E.P.L. De Santis

Evolution and identification of lactic acid bacteria isolated during the ripening of Sardinian sausages

Lactic acid bacteria (LAB) were isolated during the production and the ripening of Sardinian sausage, a typical Italian dry fermented sausage. Samples were taken at different stages, and 112 strains were isolated. The isolates were characterized using the micromethod proposed by Font de Valdez et al. [Font de Valdez, G., Savoy de Giori, G., Oliver, G., & De Ruiz Holgado, A. P. (1993). Development and optimization of an expensive microsystem for the biochemical characterization of lactobacilli. Microbiologie Aliments Nutrition, 11, 215-219]. Schillinger and Lückes [Schillinger, U., & Lücke, F. K. (1987). Identification of lactobacilli from meat and meat products. Food Microbiology. (4), 199-208] scheme and the biochemical patterns given by Bergeys Manual of Systematic Bacteriology [Bergeys Manual of Systematic Bacteriology (1986). Baltimore: William and Wilkins] were used for preliminary identification. A PCR-based method was then used to confirm the results. LAB were the dominant flora during ripening. They consisted mainly of homofermentative mesophilic rods. Lactobacillus sakei (43,3%), Lactobacillus plantarum (16,6%) and Lactobacillus curvatus (13,3%) were the main isolates. The results of the biochemical identification methods agreed well with those of PCR-based identification (91% agreement).

Massive osteolysis induced by high molecular weight polyethylene wear debris

Summary. We investigated the mechanism by which particulate wear debris of polyethylene may induce bone resorption using an in vivo model. Two uncemented total hip prostheses, in which the socket was directly in contact with acetabular bone, were selected because there was massive bone loss around the implant. A thick synovium-like layer was found at the polyethylene-bone interface during revision operations. Samples were examined by transmitted and polarised light microscopy, and by transmission electron microscopy. This study demonstrates that polyethylene wear products alone can cause massive osteolysis by triggering the formation of foreign body granuloma at the bone-implant interface.Résumé. Nous avons étudié le méchanisme par lequel les débris de polyethylène peuvent produire la résorption de l’os, en utilisant un model particulier in vivo. Deux patients traités par protheses de hanche sans ciment, avec cotyle en polyethylène en contact direct avec l’os, ont été choisis a cause de la présence d’une large érosion au niveau du cotyle. Une membrane pseudo-synoviale, présente entre l’os et le polyethylène, a été observée pendant la reprise; des échantillons de ce tissu ont étéétudiés par microscopie polarisée et par microscopie éléctronique à transmission. Les résultats de cet étude démontrent que les débris du polyethylène peuvent eux-même produir une large érosion par formation d’une membrane d’interface.

Multiplex PCR for the Identification and Serotyping of L. monocytogenes Isolated from Sheep Cheese-Processing Plants

De Santis, E.P.L., Pilo, A.L., Cosseddu, A.M., Canu, N.A., Scarano and C., Marongiu, P., 2007. Multiplex PCR for the identification and serotyping of L. monocytogenes isolated from sheep cheese-processing plants. Veterinary Research Communications, 31(Suppl. 1), 359–363

Levels and congener profiles of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) in sheep milk from an industrialised area of Sardinia, Italy

Concentrations of 7 polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs) and 22 polychlorinated biphenyls (PCBs), including 12 dioxin like-PCBs (non- and mono-ortho PCBs) were measured in 80 sheep milk samples from farms located in an industrialized area of Sardinia, Italy. PCDDs and PCDFs mean concentrations were 2.45 and 3.69 pgg(-1) fat basis, respectively. The mean dl-PCB concentration was 2.01 ngg(-1) fat basis, while cumulative ndl-PCB levels ranged from 1.02 to 20.42, with a mean of 4.92 ngg(-1) fat. The results expressed in pg WHO-TEQ/g fat showed that contamination level of milk was below the limit values for human consumption established by EC legislation. In the same way, all the investigated milk exhibited PCDD/Fs concentrations below EU action levels, while dl-PCBs concentrations exceeded the action level of 2.0 pg WHO-TEQ/g fat. These findings point to the need to continue to conduct general monitoring programmes, including also milk samples from areas not close to the contaminant-emitting industries, in order to better evaluate the impact of industrial activities on surrounding environment.

Toxin gene pattern in Bacillus cereus group strains isolated from sheep ricotta cheese

Milk and milk whey products processed at high temperatures and then stored by chilling are particularly sensitive to the health risks associated with microorganisms of the Bacillus cereus group (Heyndrickx and Scheldeman 2002). Sheep ricotta cheese is often contaminated by these spore forming microorganisms and it provides a substrate in which they can develop (Corona et al. 2002). B. cereus may cause diarrhoeic or emetic food poisoning outbreaks. Diarrhoeic syndrome is mainly due to HBL and NHE enterotoxins produced when the micro organisms multiply in the small intestine (Granum and Lund 1997; Hansen and Hendriksen 2001). Emetic syndrome is caused by emetic toxins or cereulide, a dodecapeptide produced when the microorganisms multiply in the food (Kramer and Gilbert 1989; Agata et al. 1996). Their pathogenic profile is also characterized by the ability to produce enterotoxin T, which shows toxic activity only in vitro (Agata et al. 1995), and cytotoxin K, which is necrotizing and hemolytic. Sporadic cases of death from food poisoning have been attributed to cytotoxin K producing strains (Lund et al. 2000). These microorganisms also produce hemolysins (cereolysins, hemolysin II, sphingomyelinases) and different phospholipases C (PIH, PCH and SM) (Granum 1994; Beecher and Wong 2000). The present work reports the molecular profile of the pathogenicity factors of “ B. cereus ” group strains isolated from fresh and ripened sheep ricotta-cheese. The ability of these strains to produce the hemolytic fraction L 2 of HBL and the fraction A of NHE was also tested.

A new multiplex PCR for the detection of hbl genes in strains of the 'Bacillus cereus group'

The Bacillus cereus group includes the species B. cereus , B. thuringensis , B. anthracis , B. mycoides , B. pseudomycoides and B. weihenstephanensis which are characterized by remarkable genetic affinities (Ash et al ., 1991; Carlson et al ., 1994; Henderson et al ., 1995; Stephan, 1996; Borin et al ., 1997; Nilsson et al ., 1998; Shangkuan et al ., 2000). These micro-organisms are responsible for foodborne disease through the production of several enterotoxins (Prus et al ., 1999) of which haemolysin BL (HBL) is the most studied. HBL is a three-component protein, including two lytic (L 1 and L 2 ) and one binding protein (B) and all three components must be simultaneously present to consider B. cereus group strains as potential pathogens (in’t Veld et al ., 2001). HBL has haemolytic and dermonecrotic activity which is responsible for the increase in vascular permeability and it is considered the primary virulence factor in the diarrhoic syndrome (Beecher and Wong, 1994). The transcription of complex HBL is codified from the operone hbl . Traditional PCR, by means of specific primers (Hansen and Hendriksen, 2001), can only detect single genes ( hblA , hblD , hblC ) encoding the components of HBL (B, L 1 e L 2 ). The identification of the genes could be faster using only one PCR reaction. In the present study we developed a multiplex PCR for the detection of operon hbl genes, both on reference and wild strains isolated from ovine milk and ‘ricotta’ cheese.

Multilocus sequence typing of Arcobacter butzleri isolates collected from dairy plants and their products, and comparison with their PFGE types.

The present study aimed to determine, by multilocus sequence type (MLST), the heterogeneity level of Arcobacter butzleri isolates and to compare MLST and pulsed‐field gel electrophoresis (PFGE) in terms of discriminatory power (DI) as well as unidirectional and bi‐directional concordance.

The pattern of toxin genes and expression of diarrheal enterotoxins in Bacillus thuringiensis strains isolated from commercial bioinsecticides.

Bacillus thuringiensis (Bt) is a gram positive sporeforming bacillus included in the Bacillus cereus group. Bt produces a parasporal insecticidal protein crystal, a protoxin of the δ-endotoxin that has insecticidal properties (Schnepf et al . 1998). Bt has been used in bioinsecticidal formulations for decades and it is either sprayed directly on plants or else used to coat the seeds. However, some outbreaks of foodborne disease due to Bt have been reported and a large number of its strains can produce enterotoxins (EFSA 2007). Bacillus cereus (Bc) is the most representative species of the Bacillus cereus group and is identified as the species of this group that typically causes foodborne disease in humans. Bc does not produce the parasporal crystal. It can develop and produce toxins in food, causing emetic syndrome, or in the human intestine, causing gastroenteritis (Schoeni and Wong 2005).

Characterization of Sarda sheep carcasses used in the processing of meat products.

Meat quality is perceived by consumers as being connected with certain sensory characteristics. These are colour, texture, fragrance and tenderness (Ruiz De Huidobro et al ., 2003). Some authors have studied the relationship between the different productive parameters such as rearing system, age, sex, etc., and the biological characteristics of muscles, sensory parameters, or those of economic interest such as carcass conformation and slaughter yield (Mandell et al ., 1998; Byrne et al ., 2000; Maltin et al ., 2001; Maher et al ., 2004; Sami et al ., 2004). The research on sheep has concentrated on evaluating the quality of lamb meat (Arsenos et al ., 2002; Priolo et al ., 2002; Santos-Silva et al ., 2002). There is a dearth of information on adult ovine meat, which is little appreciated and economically undervalued. This study is designed to evaluate some quality aspects of Sarda sheep carcasses used in the processing of meat products, in order to meet producer demand for a system that is able to measure meat quality. The technology and characteristics of adult sheep meat products have been described in previous studies (Mazzette et al ., 2001).

Antibiotic resistance of Aeromonas ssp. strains isolated from Sparus aurata reared in Italian mariculture farms

Selective pressure in the aquatic environment of intensive fish farms leads to acquired antibiotic resistance. This study used the broth microdilution method to measure minimum inhibitory concentrations (MICs) of 15 antibiotics against 104 Aeromonas spp. strains randomly selected among bacteria isolated from Sparus aurata reared in six Italian mariculture farms. The antimicrobial agents chosen were representative of those primarily used in aquaculture and human therapy and included oxolinic acid (OXA), ampicillin (AM), amoxicillin (AMX), cephalothin (CF), cloramphenicol (CL), erythromycin (E), florfenicol (FF), flumequine (FM), gentamicin (GM), kanamycin (K), oxytetracycline (OT), streptomycin (S), sulfadiazine (SZ), tetracycline (TE) and trimethoprim (TMP). The most prevalent species selected from positive samples was Aeromonas media (15 strains). The bacterial strains showed high resistance to SZ, AMX, AM, E, CF, S and TMP antibiotics. Conversely, TE and CL showed MIC90 values lower than breakpoints for susceptibility and many isolates were susceptible to OXA, GM, FF, FM, K and OT antibiotics. Almost all Aeromonas spp. strains showed multiple antibiotic resistance. Epidemiological cut-off values (ECVs) for Aeromonas spp. were based on the MIC distributions obtained. The results showed a high frequency of Aeromonas spp. contamination in Sparus aurata reared on the Italian coast and an elevated biodiversity in isolated bacterial strains. Aeromonas isolates comprise potentially pathogenic species for humans, often resistant to several antibiotics and able to transfer the genes responsible for antibiotic resistance to microorganisms pathogenic for humans throughout the food chain. The few ECV studies available on many antibiotics against Aeromonas spp. strains isolated from the aquaculture environment highlight the need for further research in this area, while regular monitoring programmes should be stepped up to check for antibiotic resistance.