Anthony Pinon

Development and Validation of Experimental Protocols for Use of Cardinal Models for Prediction of Microorganism Growth in Food Products

ABSTRACT An experimental protocol to validate secondary-model application to foods was suggested. Escherichia coli, Listeria monocytogenes, Bacillus cereus, Clostridium perfringens, and Salmonella were observed in various food categories, such as meat, dairy, egg, or seafood products. The secondary model validated in this study was based on the gamma concept, in which the environmental factors temperature, pH, and water activity (aw) were introduced as individual terms with microbe-dependent parameters, and the effect of foodstuffs on the growth rates of these species was described with a food- and microbe-dependent parameter. This food-oriented approach was carried out by challenge testing, generally at 15 and 10°C for L. monocytogenes, E. coli, B. cereus, and Salmonella and at 25 and 20°C for C. perfringens. About 222 kinetics in foods were generated. The results were compared to simulations generated by existing software, such as PMP. The bias factor was also calculated. The methodology to obtain a food-dependent parameter (fitting step) and therefore to compare results given by models with new independent data (validation step) is discussed in regard to its food safety application. The proposed methods were used within the French national program of predictive microbiology, Sym′Previus, to include challenge test results in the database and to obtain predictive models designed for microbial growth in food products.

Design of challenge testing experiments to assess the variability of Listeria monocytogenes growth in foods.

The assessment of the evolution of micro-organisms naturally contaminating food must take into account the variability of biological factors, food characteristics and storage conditions. A research project involving eight French laboratories was conducted to quantify the variability of growth parameters of Listeria monocytogenes obtained by challenge testing in five food products. The residual variability corresponded to a coefficient of variation (CV) of approximately 20% for the growth rate (μ(max)) and 130% for the parameter K = μ(max) × lag. The between-batch and between-manufacturer variability of μ(max) was very dependent on the food tested and mean CV of approximately 20 and 35% were observed for these two sources of variability, respectively. The initial physiological state variability led to a CV of 100% for the parameter K. It appeared that repeating a limited number of three challenge tests with three different batches (or manufacturers) and with different initial physiological states seems often necessary and adequate to accurately assess the variability of the behavior of L. monocytogenes in a specific food produced by a given manufacturer (or for a more general food designation).

Variable adhesion of Listeria monocytogenes isolates from food-processing facilities and clinical cases to inert surfaces

One hundred one strains of Listeria monocytogenes isolated from seafood and cheese industry samples and from patients with listeriosis were assessed using a microtiter plate method for adhesion to polystyrene and stainless steel surfaces. The adhesion rate for these strains ranged from 3.10 to 35.29% with an inoculum of 8 x 10(8) cells per well. A strong correlation was found between adhesion to polystyrene and stainless steel microtiter plates, indicating that the intrinsic ability of L. monocytogenes to adhere to inert surfaces is stronger than the influence of the surfaces physicochemical properties. The clinical strains were less adherent to inert surfaces than were the industrial strains. By integrating other factors such as location of the industrial strains, contamination type of the clinical strains, serotype, and pulsotype into the analysis, some weak but significant differences were noted. For the industrial isolates, the number of cells attached to both surfaces differed significantly depending on whether they were isolated from food or food-processing environments in the seafood and cheese industry. For clinical isolates, sporadic strains exhibited greater adhesion to polystyrene than did epidemic strains. Strains belonging to the pulsed-field gel electrophoretype clusters A and M (lineages II and I, respectively) were less able to adhere to polystyrene and stainless steel than were strains in the more common clusters.

Validation of a stochastic modelling approach for Listeria monocytogenes growth in refrigerated foods

A stochastic modelling approach was developed to describe the distribution of Listeria monocytogenes contamination in foods throughout their shelf life. This model was designed to include the main sources of variability leading to a scattering of natural contaminations observed in food portions: the variability of the initial contamination, the variability of the biological parameters such as cardinal values and growth parameters, the variability of individual cell behaviours, the variability of pH and water activity of food as well as portion size, and the variability of storage temperatures. Simulated distributions of contamination were compared to observed distributions obtained on 5 day-old and 11 day-old cheese curd surfaces artificially contaminated with between 10 and 80 stressed cells and stored at 14°C, to a distribution observed in cold smoked salmon artificially contaminated with approximately 13 stressed cells and stored at 8°C, and to contaminations observed in naturally contaminated batches of smoked salmon processed by 10 manufacturers and stored for 10 days a 4°C and then for 20 days at 8°C. The variability of simulated contaminations was close to that observed for artificially and naturally contaminated foods leading to simulated statistical distributions properly describing the observed distributions. This model seems relevant to take into consideration the natural variability of processes governing the microbial behaviour in foods and is an effective approach to assess, for instance, the probability to exceed a critical threshold during the storage of foods like the limit of 100 CFU/g in the case of L. monocytogenes.

Long-Term Colonization with Pneumocystis jirovecii in Hospital Staffs: A Challenge to Prevent Nosocomial Pneumocystosis

In a recent work we explored the ability of healthy hosts to contract Pneumocystis transient infections from hosts with Pneumocystis pneumonia (PcP) for transmitting it to immunocompromised hosts [3]. We showed that Balb/c mice transiently infected by contact with Pneumocystis-infected SCID mice were able to transmit Pneumocystis to other SCID mice, who developed PcP. Using the same model, another team has recently confirmed these results [2,5]. Since Pneumocystis DNA has been detected in health care workers in contact with PcP patients [6,10], this finding is relevant for at least two reasons. Firstly, these health care workers, like the Balb/c mouse carriers [2,3,5], may play a role in the circulation of P. jirovecii in the hospital environment. Secondly, health care worker carriers could play a role as reservoirs that contributed to the survival of Pneumocystis within human populations. In the present work we explored the impact of P. jirovecii carriage among the staff members of our teaching hospital. We examined the prevalence of Pneumocystis carriage, evaluated the duration of carriage as well as the potential influence of barrier systems. The methodology described herein provides a way for approaching the question of the source of Pneumocystis infection in hospitals.

Cryptosporidium parvum, a potential cause of colic adenocarcinoma

BackgroundCryptosporidiosis represents a major public health problem. This infection has been reported worldwide as a frequent cause of diarrhoea. Particularly, it remains a clinically significant opportunistic infection among immunocompromised patients, causing potentially life-threatening diarrhoea in HIV-infected persons. However, the understanding about different aspects of this infection such as invasion, transmission and pathogenesis is problematic. Additionally, it has been difficult to find suitable animal models for propagation of this parasite. Efforts are needed to develop reproducible animal models allowing both the routine passage of different species and approaching unclear aspects of Cryptosporidium infection, especially in the pathophysiology field.ResultsWe developed a model using adult severe combined immunodeficiency (SCID) mice inoculated with Cryptosporidium parvum or Cryptosporidium muris while treated or not with Dexamethasone (Dex) in order to investigate divergences in prepatent period, oocyst shedding or clinical and histopathological manifestations. C. muris-infected mice showed high levels of oocysts excretion, whatever the chemical immunosuppression status. Pre-patent periods were 11 days and 9.7 days in average in Dex treated and untreated mice, respectively. Parasite infection was restricted to the stomach, and had a clear preferential colonization for fundic area in both groups. Among C. parvum-infected mice, Dex-treated SCID mice became chronic shedders with a prepatent period of 6.2 days in average. C. parvum-inoculated mice treated with Dex developed glandular cystic polyps with areas of intraepithelial neoplasia, and also with the presence of intramucosal adenocarcinoma.ConclusionFor the first time C. parvum is associated with the formation of polyps and adenocarcinoma lesions in the gut of Dex-treated SCID mice. Additionally, we have developed a model to compare chronic muris and parvum cryptosporidiosis using SCID mice treated with corticoids. This reproducible model has facilitated the evaluation of clinical signs, oocyst shedding, location of the infection, pathogenicity, and histopathological changes in the gastrointestinal tract, indicating divergent effects of Dex according to Cryptosporidium species causing infection.

Development and Validation of a Concentration Method for the Detection of Influenza A Viruses from Large Volumes of Surface Water

ABSTRACT Contamination of lakes and ponds plays an essential role as a reservoir of avian influenza A virus (AIV) in the environment. A method to concentrate waterborne AIV is a prerequisite for the detection of virus present at low levels in water. The aim of this study was to develop and validate a method for the concentration and detection of infectious AIV from large volumes of surface water samples. Two filtration systems, glass wool and electropositive NanoCeram filter, were studied. The individual effects of filtration-elution and polyethylene glycol (PEG) concentration parameters on the recovery efficiency of the H1N1 strain from 10-liter surface water samples were assessed. An ultimate 1% recovery rate of infectious viruses was achieved with the optimal protocol, corresponding to filtration through glass wool, followed by a viral elution step and then a PEG concentration. This method was validated for the detection of highly pathogenic H5N1 strains from artificially contaminated larger water volumes, from 10 to up to 50 liters, from different sources. The viral recovery efficiencies ranged from 0.01% to 7.89% and from 3.63% to 13.79% with lake water and rainwater, respectively. A theoretical detection threshold of 2.25 × 102 TCID50 (50% tissue culture infectious dose) in the filtered volume was obtained for seeded lake waters by M gene reverse transcriptase PCR (RT-PCR). Moreover, the method was used successfully in field studies for the detection of naturally occurring influenza A viruses in lake water in France.

Adhesion of human pathogenic enteric viruses and surrogate viruses to inert and vegetal food surfaces

Enteric viruses, particularly human Noroviruses (NoV) and hepatitis A virus (HAV), are key food-borne pathogens. The attachment of these pathogens to foodstuff and food-contact surfaces is an important mechanism in the human contamination process. Studies were done to investigate the nature of the physicochemical forces, such as hydrophobic and electrostatic ones, involved in the interaction virus/matrix but, at this day, only few data are available concerning surface properties of viruses and prediction of the adhesion capacity of one specific virus onto matrices is still very difficult. The purpose of this study was to propose a reference system, including a representative virus surrogate, able to predict as close as possible behaviour of pathogenic viruses in term of adhesion on inert (stainless steel and polypropylene) and food surfaces (lettuce leaves, strawberries and raspberries). The adhesion of human pathogenic enteric viruses, cultivable strain of HAV and non-cultivable strains of human NoV (genogroups I and II), have been quantified and compared to these of human enteric viruses surrogates, included the MNV-1 and three F-specific RNA bacteriophages (MS2, GA and Qβ). A standardized approach was developed to assess and quantify viral adhesion on tested matrices after a contact time with each virus using real-time RT-PCR. Methods used for virus recovery were in accordance with the CEN recommendations, including a bovine Enterovirus type 1 as control to monitor the efficiency of the extraction process and amplification procedure from directly extracted or eluted samples. The adhesion of human pathogenic viruses, ranging from 0.1 to 2%, could be comparable for all matrices studied, except for NoV GII on soft fruits. Adhesion percentages obtained for the studied surrogate virus and phages were shown to be comparable to those of HAV and NoV on inert and lettuce surfaces. The MNV-1 appeared as the best candidate to simulate adhesion phenomena of all human pathogenic enteric viruses on all studied surfaces, while MS2 and GA bacteriophages could be a good alternative as model of viral adhesion on inert and lettuce surfaces. These results will be usable to design relevant experimental systems integrating adhesion behaviour of enteric viruses in the assessment of the efficiency of a technological or hygienic industrial process.

Prevalence and Risk Factors for Intestinal Protozoan Infections with Cryptosporidium, Giardia, Blastocystis and Dientamoeba among Schoolchildren in Tripoli, Lebanon

Background Intestinal protozoan infections are confirmed as major causes of diarrhea, particularly in children, and represent a significant, but often neglected, threat to public health. No recent data were available in Lebanon concerning the molecular epidemiology of protozoan infections in children, a vulnerable population at high risk of infection. Methodology and Principal Findings In order to improve our understanding of the epidemiology of intestinal pathogenic protozoa, a cross-sectional study was conducted in a general pediatric population including both symptomatic and asymptomatic subjects. After obtaining informed consent from the parents or legal guardians, stool samples were collected in January 2013 from 249 children in 2 schools in Tripoli, Lebanon. Information obtained from a standard questionnaire included demographic characteristics, current symptoms, socioeconomic status, source of drinking water, and personal hygiene habits. After fecal examination by both microscopy and molecular tools, the overall prevalence of parasitic infections was recorded as 85%. Blastocystis spp. presented the highest infection rate (63%), followed by Dientamoeba fragilis (60.6%), Giardia duodenalis (28.5%) and Cryptosporidium spp. (10.4%). PCR was also performed to identify species and genotypes of Cryptosporidium, subtypes of Blastocystis, and assemblages of Giardia. Statistical analysis using a logistic regression model showed that contact with family members presenting gastrointestinal disorders was the primary risk factor for transmission of these protozoa. Conclusions This is the first study performed in Lebanon reporting the prevalence and the clinical and molecular epidemiological data associated with intestinal protozoan infections among schoolchildren in Tripoli. A high prevalence of protozoan parasites was found, with Blastocystis spp. being the most predominant protozoans. Although only 50% of children reported digestive symptoms, asymptomatic infection was observed, and these children may act as unidentified carriers. This survey provides necessary information for designing prevention and control strategies to reduce the burden of these protozoan infections, especially in children.

A predictive microbiology approach for thermal inactivation of Hepatitis A virus in acidified berries

Hepatitis A virus (HAV) is a food-borne enteric virus responsible for outbreaks of hepatitis associated with consumption of raw vegetables. Soft fruits, such as red berries, exposed to faecal contamination are increasingly responsible for collective food-borne illnesses associated with HAV, when eaten raw or used in unprocessed foods. Heat is the most effective measure for the inactivation of HAV. Thermal treatments are used on fruits as a decontamination method, but they have to be adapted to product characteristics; indeed, factors such as sugar or pH may have an impact on the viral sensitivity to thermal treatments. A model was developed for the inactivation of HAV in red berries without supplemented sugar and with different pH values. Nonlinear inactivation curves in acidified raspberries were modelled using an integrated model, with a single equation nesting secondary models of temperature and pH in the primary model. Model predictions were then confronted to experimental results obtained in another laboratory on other berries with different pH values. Excellent predictions were obtained in most cases, while failed predictions provided safe results, with the model predicting higher residual virus titres than what was observed.