Tiziana Pepe

A Multiplex Polymerase Chain Reaction Assay for Rapid Detection and Identification of Escherichia coli O157:H7 in Foods and Bovine Feces†

A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly933), and chromosomal flagella (fliCh7; flagellar structural gene of H7 serogroup), Shiga toxins (stx1, stx2), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E. coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was < or = 1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.

Distribution and behavior of Listeria monocytogenes in three lots of naturally-contaminated vacuum-packed smoked salmon stored at 2 and 10°C

The incidence, the number and the behavior of L. monocytogenes in three lots of naturally-contaminated vacuum-packed sliced smoked salmon, processed in different plants, were investigated during storage at 2 and 10 degrees C. L. monocytogenes was isolated from 20 of the 100 packages stored at 2 degrees C (16 from lot 1, 1 from lot 2 and 3 from lot 3) and from 12 of the 65 packages stored at 10 degrees C (all from lot 1). The levels detected were 15, 20, 290, 1100 and > 1100 cfu/g in 5 packages, all belonging to lot 1, and < 10 cfu/g in the remaining samples. The high incidence of L. monocytogenes in lot 1 was assumed to be due mainly to the use of causal workers for the slicing and packing operations.

Qualitative and quantitative assessment of viral contamination in bivalve molluscs harvested in Italy.

Bivalve molluscs are a well documented source of viral infection. Further data on shellfish viral contamination are needed to implement European Regulations with sanitary measures more effective against viral pathogens. To this aim, 336 samples of bivalve molluscs (185 mussels, 66 clams, 23 oysters and 62 samples from other species) collected in harvesting areas of class A and B of four Italian Regions were analyzed for qualitative and quantitative determination of hepatitis A virus (HAV) and Norovirus (NoV) GI and GII, using real time RT-PCR. The results showed a wide diffusion of viral contamination in the shellfish production areas considered. HAV prevalence was low (0.9%) with contamination levels that varied from 5 to 7 × 10(2)copies/g. On the contrary, NoV showed a high prevalence (51.5%), with a large variability according to the group considered (e.g. 47.8% for Crassostrea in Veneto, 79.7% for Mytilus in Campania, 84.6% for Tapes in Sardinia). NoV contamination affected class A and class B production areas to a different extent, with a statistically significant difference in both contamination prevalence (22.1% vs. 66.3%; p<0.0001) and quantity (average contamination level of 3.1 × 10(2) vs. 1.9 × 10(3) copies/g; p<0.05). The different species analyzed from class B harvesting areas (Mytilus, Tapes/Ruditapes and Crassostrea) showed a NoV prevalence respectively of 70.3%, 66.0% and 47.8% but comparable NoV contamination levels (between 8.4 × 10(2) and 4.9 × 10(3)copies/g). Other two bivalve species considered in the study (Donax spp. and Solen spp.) showed a relevant NoV presence (40.0% and 34.4% of samples). Finally, samples analyzed before and after commercial purification treatment showed a decrease of contamination prevalence after the treatment, but inconsistent results were recorded on NoV levels. The data obtained, together with other quantitative information to estimate consumer exposure, in association with studies on dose-response and on the effectiveness of post-harvest treatments, will provide a useful tool for the definition of microbiological criteria related to the different shellfish species.

Mitochondrial Cytochrome b DNA Sequence Variations: An Approach to Fish Species Identification in Processed Fish Products

The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.

The complete DNA sequence and analysis of the virulence plasmid and of five additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili (pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.

Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

Detection of Campylobacter from poultry carcass skin samples at slaughter in Southern Italy.

Campylobacter is a major foodborne pathogen responsible for acute gastroenteritis characterized by diarrhea that is sometimes bloody, fever, cramps, and vomiting. Campylobacter species are carried in the intestinal tracts of mammals and birds, and sources of human infection include raw milk, contaminated water, direct contact with pets, and foods, particularly poultry. Campylobacter jejuni and C. coli are the species that account for the majority of human infections. The aim of this work was to determine the prevalence of Campylobacter in 190 poultry carcasses sampled at slaughter and to use a multiplex PCR assay to determine if the isolates were C. jejuni or C. coli. C. coli was not isolated, while C. jejuni was recovered from 52 (37.1%) of 140 carcasses for which pools of four sampling sites (neck, cloaca, breast, and back) were examined. In the remaining 50 carcasses, the four sites were analyzed separately, and C. jejuni was recovered from the samples in the following order: neck (n = 20), cloaca (n = 16), breast (n = 14), and back (n = 11). The results are in agreement with those of other studies, which showed that C. jejuni is more commonly associated with poultry than is C. coli. Control strategies for Campylobacter should include interventions to eliminate C. jejuni in poultry at various stages of production and processing, including at slaughter.

Development of biogenic amines during the ripening of Italian dry sausages.

The effect of modification of different chemical and microbiological parameters and the production of biogenic amines (histamine, cadaverine, putrescine, and tyramine) was examined during ripening of various types of typical Italian dry sausages (salami). Water activity decreased from 0.97 to 0.87, and pH reached the lowest value between the 13th and the 20th day of the ripening period, and then increased. Putrescine (up to 122.7 mg/kg) and tyramine (up to 105.9 mg/kg) mean levels showed dominance in comparison with cadaverine (up to 26.1 mg/kg) and histamine (up to 6.2 mg/kg) mean values in all sausage types. The highest putrescine and tyramine concentrations were observed in salami with the largest diameters. This comparative study suggests a good correlation between microbial behavior and amine evolution, particularly tyramine and putrescine, in dry sausage production.

Norovirus monitoring in bivalve molluscs harvested and commercialized in southern Italy.

Norovirus (NoV) is the main cause of human nonbacterial gastroenteritis throughout the world. NoVs are classified into five genogroups: GI, GII, GIII, GIV, and GV. NoVs from GI and GII are the most commonly reported NoVs associated with human infections, and raw or undercooked shellfish have been identified as the main potential infection vehicle. European Commission Regulation 2073/2005 defines only bacteriological parameters for use as safety criteria for shellfish because reference methods for detection of viruses are lacking. From July 2007 to April 2010, 163 shellfish samples were collected in southern Italy from harvesting areas, authorized or nonauthorized retailers, and a restaurant after an outbreak of human gastroenteritis. The shellfish were analyzed for the presence of NoVs from GI and GII using the one-step real-time reverse transcription PCR protocol. A total of 94 shellfish samples (57.7%) were positive for the presence of NoV, and GII was the most frequently identified genogroup.

Construction of Listeria monocytogenes Mutants with In-Frame Deletions in Putative ATP-Binding Cassette (ABC) Transporters and Analysis ofTheir Growth under Stress Conditions

Listeria monocytogenes is a food-borne pathogen that is difficult to eliminate since it can survive under different stress conditions such as low pH and high salt. Understanding its survival under stress conditions is important in controlling this pathogen in food. ABC transporters have been shown to be induced in L. monocytogenes subjected to high pressure and nisin treatments; therefore, we hypothesized that genes encoding the ABC transporters may be involved in general stress responses. To study the function of these genes, deletion mutants of ABC transporter genes (LMOf2365_1875, LMOf2365_1877) were created in L. monocytogenes F2365, and these deletion mutants were tested under different stress conditions. Compared to the wild type, ΔLMOf2365_1875 and ΔLMOf2365_1877 showed slower growth under nisin (250 μg/ml) and acid (pH 5) treatments. Under salt treatment (5% NaCl in minimal medium), ΔLMOf2365_1877 showed slower growth whereas ΔLMOf2365_1875 had growth similar to the wild type. Moreover, ΔLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type. Our results indicate that these deletion mutants may be more sensitive to multiple stress conditions compared to the wild type, suggesting that LMOf2365_1875 and LMOf2365_1877 may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions and their ability to form biofilms may help in the development of intervention strategies to control L. monocytogenes in food and in the environment.