Laurent Guillier

Comparison of chlorine and peroxyacetic-based disinfectant to inactivate Feline calicivirus, Murine norovirus and Hepatitis A virus on lettuce

In recent years, raw fruits and vegetables have frequently been involved in foodborne transmission to humans of enteric viruses, particularly noroviruses and hepatitis A virus (HAV). Although viral contamination can occur during all steps of food processing, primary production is a critical stage on which prevention measures must be focused to minimize the risk of infection to consumers. Postharvest sanitation may be a valid technological solution for decreasing the bacterial load on fresh raw material, but there is a lack of data concerning the effectiveness of this process on enteric viruses. In this study, we compared the survival of two human norovirus surrogates, the feline calicivirus (FCV), and the murine norovirus (MNV-1), and of HAV on lettuce after water washing with bubbles and with or without ultrasound, and washing with bubbles in the presence of active chlorine (15 ppm) or peroxyacetic acid-based disinfectant (100 ppm). Cell culture and quantitative RT-PCR assays were used to detect and quantify the viruses on the surface of the lettuce after the sanitizing treatments. Levels of viral inactivation on the lettuce leaves were not significantly different between washing with bubbles and washing with bubbles plus ultrasound and were not dependant on the quantification method. A simple washing without disinfectant resulted in a decrease of approximately 0.7 log units in the quantity of virus detected for HAV and FCV and of 1.0 log unit for MNV-1. In the experimental set-up including a washing step (with or without ultrasound) followed by washing for 2 min in the presence of disinfectants, 15 ppm of active chlorine was found more effective for inactivating FCV (2.9 log units) than HAV and MNV-1 (1.9 log units and 1.4 log units, respectively) whereas 100 ppm of peroxyacetic-based biocide was found effective for inactivating FCV (3.2 log units) and MNV-1 (2.3 log units), but not HAV (0.7 log units). Quantitative RT-PCR results indicated that the presence of viral RNA did not correlate with the presence of infectious viruses on disinfected lettuce, except for MNV-1 processed with chlorine (15 ppm). In comparison with water washing, a substantial additional decrease of genomic FCV titer (1.1 log units) but no significant reduction of the genomic titers of HAV and MNV-1 were found on lettuce treated with chlorine (15 ppm). No significant effect of the disinfection step of lettuce with peroxyacetic-based biocide (100 ppm peracetic acid) was found by qRT-PCR on all genomic viral titers tested. This study illustrates the necessity of determining the effectiveness of technological processes against enteric viruses, using a relevant reference such as HAV, in order to reduce the risk of hepatitis and gastroenteritis by exposure to vegetables.

Duplex real-time qRT-PCR for the detection of hepatitis A virus in water and raspberries using the MS2 bacteriophage as a process control

Hepatitis A virus (HAV) infection is the leading worldwide cause of acute viral hepatitis. An important aspect of viral control is rapid diagnosis. Epidemiological studies have linked hepatitis A outbreaks to the consumption of drinking water or soft fruits exposed to faecal contamination. Real-time reverse transcriptase PCR (qRT-PCR) is now widely used for detecting RNA viruses in food samples. Efficiency of viral concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction must be monitored to prevent false negative results. In this study, the MS2 bacteriophage used as a process control was detected simultaneously with HAV in a one-step duplex real-time qRT-PCR. The assay was developed for testing water and raspberries. Adding MS2 showed no loss of sensitivity for HAV detection in water and raspberry samples. The limit of detection of HAV with this new approach was 10PFU for 1.5L of bottled water, 100PFU for 1.5L of tap water, 50PFU for 25g of fresh raspberries and 100PFU for 25g of frozen raspberries. The data show that the MS2 offers a very reliable and simple way to monitor false-negative results, making it a valuable tool in the routine diagnostics laboratory.

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.

Prevalence and characterization of Campylobacter jejuni from chicken meat sold in French retail outlets.

Campylobacter was detected in 76% of broiler meat products collected in retail outlets during a monitoring plan carried out in France throughout 2009. Campylobacter jejuni was the most prevalent species (64.7% of products being contaminated). The 175 C. jejuni isolates collected were characterized. MLST typing results confirmed substantial genetic diversity as the 175 C. jejuni isolates generated 76 sequence types (STs). The ST-21, ST-45 and ST-464 complexes predominated accounting for 43% of all isolates. A class-specific PCR to screen the sialylated lipooligosaccharide (LOS) locus classes A, B and C showed that 50.3% of the C. jejuni isolates harbored sialylated LOS. The antimicrobial resistance profiles established using a subset of 97 isolates showed that resistance to tetracycline was the most common (53.6%), followed with ciprofloxacin and nalidixic acid (32.9%, and 32.0% respectively). All the tested isolates were susceptible to erythromycin, chloramphenicol and gentamicin. Clear associations were demonstrated between certain clonal complexes and LOS locus classes and between certain clonal complexes and antimicrobial resistance. This work paints a representative picture of C. jejuni isolated from poultry products circulating in France, providing data on STs, LOS locus classes and antibiotic resistance profiles in isolates recovered from products directly available to the consumer.

A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples

Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples.

Method for HEV detection in raw pig liver products and its implementation for naturally contaminated food.

It is now recognized that Hepatitis E virus (HEV) infection is not confined to developing countries. HEV infection is a growing public health concern in industrialized countries where the disease is mainly autochthonous, caused by HEV genotypes 3 and 4 and is today considered to be zoonotic. HEV causes acute hepatitis in humans, predominantly through contamination of food and water. Due to the low concentrations found in food and water samples, an efficient and rapid virus concentration method is required for routine control. Because of the absence of a reliable cell culture method for the main enteric viruses most commonly involved in the outbreaks, reverse transcription quantitative real time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in all types of samples. The aim of this study was to provide a rapid and sensitive method for detecting HEV in pig liver products. A method which includes a virus concentration step by PEG has been chosen from 9 protocols to be further validated. We used a one-step duplex RT-qPCR for detecting HEV and the murine norovirus (MNV-1) used as a process control for monitoring the quality of the whole extraction procedure. The mean recovery rates of the HEV and MNV-1 obtained from pig liver sausages were respectively 3.94% and 2.92%, increasing in figatelli to 18.38% and 13.11% respectively. This method also proved to be effective for HEV detection in naturally contaminated foodstuffs containing raw pig liver.

Comparison of two extraction methods for the detection of hepatitis A virus in lettuces using the murine norovirus as a process control.

Enteric viruses are important agents of foodborne diseases. In recent years, raw fruits and vegetables have frequently been involved in foodborne transmission of enteric viruses to humans, particularly noroviruses and hepatitis A virus (HAV). Although viral contamination can occur at any stage of food processing, primary production is a critical stage in which prevention measures are essential to minimise the risk of infection to consumers. Due to the low infectious doses and low concentrations of enteric viruses in food samples, an efficient and rapid virus concentration method is required for routine control and risk assessment. In this study, the virus concentration reference method proposed by the CEN/TC275/WG6/TAG4 working group for samples of soft fruits and salad vegetables was compared with a method including a filtration step in order to recover hepatitis A virus (HAV) on lettuces. Murine norovirus (MNV-1) was used as a process control and detected simultaneously with HAV in a one-step duplex RT-qPCR following both procedures. The HAV LOD ranged from 10 to 100 PFU/25g of lettuce in the presence or absence of MNV-1, regardless of method used. In conclusion, MNV-1 offers a very reliable and simple way to monitor the quality of the detection procedures. Although it has been found that both methods achieved an identical limit of detection, the method including a filtration step requires less processing and could be proposed as an alternative method.

Spatial competition with Lactococcus lactis in mixed-species continuous-flow biofilms inhibits Listeria monocytogenes growth

Surfaces in industrial settings provide a home for resident biofilms that are likely to interact with the attachment, growth and survival of pathogens such as Listeria monocytogenes. Experimental results have indicated that L. monocytogenes cells were inhibited by the presence of a model resident flora (Lactococcus lactis) in dual-species continuous flow-biofilms, and are spatially restricted to the lower biofilm layers. Using a new, simplified individual-based model (IBM) that simulates bacterial cell growth in a three-dimensional space, the spatial arrangements of the two species were reconstructed and their cell counts successfully predicted. This model showed that the difference in generation times between L. monocytogenes and L. lactis cells during the initial stages of dual-species biofilm formation was probably responsible for the species spatialization observed and the subsequent inhibition of growth of the pathogen.

Determination of which virus to use as a process control when testing for the presence of hepatitis A virus and norovirus in food and water

Noroviruses (genogroup I (NoV GI) and genogroup II (NoV GII)) and the hepatitis A virus (HAV) are frequently involved in foodborne infections worldwide. They are mainly transmitted via the fecal-oral route, direct person-to-person contact or consumption of contaminated water and foods. In food virology, detection methods are currently based on identifying viral genomes using real-time reverse transcriptase PCR (RT-qPCR). One of the general requirements for detecting these viruses in food involves the use of a process control virus to monitor the quality of the entire viral extraction procedure as described in the ISO/TS 15216-1 and 15216-2 standards published in 2013. The selected process control virus should have similar morphological and physicochemical properties as the screened pathogenic virus and thus have the potential to provide comparable extraction efficiency. The aim of this study was to determine which virus should be used for process control, murine norovirus (MNV-1) or Mengovirus, when testing for the presence of HAV, NoV GI and NoV GII in bottled water, lettuce and semi-dried tomatoes. Food samples were spiked with HAV, NoV GI or NoV GII alone or in the presence of MNV-1 or Mengovirus. Recovery rates of each pathogenic virus were compared to those of both process control viruses using a multiple comparison procedure. Neither process control virus influenced the recovery of pathogenic virus regardless of the type of food matrix. MNV-1 was the most appropriate virus for validating the detection of HAV and NoV GII in all three food matrices as well as NoV GI in lettuce. Mengovirus proved to be the most appropriate control for NoV GI detection in bottled water and semi-dried tomatoes. The process control virus is essential for validating viral detection in food and the choice of virus depends on food type and the screened pathogenic virus.

Duplex RT-qPCR for the detection of hepatitis E virus in water, using a process control

Human hepatitis E virus (HEV) causes acute hepatitis in humans, predominantly by contamination of food and water. HEV, in particular genotype III, is currently considered to be an emerging pathogen in industrialized countries. Because of the low infectious dose, an efficient and rapid virus concentration method is required to detect low amounts of HEV in food and water samples for routine control. Because of the absence of a reliable cell culture method for the main enteric viruses most involved in the outbreaks, reverse transcription quantitative real time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in food and water samples. One of the general requirements for viral diagnosis concerns the use of a process control to monitor the efficiency of the concentration of viral particles, the extraction of nucleic acid and the presence of the potential inhibitors of the RT-qPCR reaction. The aim of this study was to provide a rapid and sensitive method for detecting HEV in water. The method is based on viral concentration by filtration on membrane filters and direct lysis of adsorbed viruses from filters before RNA extraction and RT-qPCR amplification. We developed a one-step duplex RT-qPCR for detecting HEV and the murine norovirus (MNV-1) was used as a process control. The data show that MNV-1 offers a very reliable and simple way of monitoring false-negative results and is a valuable tool in the routine diagnostic laboratory. The limit of detection (LOD) was in the range of 700 to 3500 HEV genome copies/0.5L bottled water and 3500 HEV genome copies/0.5L tap water.