Frédérique Pasquali

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

Occurrence of Helicobacter pullorum in turkeys.

In order to investigate the occurrence of Helicobacter pullorum in turkeys, caecum contents collected at the slaughterhouse from 55 animals intensively reared in 11 farms were sampled. Gram-negative curved rod bacteria were isolated by a modified Steele and McDermott filter technique and further identified as H. pullorum by polymerase chain reaction (PCR). Eleven and 31 isolates, randomly selected from each positive farm, underwent phenotypic (biochemical and antibiotic susceptibility tests) and genotypic characterization (PFGE and AFLP analysis), respectively. Forty-two out of 55 animals (76.4%) and all the 11 farms sampled were positive for H. pullorum. Isolates showed similar biochemical characteristics and whole cell protein profiles but showed a high degree of genetic heterogeneity. Ten out of 11 isolates were resistant to one or more antibiotics with erythromycin, ciprofloxacin and nalidixic acid resistance being the most frequently detected. This is the first description of H. pullorum in turkeys. H. pullorum is a frequent intestinal colonizer in turkeys; therefore, attention should be given to clarify the food-borne risk linked to carcass contamination. Antibiotic resistance is a concern since high values of resistance rates were observed.

Isolation of Arcobacter Species in Water Buffaloes (Bubalus bubalis)

This is the first report of Arcobacter spp. in rectal fecal samples from healthy water buffaloes (Bubalus bubalis) reared on a dairy farm. Arcobacter species were isolated after enrichment, and isolates were identified at species level by multiplex-polymerase chain reaction assay. Thirty samples were examined and Arcobacter spp. were isolated from 96.7% of water buffaloes tested: 38 Arcobacter spp. isolates were obtained, with A. cryaerophilus as the dominant species followed by A. butzleri and A. skirrowii. Nine animals (31%) were colonized by more than one Arcobacter species. The present study indicates that water buffaloes can harbor a variety of Arcobacter spp. and that healthy buffaloes may act as hosts. Water buffalo fecal shedding of Arcobacter spp. may be of significance to human health, considering the potential fecal contamination during harvesting of raw milk and slaughtering.

Enumeration of Salmonellae in Table Eggs, Pasteurized Egg Products, and Egg-Containing Dishes by Using Quantitative Real-Time PCR

ABSTRACT Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (Cq ) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products.

Campylobacter control strategies in European poultry production

In 2008 as in previous four years, campylobacteriosis was the most frequently reported zoonotic disease in humans in the European Union (EU) with fresh poultry meat as one of the most important reservoir of human infection (EFSA, 2010a). The reduction of campylobacter prevalence and load in live poultry is believed to be one of the most effective ways of reducing the contamination of foodstuffs and the number of human campylobacter cases. On this purpose some European Member States adopted national campylobacter control or monitoring programs but a European strategy to reduce campylobacter is still missing. The first step in this direction has been a European Union-wide baseline survey carried out in 2008 at slaughterhouses to obtain comparable values of prevalence of campylobacter in broiler batches and on broiler carcasses for all Member States. Current pre-harvest strategies available to reduce campylobacter contamination in poultry production include the application of on-farm biosecurity measures, the decontamination of litter, and the supplementation of feed with compounds inhibiting campylobacter and the treatment of drinking water. Moreover, novel strategies, specifically targeting campylobacter control at pre-harvest level, are in progress, including administration of probiotics, vaccination, antibiotics used in combination with molecule able to prevent the emergence of antibiotic resistance and antimicrobial alternatives (i.e. bacteriophages, bacteriocins). This paper is an overview on pre-harvest control strategies.

Genetic diversity of Escherichia coli isolates of animal and environmental origins from an integrated poultry production chain

Escherichia coli is a normal inhabitant of the intestinal tract of chickens, but when an imbalance in the gut microbiota occurs, E. coli may overgrow and cause extraintestinal infections. The aims of this study were to assess the distribution and spread of E. coli isolates with specific phylogenetic groups and antimicrobial resistance characters among asymptomatic breeder flocks and their broiler progenies with early symptoms of colibacillosis. Broiler flocks were treated with lincospectin during the first week of life and sampled at one, 21 and 42 days. The majority of the 363 E. coli isolates belonged to phylogenetic group A (53.17%), followed by groups D (23.14%), B1 (19.28%) and B2 (4.41%). In broilers, group A was the most represented in birds of 21 and 42 days of age whereas group B1 was the most represented phylogroup in one-day old chicks. More than 90.00% of the isolates were resistant to one or more antimicrobials. Along the life-time of broilers, no differences were found on the occurrence of resistant isolates except for the number of E. coli with elevated MIC to spectinomycin, which increased significantly after the lincospectin treatment. According to XbaI-macrorestriction analysis, a high genetic diversity among E. coli isolates was underlined. Four antimicrobial resistant E. coli isolates of phylogroups A, B1 and D collected from breeders showed similar PFGE patterns to five isolates collected from the respective broiler progenies suggesting a potential spread of these isolates from breeders to broilers.

Improvement of sampling plans for Salmonella detection in pooled table eggs by use of real-time PCR.

Eggs and egg products have been described as the most critical food vehicles of salmonellosis. The prevalence and level of contamination of Salmonella on table eggs are low, which severely affects the sensitivity of sampling plans applied voluntarily in some European countries, where one to five pools of 10 eggs are tested by the culture based reference method ISO 6579:2004. In the current study we have compared the testing-sensitivity of the reference culture method ISO 6579:2004 and an alternative real-time PCR method on Salmonella contaminated egg-pool of different sizes (4-9 uninfected eggs mixed with one contaminated egg) and contamination levels (10°-10(1), 10(1)-10(2), 10(2)-10(3)CFU/eggshell). Two hundred and seventy samples corresponding to 15 replicates per pool size and inoculum level were tested. At the lowest contamination level real-time PCR detected Salmonella in 40% of contaminated pools vs 12% using ISO 6579. The results were used to estimate the lowest number of sample units needed to be tested in order to have a 95% certainty not falsely to accept a contaminated lot by Monte Carlo simulation. According to this simulation, at least 16 pools of 10 eggs each are needed to be tested by ISO 6579 in order to obtain this confidence level, while the minimum number of pools to be tested was reduced to 8 pools of 9 eggs each, when real-time PCR was applied as analytical method. This result underlines the importance of including analytical methods with higher sensitivity in order to improve the efficiency of sampling and reduce the number of samples to be tested.

Model approach to estimate the probability of accepting a lot of heterogeneously contaminated powdered food using different sampling strategies

Current sampling plans assume a random distribution of microorganisms in food. However, food-borne pathogens are estimated to be heterogeneously distributed in powdered foods. This spatial distribution together with very low level of contaminations raises concern of the efficiency of current sampling plans for the detection of food-borne pathogens like Cronobacter and Salmonella in powdered foods such as powdered infant formula or powdered eggs. An alternative approach based on a Poisson distribution of the contaminated part of the lot (Habraken approach) was used in order to evaluate the probability of falsely accepting a contaminated lot of powdered food when different sampling strategies were simulated considering variables such as lot size, sample size, microbial concentration in the contaminated part of the lot and proportion of contaminated lot. The simulated results suggest that a sample size of 100g or more corresponds to the lower number of samples to be tested in comparison with sample sizes of 10 or 1g. Moreover, the number of samples to be tested greatly decrease if the microbial concentration is 1CFU/g instead of 0.1CFU/g or if the proportion of contamination is 0.05 instead of 0.01. Mean contaminations higher than 1CFU/g or proportions higher than 0.05 did not impact on the number of samples. The Habraken approach represents a useful tool for risk management in order to design a fit-for-purpose sampling plan for the detection of low levels of food-borne pathogens in heterogeneously contaminated powdered food. However, it must be outlined that although effective in detecting pathogens, these sampling plans are difficult to be applied since the huge number of samples that needs to be tested. Sampling does not seem an effective measure to control pathogens in powdered food.

Modified-atmosphere packaging of hen table eggs: Effects on pathogen and spoilage bacteria

As part of a more comprehensive research activity on the use of modified-atmosphere packaging for the improvement of quality and functional properties of table eggs, the effects of air, 100% CO(2), and 100% O(2) packaging were also evaluated on the survival of experimentally inoculated pathogen bacteria (Salmonella Enteritidis, Escherichia coli, and Listeria monocytogenes) as well as on spoilage bacteria (total aerobic mesophilic bacteria) on table eggs during 30 d of storage at 4, 25, and 37°C by colony count method. In general, temperatures played a major role, rather than gasses, in influencing the bacterial survival. In particular, the lowest microbial loads were registered at 4°C on E. coli and spoilage bacteria, whereas 37°C was the best storage temperature to avoid the psychrotropic microorganism L. monocytogenes development regardless of the gas used. One hundred percent CO(2) packaging, in association with a low storage temperature (4°C), had a significant positive effect in reducing Salmonella loads. On eggs inoculated with L. monocytogenes and stored at 4°C as well as on eggs containing only spoilage bacteria and stored at 25°C, 100% CO(2) resulted the best gas in comparison with air and O(2). One hundred percent CO(2) packaging showed no negative effect on pathogen survival compared with air. Although further improvements are required to control RH within packaging to limit bacteria growth/survival, in view of the positive effects of CO(2) packaging on quality traits of table eggs, 100% CO(2) packaging might represent a promising innovative technique for the maintenance of egg characteristics during transport, retail, and domestic storage.