G. Normanno

Amplified Fragment Length Polymorphism and Multi-Locus Sequence Typing for high-resolution genotyping of Listeria monocytogenes from foods and the environment.

Standardized tools for typing Listeria monocytogenes isolates are required in epidemiological surveys investigating food-borne disease outbreaks and in the food-processing environment to identify the sources of contamination and routes by which the organisms are spread. In this survey Amplified Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Typing (MLST) have been used to study 103 L. monocytogenes isolates from food and environmental sources. A total of 62 AFLP types and 66 MLST Sequence Types were identified. AFLP and MLST produced similar results in terms of discriminating power. The Discrimination Index calculated for the two techniques was 0.976 for AFLP and 0.972 for MLST. These values were appreciably higher compared to serotyping (0.739). A good congruence was observed between AFLP and MLST. The present study demonstrated that AFLP and MLST subtyping are suitable tools for studying the epidemiology of L. monocytogenes. The great advantage of MLST over AFLP and other molecular typing methods based on fragment fingerprinting lies in the unambiguity of sequence data while AFLP is less costly and highly processive. In conclusion the two methods can be perfectly integrated for high-resolution genotyping of L. monocytogenes.

EFFECT OF REDUCING DIETARY PROTEIN LEVEL ON PERFORMANCE RESPONSES AND SOME MICROBIOLOGICAL ASPECTS OF BROILER CHICKENS UNDER SUMMER ENVIRONMENTAL CONDITIONS

This study examined the effect of different crude protein (CP) levels in the diet on growth performance of broilers as well as the total aerobic mesophilic count (TAMC) and Escherichia coli count (ECC) isolated from chicken faeces. A total 150 day-old broilers (Hubbard) were allocated to three treatments with five replicates containing 10 birds. The dietary treatments consisted of three diets with different CP levels for 42 days: high-protein (HCP, 22.5%), medium-protein (MCP, 20.5%), and low-protein (LCP, 18.5%). Body weight and feed intake were determined and the feed conversion ratio was calculated. Faecal samples were collected at 14, 21 and 42 days. Dietary CP did not affect the growth performance of broilers. Reducing CP level was effective at beneficially modulating the composition of the faecal microflora, in particular TAMC and ECC concentrations (log10 CFU g−1) were significant lower for the LCP birds. During hot temperature conditions (33°C and 70% relative humidity) a low protein diet may help to control the characteristics of the faecal microbial community without negative effects on broiler performance.

Methicillin-resistant Staphylococcus aureus (MRSA) in slaughtered pigs and abattoir workers in Italy

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen present in the hospital environment (HA-MRSA), in the community (CA-MRSA) and in livestock, including pigs (LA-MRSA). MRSA may enter the human food chain during slaughtering and may infect humans coming into direct contact with pigs or pork products. This study aimed to determine the prevalence and characteristics of MRSA isolated from pigs and workers at industrial abattoirs in southern Italy. A total of 215 pig nasal swabs were screened for the presence of MRSA using PCR. An MRSA isolate was detected from each mecA/nuc PCR-positive sample and characterized by spa-typing, Multi-Locus Sequence Typing, SCC-mec and Panton-Valentine Leukocidin (PVL), and also tested for the production of staphylococcal enterotoxins (SEs). Eighty-one MRSA isolates (37.6%) were obtained from the 215 pig nasal swabs; 37 of these isolates were further characterized, and showed 18 different spa-types and 8 different STs. The most frequently recovered STs were ST398 (CC398-t034, t011, t899, t1939 - 43.2%) followed by ST8 (CC8-t008, t064, t2953, t5270 - 24.3%) and ST1 (CC1-t127, t174, t2207 - 10.8%). Nine MRSA isolates were obtained from the 113 human swabs; the isolates showed 5 different spa-types and 5 different STs, including the novel ST2794 (t159). The most representative STs recovered were ST1 (CC1-t127) and ST398 (CC398-t034) (33.3%). None of the MRSA isolates showed the ability to produce SEs and PVL and all resulted resistant to two or more classes of antimicrobials. This study shows the great genetic diversity of MRSA strains in slaughtered pigs and in abattoir employees in Italy, and clearly demonstrates the need for improved hygiene standards to reduce the risk of occupational and food-borne infection linked to the handling/consumption of raw pork containing MRSA.

Comparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy.

Listeria monocytogenes ranks among the most frequent causes of death due to foodborne illness (20-30% case fatality rate). Discriminative subtyping methods are important to detect the relatedness of isolates and verify epidemiologic associations. AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. In this study, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance. A total of 103 unrelated L. monocytogenes strains isolated from different environmental and food sources were analyzed. Strains were isolated from samples obtained from food-production plants, supermarkets and small food markets in Piedmont, Italy. All methods clustered L. monocytogenes strains into two genetic lineages, Lineage I and II. The three methods were compared using the 82 isolates which were typeable with all techniques. The calculated pair-wise Pearsons correlation coefficients (r) showed close agreement between all three methods. Our findings suggest that the AFLP II method can be successfully used to subtype L. monocytogenes strains isolated from foods and food processing facilities.

Use of Lactobacillus plantarum Strains as a Bio-Control Strategy against Food-Borne Pathogenic Microorganisms

Lactobacillus plantarum is one of the most versatile species extensively used in the food industry both as microbial starters and probiotic microorganisms. Several L. plantarum strains have been shown to produce different antimicrobial compounds such as organic acids, hydrogen peroxide, diacetyl, and also bacteriocins and antimicrobial peptides, both denoted by a variable spectrum of action. In recent decades, the selection of microbial molecules and/or bacterial strains able to produce antagonistic molecules to be used as antimicrobials and preservatives has been attracting scientific interest, in order to eliminate or reduce chemical additives, because of the growing attention of consumers for healthy and natural food products. The aim of this work was to investigate the antimicrobial activity of several food-isolated L. plantarum strains, analyzed against the pathogenic bacteria Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7 and Staphylococcus aureus. Antagonistic activity was assayed by agar spot test and revealed that strain L. plantarum 105 had the strongest ability to contrast the growth of L. monocytogenes, while strains L. plantarum 106 and 107 were the most active microorganisms against E. coli O157:H7. The antimicrobial ability was also screened by well diffusion assay and broth micro-dilution method using cell-free supernatants (CFS) from each Lactobacillus strain. Moreover, the chemical nature of the molecules released in the CFS, and possibly underlying the antagonistic activity, was preliminary characterized by exposure to different constraints such as pH neutralization, heating, catalase, and proteinase treatments. Our data suggest that the ability of L. plantarum cultures to contrast pathogens growth in vitro depends, at least in part, on a pH-lowering effect of supernatants and/or on the presence of organic acids. Cluster analysis was performed in order to group L. plantarum strains according to their antimicrobial effect. This study emphasizes the tempting use of the tested L. plantarum strains and/or their CFS as antimicrobial agents against food-borne pathogens.

Prevalence, antimicrobial susceptibility and molecular typing of Methicillin-Resistant Staphylococcus aureus (MRSA) in bulk tank milk from southern Italy

This paper assesses the prevalence of MRSA in bulk tank milk (BTM) samples from southern Italy, and the relationship between the Coagulase Positive Staphylococci count (CPS) and MRSA prevalence. Of 486 BTM samples tested, 12 samples (2.5%) resulted positive for the presence of MRSA. Great genetic diversity was found among the isolates: ST1/t127 and t174/IVa, ST5/t688/V, ST8/t unknown/IVa/V, ST45/t015/IVa, ST71/t524/V, ST88/t786/Iva, ST398/t011 and t899/IVa/V and ST2781/t1730/V. All isolates were pvl-negative and icaA positive. The majority of strains (58%) carried the ses (sec, seh, seg, seo, sem and sen) genes. All tested strains resulted susceptible to amikacin, cephalotin, cloramphenicol, gentamycin, trimethoprim - sulfamethoxazole, tobramycin and vancomycin, and variably resistant to ampicillin, oxacillin and tetracycline. No statistical association between the CPS count and MRSA detection was found in the MRSA-positive samples. Although some of the spa-types and STs detected in our survey are known to cause human infections, raw milk from Italian herds in the considered area is not a common source of MRSA. Nonetheless, it is necessary to assess the risk of foodborne infection and the risk related to the handling of milk.

Toxic Metals (Hg, Cd, and Pb) in Fishery Products Imported into Italy: Suitability for Human Consumption

Mercury, cadmium, and lead concentrations were determined in various fishery products (fishes, cephalopod molluscs, and crustaceans) imported into Italy from many European and non-European coastal countries. Considerable differences were found in the concentrations of these metals among the products tested. The highest mean Hg concentration was found in fishes (0.21 μg g(-1) wet weight), whereas cephalopods had the highest mean Cd concentration (0.35 μg g(-1) wet weight). Swordfish (0.80 μg g(-1) wet weight), longtail tuna (0.53 μg g(-1) wet weight), and thornback ray (0.52 μg g(-1) wet weight) had the highest concentrations of Hg, whereas maximum Cd concentrations were found in samples of common cuttlefish (0.85 μg g(-1) wet weight) and common octopus (0.64 μg g(-1) wet weight). The majority of the samples analyzed were in compliance with European Union legislation, except for a few cases. The calculated mean weekly intakes of Hg, Cd, and Pb through consumption of the fishery products tested were all below the legislated respective provisional tolerable weekly intakes. In general, the samples analyzed were considered safe to eat with regard to the metal concentrations found and the allowable intakes based on legislation. Nevertheless, the consumption of some species may be of significant importance for consumer health.

Verocytotoxin-producing Escherichia coli O26 in raw water buffalo (Bubalus bubalis) milk products in Italy.

Escherichia coli 026 is known as a verocytotoxin-producing E. coli (VTEC) organism that causes severe foodborne diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Although cattle are the most important reservoir of VTEC, only a few reports on the role of water buffalo (Bubalus bubalis) as a reservoir of VTEC and on the presence of these organisms in their milk are available. However, in Southern Italy, where water buffalo are intensively reared, an outbreak of hemolytic uremic syndrome due to E. coli 026 has recently been reported, in which the consumption of typical dairy products was considered to be a common risk factor. The aims of this work were to assess the prevalence of E. coli O26 in raw water buffalo milk, to characterize the virulence gene profiles of the isolates, and to evaluate their phenotypic antimicrobial resistance pattern. Of 160 analyzed samples, 1 (0.6%) tested positive for E. coli O26, and the isolate showed the stx1+/stx2+/eae-/hlyA+ genotypic profile. The strain showed resistance against glycopeptides, macrolides, and penicillins. The presence of VTEC organisms in raw water buffalo milk could be considered to be a potential threat to consumers; however, the strict adherence to the processes used in the preparation of the most common buffalo dairy products could strongly mitigate the foodborne risk. To our knowledge, this article reports the first isolation and characterization of E. coli O26 VTEC in raw water buffalo milk.

Typing of Escherichia coli O157 strains isolated from fresh sausage

Abstract E. coli O157 is a foodborne pathogen responsible for serious human illnesses, such as hemorrhagic colitic and hemolytic uremic syndrome. Ground beef products are among the foods that are more commonly contaminated, and the strains isolated have been frequently found to carry virulence factors of this pathotype. This paper reports the results of serotyping assays and of investigations performed to screen for virulence factors of 10 E. coli O157 strains isolated from fresh sausages purchased at retail meat outlets in various provinces of Apulia (southern Italy). The presence of verocytotoxins was assessed on VERO cells and ELISA tests. Multiplex PCR assays were performed for the eae, stx1, stx2 and hlyA genes. Six of the 10 strains examined presented the H7 antigen and all of them proved to be potentially pathogenic due to the presence of individual or multiple virulence factors.

Microbiological quality of Burrata cheese produced in Puglia region: southern Italy.

Burrata cheese is a popular typical Italian food product, produced in Puglia (an administrative region of southern Italy), and this study investigated the microbiological quality of 404 samples of this cheese. The samples were analyzed in order to quantify Escherichia coli and to detect the presence of Staphylococcus aureus, Salmonella spp., and Listeria monocytogenes. No sample exceeded the values of E. coli set by EC Regulation 1441/07 for some dairy products, while 15 (3.7%) samples tested coagulase-positive staphylococci positive, with values greater than 10(3) CFU/g. One strain of S. aureus was identified and characterized from each of these positive samples, and of these strains, 7 (46.6%) produced staphylococcal enterotoxin A, 5 (33.3%) produced staphylococcal enterotoxin C, 2 (13.3%) produced staphylococcal enterotoxin D, and 1 (6.6%) produced both staphylococcal enterotoxins A and D. All strains were mecA negative. The 15 S. aureus isolates were tested for their antimicrobial resistance patterns, and all analyzed strains showed antimicrobial resistance properties for at least one of the tested antibiotics. Testing for the other pathogens mentioned above gave negative results. The results of our study mean that the microbiological quality of Burrata cheese can be assumed to be good, although care must be taken with raw materials and good hygiene during processing in order to guarantee greater food safety.