Alessandra De Cesare

Automated Ribotyping Using Different Enzymes To Improve Discrimination of Listeria monocytogenes Isolates, with a Particular Focus on Serotype 4b Strains

ABSTRACT To develop improved automated subtyping approaches forListeria monocytogenes, we characterized the discriminatory power of different restriction enzymes for ribotyping. When 15 different restriction enzymes were used for automated ribotyping of 16 selected L. monocytogenes isolates, the restriction enzymesEcoRI, PvuII, and XhoI showed high discriminatory ability (Simpsons index of discrimination > 0.900) and produced complete and reproducible restriction cut patterns. These three enzymes were thus evaluated for their ability to differentiate among isolates representing the two major serotype 4b epidemic clones, those having ribotype reference pattern DUP-1038 (51 isolates) and those having pattern DUP-1042 (20 isolates). Among these isolates, PvuII provided the highest discrimination for a single enzyme (nine different subtypes; index of discrimination = 0.518). A combination of PvuII and XhoI showed the highest discriminatory ability (index of discrimination = 0.590) for these isolates. A group of 44 DUP-1038 isolates and a group of 12 DUP-1042 isolates were identical to each other even when the combined data for all three enzymes were used. We conclude that automated ribotyping using different enzymes allows improved discrimination of L. monocytogenes isolates, including epidemic serotype 4b strains. We furthermore confirm that most of the isolates representing the genotypes linked to the two major epidemic L. monocytogenes clonal groups form two genetically homogeneous groups.

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.

Survival kinetics of Listeria monocytogenes on raw sheep milk cured cheese under different storage temperatures

Raw sheep milk cured cheese produced in the Castilla y Leon region (Spain) constitutes a traditional semi-hard aromatic cheese typically aged for three to six months. This product is catalogued as ready-to-eat since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens such as Listeria monocytogenes can represent a health concern for susceptible consumers. This study was aimed at evaluating the survival of L. monocytogenes on raw sheep milk cured cheese under different storage temperatures. Log-linear+shoulder and Weibull type models were fitted to data observed in order to estimate kinetic parameters. The Arrhenius relationship was further used to predict the impact of temperature on L. monocytogenes behavior during storage at 4, 12 and 22°C. Additionally, growth of lactic acid bacteria (LAB) as a representative group of the indigenous microbiota was evaluated. Results obtained indicated that the time to eradication (time when absence of L. monocytogenes in the analyzed samples was observed) was 114, 104, and 77 days for cheese samples stored at 4, 12 and 22°C, respectively. The LAB population showed an increase at 12 and 22°C during storage. However, an increase of 1 log CFU/g was observed during the first 2 weeks irrespectively of the storage temperature. The log-linear+shoulder model indicated a good fit to observed data. Likewise, the Arrhenius relationship explained sufficiently the dependency of temperature on L. monocytogenes behavior. This study demonstrated that cheese storage at ambient temperatures could lead to the preservation of its quality properties as well as its safety against L. monocytogenes.

Prevalence of Helicobacter pullorum in Conventional, Organic, and Free-Range Broilers and Typing of Isolates

ABSTRACT Helicobacter pullorum represents a potential food-borne pathogen, and avian species appear to be a relevant reservoir of this organism. In this study, the prevalence of H. pullorum was investigated at 30 conventional farms where 169 ceca from 34 flocks were tested, at eight organic farms where 39 ceca from eight flocks were tested, and at seven free-range farms where 40 ceca from eight flocks were tested. All of the ceca were obtained from healthy broiler chickens. Moreover, amplified fragment length polymorphism, pulsed-field gel electrophoresis, and automated ribotyping were employed to estimate the levels of genetic variability of H. pullorum broiler isolates within and between flocks. Overall, Gram-negative, slender, curved rods, identified as H. pullorum by PCR, were isolated at 93.3% of the farms tested. The percentage of positive free-range farms (54.2%) was significantly lower than that of conventional (100%) or organic (100%) farms (P < 0.001). The level of within-flock genetic variability, calculated as the number of flocks colonized by isolates genetically different by all of the typing methods, was 34.9%. Isolates showing identical profiles by each typing method were observed in 11.6% of the flocks, but they were never detected between flocks. However, groups of isolates clustered together with an overall similarity level of ≥85%. Our results suggest that even though a high level of genetic variability is attributable to H. pullorum broiler isolates, their hierarchical genotyping produces data useful for epidemiological investigations.

The challenge of defining risk-based metrics to improve food safety: Inputs from the BASELINE project

In 2002, the Regulation (EC) 178 of the European Parliament and of the Council states that, in order to achieve the general objective of a high level of protection of human health and life, food law shall be based on risk analysis. However, the Commission Regulation No 2073/2005 on microbiological criteria for foodstuffs requires that food business operators ensure that foodstuffs comply with the relevant microbiological criteria. Such criteria define the acceptability of a product, a batch of foodstuffs or a process, based on the absence, presence or number of micro-organisms, and/or on the quantity of their toxins/metabolites, per unit(s) of mass, volume, area or batch. The same Regulation describes a food safety criterion as a mean to define the acceptability of a product or a batch of foodstuff applicable to products placed on the market; moreover, it states a process hygiene criterion as a mean indicating the acceptable functioning of the production process. Both food safety criteria and process hygiene criteria are not based on risk analysis. On the contrary, the metrics formulated by the Codex Alimentarius Commission in 2004, named Food Safety Objective (FSO) and Performance Objective (PO), are risk-based and fit the indications of Regulation 178/2002. The main aims of this review are to illustrate the key differences between microbiological criteria and the risk-based metrics defined by the Codex Alimentarius Commission and to explore the opportunity and also the possibility to implement future European Regulations including PO and FSO as supporting parameters to microbiological criteria. This review clarifies also the implications of defining an appropriate level of human protection, how to establish FSO and PO and how to implement them in practice linked to each other through quantitative risk assessment models. The contents of this review should clarify the context for application of the results collected during the EU funded project named BASELINE (www.baselineeurope.eu) as described in the papers of this special issue. Such results show how to derive POs for specific food/biological hazard combinations selected among fish, egg, dairy, meat and plant products.

Comparison of the BAX® System with a multiplex PCR method for simultaneous detection and identification of Campylobacter jejuni and Campylobacter coli in environmental samples

The Campylobacter detection is performed by conventional culture methods and the identification of Campylobacter jejuni and Campylobacter coli is principally based on the hippurate hydrolysis test. The two major drawbacks of this biochemical test for species identification include the inconsistency of the results and the presence of atypical strains, which can lead to the misidentification of an isolate. As an alternative, multiplex polymerase chain reaction (mPCR) protocols for the simultaneous detection and identification of different Campylobacter species have been developed. This study examined the performances of an experimental BAX System assay for the C. jejuni and C. coli identification in comparison to a multiplex PCR protocol recently published. The samples tested were represented by 106 environmental swabs collected on Teflon strips and tables, stainless steel saws, hooks and trays, ceramic floors and walls, as well as equipment surfaces, located in a swine (N=50) and a poultry (N=56) slaughterhouse. The highest Campylobacter detection rate was obtained after 48 h of enrichment by using both the PCR procedures. After 24 h, the BAX System provides a more rapid and accurate Campylobacter detection and identification assay than the multiplex PCR. Except for two samples, all the broths where Campylobacter cells were detected after 24 or 48 h of enrichment, with at least one of the PCR protocols, gave Campylobacter colonies using the culture method.

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.

Ribotyping characterisation of campylobacter isolates randomly collected from different sources in Italy

In this study the potential for using the automated PstI ribotyping as a primary library typing method to survey Campylobacter and for identification of two thermophilic Campylobacter jejuni and Campylobacter coli species was evaluated. A total of 158 isolates randomly collected in Italy from different sources were analyzed. A large percentage of chicken (28%), turkey (27%) and turkey meat (25%) isolates shared their ribotyping profiles (ribotypes) with those of humans, whereas the swine isolates had unique profiles. The identification results obtained by ribotyping corresponded to those collected by using a multiplex PCR protocol specifically designed for C. jejuni and C. coli detection. The comparison of the PstI ribotyping profiles obtained in this research with those of the isolates collected over time will facilitate determining the ribotypes that are more frequently transmitted to humans in comparison to those that are normally harboured only in animals, foods and in the environment.

Fate of Salmonella enterica in a Mixed Ingredient Salad Containing Lettuce, Cheddar Cheese, and Cooked Chicken Meat

Food service and retail sectors offer consumers a variety of mixed ingredient salads that contain fresh-cut vegetables and other ingredients such as fruits, nuts, cereals, dairy products, cooked seafood, cooked meat, cured meats, or dairy products obtained from external suppliers. Little is known about the behavior of enteric bacterial pathogens in mixed ingredient salads. A model system was developed to examine the fate of Salmonella enterica (inoculum consisting of S. enterica serovars Agona, Typhimurium, Enteritidis, Brandenberg, and Kentucky) on the surface of romaine lettuce tissues incubated alone and in direct contact with Cheddar cheese or cooked chicken. S. enterica survived but did not grow on lettuce tissues incubated alone or in contact with Cheddar cheese for 6 days at either 6 or 14°C. In contrast, populations increased from 2.01 ± 0.22 to 9.26 ± 0.22 CFU/cm(2) when lettuce washed in water was incubated in contact with cooked chicken at 14°C. Populations on lettuce leaves were reduced to 1.28 ± 0.14 CFU/cm(2) by washing with a chlorine solution (70 ppm of free chlorine) but increased to 8.45 ± 0.22 CFU/cm(2) after 6 days at 14°C. Experimentation with a commercial product in which one third of the fresh-cut romaine lettuce was replaced with inoculated lettuce revealed that S. enterica populations increased by 4 log CFU/g during storage for 3 days at 14°C. These findings indicate that rapid growth of bacterial enteric pathogens may occur in mixed ingredient salads; therefore, strict temperature control during the manufacture, distribution, handling, and storage of these products is critical.