Detection and characterisation of potentially pathogenic species of Vibrio in the Vibrata river, Abruzzo Region, Italy.

This study aimed to isolate, define the genetic profile, assess potential pathogenicity and evaluate the seasonal distribution of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus strains isolated from the Vibrata river (Abruzzo Region, Italy) during a monitoring period of one year. Detection was performed according to ISO/TS 21872-1-2:2007. Species identification and characterisation were achieved using molecular methods. Vibrio spp. were detected in 50% (23) of the water samples. In particular, V. cholerae, V. parahaemolyticus, and V. vulnificus were isolated in 18 (39.1%), 4 (8.7%), and 2 (4.3%) samples, respectively. All V. parahaemolyticus strains were tdh gene negative, 75% were positive for trh gene. In 30 V. cholerae isolates, the polymerase chain reaction (PCR) assay for detecting virulence and regulatory genes (ctxA, toxR, tcpA, ompU, hlyA, tcpI, zot, and stn/sto) revealed 6 genotypes associated to different levels of pathogenicity. Pulsed-field gel electrophoresis (PFGE) characterisation of the V. cholerae strains identified 13 different pulsotypes. A greater degree of similarity was shown for strains isolated in the same period of the year. Results of our study reveal a potential health risk associated with the waters of the Vibrata river, which are used for irrigation and next to the swimming areas of Abruzzo coastline.

The applications of bacteriophages and their lysins as biocontrol agents against the foodborne pathogens Listeria monocytogenes and Campylobacter: An updated look.

Listeria monocytogenes and Campylobacter spp. are foodborne pathogens responsible for outbreaks and disease in humans. The emerging problem of bacterial antibiotic resistance and the persistence of pathogens in the environment, especially where foods are processed, are some of the reasons that have led to a re­emerging interest in bacteriophages and their lysins as potential candidates for bio­control. This review focuses on the use of bacteriophages and their lysins as alternative strategies for controlling the foodborne pathogens L. monocytogenes and Campylobacter spp. In addition, the application of bacteriophages and their lysins in food safety and animal health, as well as phage­resistance development, legislation, and future prospects were discussed.

Multiplex PCR to detect bacteriophages from natural whey cultures of buffalo milk and characterisation of two phages active against Lactococcus lactis, ΦApr-1 and ΦApr-2.

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L. lactis, ΦApr-1 and ΦApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for ΦApr-1 and ΦApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS­PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters.

Listeria monocytogenes in Smoked Salmon and Other Smoked Fish at Retail in Italy: Frequency of Contamination and Strain Characterization in Products from Different Manufacturers.

Seven hundred seventy-eight samples of packaged smoked fish (774 smoked salmon and 4 smoked swordfish) on sale in Italy, from 50 different manufacturers located in 12 European Union countries, were purchased from the Italian market between May and December 2011. The surface temperatures of the samples on sale ranged from 0 to 13°C (3.4 ± 1.5°C, mean ± SD). Six hundred eighty (87.4%) of 778 samples were stored at ≤4°C. One hundred fifty-seven samples (20.2%, 95% confidence interval 17.5 to 23.1%) were contaminated by Listeria monocytogenes , with 26 samples (3.3%, 95% confidence interval 2.3 to 4.8%) at levels >100 CFU/g. The maximum level of contamination was 1.3 ×106 CFU/g. The differences in the level of contamination of smoked fish between countries (χ2 = 91.54, P < 0.05) and manufacturers (χ2 = 193.22, P < 0.05) were significant. The frequency of detection for products from different manufacturing premises ranged from 0 to 76.9%. Serotyping by serological agglutination revealed that the main serotypes detected were 1/2a (65.3%) and 1/2b (22.4%). Pulsed-field gel electrophoresis typing with restriction enzymes AscI and ApaI yielded 36 pulsotypes from 144 isolates, clustering into 17 groups. Eight main pulsotypes accounted for 70.8% of the isolates. Three of the main pulsotypes were exclusively from products of a single manufacturer. In general, products from the same manufacturer showed genetic homogeneity, with one strongly prevalent pulsotype. Different manufacturers usually showed very different levels of contamination of the final product, confirming the importance of the management of process hygiene for controlling L. monocytogenes contamination.

Identification and characterization of Yersinia enterocolitica strains isolated from pig tonsils at slaughterhouse in Central Italy.

Yersinia enterocolitica causes foodborne disease in humans and infections are usually acquired from contaminated raw or undercooked pork. Pigs are considered the primary reservoir of human pathogenic bio-serotypes. A total of 376 tonsil tissue samples collected after evisceration and cutting from pig carcasses were tested for Yersinia enterocolitica. Animals came from an abattoir located in the Abruzzo region, Italy. Yersinia enterocolitica was isolated from 35 out of 376 (9.31%) samples. A total of 47 strains were isolated, the prevalent bio-serotype was 4÷O:3 (95.74%), followed by bio-serotype 4÷O:9 (2.13%), and 3÷O:9 (2.13%). When characterized by DNA microarray, all strains clustered into 2 main groups. The bigger group was characterised by the presence of plasmid genes of the secretion apparatus as well as by the genes for the agellum transport machinery, while the smaller group was characterised only by genes for the agellum transport machinery. The high frequency of the pathogenic biotype 4÷O:3 able to infect humans and considered an emerging zoonotic pathogen con rms the role of pigs as natural reservoir. Since there is no official data on Yersinia enterocolitica, it is difficult to assess the implications of this food pathogen for public health. A monitoring program should be implemented for contamination in food in order to assess the risk for the consumer linked to raw or undercooked pork products.

Comparison of Multiple­Locus Variable number tandem repeat Analysis and Pulsed Field Gel Electrophoresis in molecular subtyping of Listeria monocytogenes isolates from Italian cheese.

Subtyping of Listeria monocytogenes strains is an essential epidemiological tool to trace contamination and determine evolutionary relationships among different strains. Pulsed field gel electrophoresis (PFGE) is the current gold standard method for Listeria characterization. Multiple-Locus Variable-number tandem repeat Analysis (MLVA) is a rapid subtyping method based on Polymerase Chain Reaction (PCR) amplification that has been successfully developed for subtyping bacterial genera. The purpose of this study was to evaluate MLVA for subtyping L. monocytogenes strains isolated from Italian cheese and to compare it with PFGE. The type ability and discriminatory power of MLVA was determined on a collection of 90 isolates corresponding to 5 serotypes and 29 pulsotypes with enzymes AscI and ApaI. A panel of 5 variable-number tandem repeat loci was used. MLVA and PFGE showed very similar discriminatory power (Simpson's Index of diversity 0.840 with 95% Confidence Interval [CI] 0.780/0.899 and 0.837 with 95% CI 0.768/0.906, respectively). MLVA is an easy test to perform. It is relatively fast, reproducible and could be implemented in any molecular laboratory but, according to the performed protocol, it is not sufficient for discriminating the L. monocytogenes strains isolated from cheese. This method could be combined with PFGE to increase the discrimination in molecular subtyping of these strains.

Whole-Genome Sequence of Listeria monocytogenes Serovar 4b Strain IZSAM_Lm_hs2008, Isolated from a Human Infection in Italy.

Listeria monocytogenes is one of the most important foodborne pathogens. In this report, we present the complete and annotated genome of L. monocytogenes sequence type 06 (ST06) serovar 4b strain IZSAM_Lm_hs2008, isolated from an adult immunocompetent patient who developed the disease and died.

Definition of a Standard Protocol to Determine the Growth Potential of Listeria Monotgenes and Yersinia Enterocolitica in Pork Sausage Produced in Abruzzo Region, Italy.

Pork meat products consumed raw or after a short period of fermentation can be considered at risk for food safety. Sausages (fresh sausage made from pork meat) are produced in several Italian regions, with variation in ingredients. In some Italian Regions, including Abruzzo, these products are frequently consumed raw or undercooked, after a variable period of fermentation. The European Community food regulation promotes the use of challenge tests to determine safety levels. This study is aimed to ensure safety of Abruzzo's sausages, compared with growth potential (δ) of Listeria monocytogenes and Yersinia enterocolitica, and also aims to define an experimental standard protocol document to carry out challenge tests. Guidelines classify ready-to-eat foods in categories that are able to support (δ>0.5 log10 ufc/g) and not support (δ≤0.5 log10 ufc/g) the growth of Listeria monocytogenes. The products were manufactured according to traditional recipes and were contaminated in laboratory. Results from the experiment yielded information useful to assess the ability of these products to support the growth of pathogenic microorganisms. The batches of sausages were stored at 8, 12, 18 and 20°C to get statistical evaluation. The results showed that, despite the conditioning of the storage temperature and the level of water activity, both organisms remain in the product in concentrations similar to those leading or being able to increase its charge. In particular, the period of greatest consumption of this product (7/8 days of preparation) corresponds to the period of greatest growth of pathogenic microorganisms studied, except for those stored at a temperature of 8°C, which are safer for the consumer.

Detection of Listeria monocytogenes in food samples in Avezzano, Sulmona and Castel di Sangro (province of L'Aquila, Abruzzo, Italy) between 2000-2009.

The retrospective study of the results of the analysed samples is a fundamental tool for the identification of major risk related to food and for planning future monitoring activities. The evaluation of the quality of data collected may also allow for estimating the effectiveness of the controls so to improve their efficacy. In this article, the authors evaluated the results of tests for the detection of Listeria monocytogenes performed by the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' (IZSAM) on food samples collected during the years 2000-2009 in the territory of Avezzano, Sulmona and Castel di Sangro (province of L'Aquila, Abruzzo, Italy). The comparison of the data examined with those from studies conducted in Italy and in other countries shows that the categories with higher percentages of positivity for Listeria monocytogenes are meat and fish products. Data collected do not indicate cheese as a vehicle of contamination in the sampled areas, in contrast to what reported in the national and international literature. It would therefore be necessary to promote an ad hoc sampling in the areas covered by this study to verify this aspect in more depth.

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.

Detection of Vibrio splendidus and related species in Chamelea gallina sampled in the Adriatic along the Abruzzi coastline.

Vibrio species are an important and widespread component of marine microbial communities. Some Vibrio strains are potentially pathogenic to marine vertebrates and invertebrates. The aim of this study was to identify vibrios, in particular Vibrio splendidus and related species, isolated from clams (Chamelea gallina) collected along the coasts of the Abruzzi region from May to October 2007. The isolates obtained were phenotyped and classified as belonging to the genus Vibrio. The strains underwent biochemical testing in accordance with Alsina's scheme for V. splendidus identification. Molecular analysis of the 16S-23S intergenic space region and recA gene was used to identify V. splendidus and related species. All the samples examined were found to contain halophylic Vibrio species, with V. alginolyticus, V. splendidus-related species and V. mediterranei most commonly found. A polymerase chain reaction of the 16S-23S intergenic space region and sequencing of the recA gene from isolates confirmed that phenotyping of Vibrio species is not sufficient to distinguish between different species. Differentiation of the highly related species among V. splendidus-related clusters remains an important issue. In this regard, our data suggests sequencing the recA genes was far more discriminatory than sequencing 16S rDNA for this purpose.

Characterisation of Staphylococcus aureus strains isolated from food for human consumption.

An investigation was conducted to evaluate Staphylococcus aureus contamination in various types of food of animal origin. Of the 350 samples examined, 14.0% were found to be contaminated with S. aureus. Prevalence rates varied according to type, namely: 19.3% for fresh meat products, 13.3% for fresh cheeses, 3.6% for bakery products and 7.7% for deli products. The isolated S. aureus strains then underwent 16S rDNA polymerase chain reaction (PCR) followed by reverse latex agglutination tests to identify enterotoxigenic strains. The results were compared with data obtained by subjecting the same strains to tests for the genes coding for the S. aureus enterotoxins (SEs) sea, seb, sec, sed, see, seg, seh, sei and toxic shock syndrome toxin 1 (TSST 1). Reversed passive latex agglutination (RPLA) testing revealed that 16.3% of strains (8/49) produced enterotoxins, while on PCR, 48.97% (24/49) were found to carry one or more genes for the production of SEs, and were therefore potentially enterotoxigenic.

Characterisation of Listeria monocytogenes strains isolated from soft and semi-soft cheeses sampled in a region of Italy.

A total of 47 Listeria monocytogenes strains isolated in a survey of cheeses sampled from retail outlets were characterised. Five cheeses (Gorgonzola, Taleggio, Asiago, Crescenza and Brie) were chosen from the most popular soft and semi-soft cheeses consumed in Italy and most commonly contaminated with L. monocytogenes. The serotype and antibiotic resistance pattern were determined for each strain and their macrorestriction profile was analysed with pulsed-field gel electrophoresis (PFGE). The main serotypes detected were 1/2a (76.6%) and 1/2c (21.3%). Serotype 1/2b was found in only one sample. A total of 97.9% of strains were resistant to oxacillin (OX), 80.9% to lincomycin (L) and 78.7% to clindamycin (CC). Of these strains, 17% were found to be resistant to two antibiotics (OX-CC or OX-L) while 70.2% were resistant to three antibiotics (OX-CC-L). No strains were susceptible to all the compounds tested. A combined analysis of the macrorestriction profiles AscI and ApaI identified eleven pulsotypes divided into three clusters. Two pulsotypes predominated, accounting for 57.4% and 21.3% of the isolated strains. Analysis of the PFGE profiles did not reveal any correlation between pulsotype and type of cheese, producer or retail outlet. A temporal analysis revealed that one pulsotype was persistent throughout the study period, with the exception of August and September, in which time a different pulsotype was detected. This variability suggests the influence of factors affecting the dynamics of the contamination of these products. Large-scale studies could help clarify this phenomenon.

Prevalence of thermotolerant Campylobacter in broiler flocks and broiler carcasses in Italy.

In accordance with European Union regulations, from 5 February until 15 December 2008, sampling and analysis activities were conducted in Italy to assess the extent of contamination caused by thermotolerant Campylobacter in broiler chickens farmed nationwide. The survey involved 48 poultry slaughterhouses distributed across eleven regions of Italy, where the caeca and carcasses of 393 slaughter batches were sampled. A total of 284 batches (72.3%) gave positive results for Campylobacter spp. as follows: 52.1% were contaminated by C. jejuni, 55.6% by C. coli and 1.1% by C. lari. C. jejuni and C. coli were isolated together in 37 batches (13% of positive results). Campylobacter spp. was isolated only from the caeca in 251 slaughter batches (63.9%) including caecal isolates of C. jejuni (48.2%), C. coli (50.6%), and C. lari (1.2%). Carcasses from 182 batches (46.3%) were contaminated by C. jejuni in 40.7% of cases, C. coli in 57.7% and the absence of C. lari from all batches examined. The contamination level observed in the carcasses ranged between 10 and 1.6 × 10(7) cfu/g.

The egg consumption of the average household in Italy.

A survey was conducted over a one-year period by means of telephone interviews with 7 991 Italian households to establish the domestic consumption of eggs, the distribution by source of supply, seasonal variations and storage and preparation methods used. Eggs are mainly purchased from large retailers (53%), followed by small retailers (25.2%), direct purchase from producers (16%), and local or itinerant markets (5.8%). It was found that 69.9% of households buy packaged eggs; 92% of households store them in the refrigerator, although this percentage varies considerably, according to the type of presentation (packaged or loose) and the number of eggs bought. Italian households mainly eat eggs cooked (48.9%), followed by partly cooked (35.0%) and raw (16.1%).

Assessment of hygienic quality of some types of cheese sampled from retail outlets.

The authors evaluated the prevalence of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp. and staphylococcal enterotoxin, in 2,132 samples selected from six types of cheese on the basis of recorded consumption in Italy in 2004. In L. monocytogenes-positive samples the precise level of contamination was determined. To define the physical-chemical characteristics of the selected natural cheeses, the pH values, water activity and sodium chloride content were determined. The results suggest that blue and soft cheeses (Brie, Camembert, Gorgonzola and Taleggio) are more likely to be contaminated with L. monocytogenes. The mean prevalence of L. monocytogenes in the six types of cheese was 2.4% (from 0.2% in Asiago and Crescenza to 6.5% in Taleggio), with contamination levels of up to 460 MPN/g. No presence of Salmonella spp. and E. coli O157 was found in any sample. Staphylococcus enterotoxin was found in 0.6% of the samples examined. Physical and chemical parameter values confirmed that all types of cheese are considered capable of supporting the growth of L. monocytogenes. The study confirmed the need to apply effective control at production and sales levels to reduce the probability of contamination by L. monocytogenes. This micro-organism can attain high levels of contamination in food products, such as cheeses that have a long shelf-life when associated with difficulties of maintaining appropriate storage temperatures in both sales points and in the home.

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy.

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS.

Polymerase chain reaction and bacteriological comparative analysis of raw milk samples and buffalo mozzarella produced and marketed in Caserta in the Campania region of Italy.

To help identify an epidemiological link between the consumption of buffalo mozzarella prepared with raw milk (not heat-treated) and cases of human brucellosis, 80 samples of raw buffalo milk and 315 samples of mozzarella were tested for the presence of Brucella spp. Samples originated from Caserta, the province with the highest number of Brucella-positive buffalo herds in Campania, the region in which 96.02% of all cases of human brucellosis reported in 2000-2005 were recorded. To take into account possible seasonal variations, between February 2006 and March 2007, samples were purchased directly from 72 dairy outlets that were representative of the province. Samples were tested for Brucella spp. using the polymerase chain reaction (PCR) and bacterial isolation. Samples tested negative for Brucella using both methods. Spiked samples were then prepared and tested by PCR and bacterial isolation and the sensitivity, specificity, repeatability, reproducibility and limit of detection were determined. The PCR limit of detection was below 1 colony-forming unit (cfu)/g. The repeatability and reproducibility of the method were 100% (p = 0.95), the sensitivity was 96.7% (p = 0.95) and the specificity was 100% (p = 0.95).

Gastroenteritis outbreak at holiday resort, central Italy.

During the summer of 2003, a gastroenteritis outbreak spread throughout a holiday resort in central Italy. Fecally contaminated groundwater and seawater were leaking into the non-drinking-water system, which was found to be connected to the drinking-water system of a large resort. This contamination had a primary role in the onset of the outbreak and spread of the infection.

Investigation of an outbreak of Salmonella enterica subsp. enterica serovar Hadar food illness in the Abruzzi region of Italy.

A comparative study was made of 22 strains of Salmonella Hadar isolated from victims of an outbreak of food illness in the Abruzzi region of Italy in 2000 and 21 strains of the same serotype isolated from poultry meat and human stool samples in the Abruzzi and Molise regions between 2000 and 2001. The aim of the investigation was to provide an epidemiological interpretation of the food illness outbreak to establish the degree of similarity between the S. Hadar strains isolated from victims of the outbreak and those isolated from poultry meat (identified but unconfirmed as the possible source of infection) and from other human samples received in the laboratory. Pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) were used to identify the genotypes and antimicrobial resistance patterns were determined. PFGE analysis of the restriction patterns obtained with XbaI and BlnI led to the identification of 12 pulsotypes in three groups. RAPD did not provide any information, as it was unable to differentiate between the strains isolated from food illness victims with gastroenteritis. Antimicrobial resistance tests revealed multiple resistance patterns and no strains were found to be resistant to ciprofloxacin or to the other quinolones tested. The poultry strains were found to be resistant to nalidixic acid, while the resistance in human strains was 31.8%. A combined analysis of resistance patterns and pulsotypes revealed four resistance patterns; the pattern associated with the outbreak was not correlated with the others present in the same period. This work suggests that a study of the relationship between different strains of the same serovar requires the implementation of different analytical methods.