypes Typhimurium, Enteritidis, Heidelberg, Hadar and Virchow, collected over three years 1993, 1997 and 2000, were examined in order to determine the rate of antimicrobial resistance to 12 antimicrobial drugs. The objective of the study was to describe and to compare the evolution of the main resistance types in human and non-human isolates, focusing on the poultry sector. The evolution and the rates of antimicrobial resistances for the five serotypes, with the exception of Virchow, were almost comparable in strains isolated from human and non-human sources over the period studied. The most striking result concerning single resistance was the spectacular increase of the resistance frequency to nalidixic acid for the strains belonging to serotypes Hadar and Virchow, especially in the poultry food sector (14% in 1993 vs. 72% in 2000 for Salmonella Virchow, 4% in 1993 vs. 70% in 2000 for Salmonella Hadar) and also in human isolates (24% in 1997 vs. 48% in 2000 for S. Virchow, 31% in 1997 vs. 78% in 2000 for S. Hadar). In addition to the classical resistance to ampicillin, streptomycin, sulphonamide, chloramphenicol and tetracycline (ASSuCT resistance type), which stabilized between 1997 and 2000, the emergence of a new resistance type was observed.

Outbreak of Salmonella enterica serotype Manhattan infection associated with meat products, France, 2005.

Between August 2005 and March 2006 in France, 69 cases of Salmonella enterica serotype Manhattan (Salmonella Manhattan) were reported, 51 (74%) of them from southeastern France. At the time of the alert (November 2005), 13 cases and 33 controls were interviewed. Cases were more likely than controls to have eaten pork sausages (OR=5.9, confidence interval CI [1.3; 26.9]) and beef (OR=9.3, CI [1.3; 68.6]). At the same time, 19 strains of Salmonella Manhattan isolated from meat products in southeastern France, reported to the French food safety agency (Afssa, Agence francaise de securite sanitaire des aliments) in September and November 2005, had an indistinguishable PFGE profile to the 7 human isolates of Salmonella Manhattan from the outbreak in southeastern France. Trace-back investigations revealed that pork samples came from one wholesaler whose pork products had tested positive for S. Manhattan during routine food testing in August 2005. This wholesaler supplied retail outlets in southeastern France. Additionally, a slaughterhouse supplying the wholesaler was inspected and widespread contamination with Salmonella spp. and S. Manhattan was found. Cooperation between the national agencies in charge of human health (Institut de veille sanitaire, InVS) and food safety (Afssa) allowed us to determine the most probable source of contamination and to take appropriate control measures.

Outbreak of Salmonella enterica serotype Manhattan infection associated with meat products, France, 2005.

Between August 2005 and March 2006 in France, 69 cases of Salmonella enterica serotype Manhattan (Salmonella Manhattan) were reported, 51 (74%) of them from southeastern France. At the time of the alert (November 2005), 13 cases and 33 controls were interviewed. Cases were more likely than controls to have eaten pork sausages (OR=5.9, confidence interval CI [1.3; 26.9]) and beef (OR=9.3, CI [1.3; 68.6]). At the same time, 19 strains of Salmonella Manhattan isolated from meat products in southeastern France, reported to the French food safety agency (Afssa, Agence française de sécurité sanitaire des aliments) in September and November 2005, had an indistinguishable PFGE profile to the 7 human isolates of Salmonella Manhattan from the outbreak in southeastern France. Trace-back investigations revealed that pork samples came from one wholesaler whose pork products had tested positive for S. Manhattan during routine food testing in August 2005. This wholesaler supplied retail outlets in southeastern France. Additionally, a slaughterhouse supplying the wholesaler was inspected and widespread contamination with Salmonella spp. and S. Manhattan was found. Cooperation between the national agencies in charge of human health (Institut de veille sanitaire, InVS) and food safety (Afssa) allowed us to determine the most probable source of contamination and to take appropriate control measures.

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples.

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.

Two outbreaks of Salmonella enteritidis phage type 8 linked to the consumption of Cantal cheese made with raw milk, France, 2001.

Salmonelloses are one are the main causes of foodborne infections in industrialised countries. In France, the incidence of human salmonellosis recorded by the National Reference Centre for Salmonella and Shigella (CNRSS) in 2001 was 21 cases per 100,000 inhabitants, and Salmonella serotype Enteritidis represented 39% of cases (1). This article reports the investigation results of two community outbreaks of salmonellosis that occurred simultaneously in the south west of France, and which were linked to the consumption of cheese made from raw milk.

Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments.

AIMS: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach. METHODS AND RESULTS: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an 'abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the 'abattoir' biotype were clustered in one separate sub-cluster. CONCLUSIONS: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing. SIGNIFICANCE AND IMPACT OF THE STUDY: The PFGE typing confirms the 'abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food.