V. Lorusso

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.

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.

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.

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.

Development of a multiplex PCR for rapid detection of verocytotoxin-producing Escherichia coli O26 in raw milk and ground beef.

Verocytotoxin-producing Escherichia coli (VTEC) O26 is an emergent pathotype that has caused an increasing number of sporadic cases and outbreaks of gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome in the United States and Europe. Many cases are associated with the consumption of milk and undercooked or fermented meats. The stx(2) strains of VTEC O26 seem to be more likely to cause human infections than isolates expressing only stx(1). The isolation and identification of VTEC O26 from foods is labor intensive and time-consuming. We developed a multiplex PCR (M-PCR) assay for the identification and characterization of E. coli O26 VTEC and its detection in raw milk and ground beef. The method is based on the amplification of the wzx, stx(1), and stx(2) genes for the simultaneous detection of the O26 antigen and verocytotoxin types 1 and 2. This M-PCR assay had a sensitivity of 10(8) CFU/ml when applied to a bacterial suspension and of 10(6) CFU/ml or g when applied to both inoculated milk and minced beef samples. This M-PCR assay also was highly specific, and results were consistently negative for negative controls (nonpathogenic E. coli strains, uninoculated milk and beef samples, and samples inoculated with the nontarget microorganisms). This method could be used for the rapid detection of E. coli O26 VTEC from foods and for the rapid identification and characterization of clinical and environmental isolates.