Survival of Escherichia coli O157:H7 during the manufacture and ripening of Cacioricotta goat cheese.

The aim of this study was to assess the growth and survival of Escherichia coli O157:H7 during the manufacturing and ripening of Cacioricotta goat cheese. Goat milk was artificially contaminated with E. coli O157:H7 and the bacterial load was monitored from production up to 90 days of ripening. Goat milk was inoculated with 102 cfu ml-1 of E. coli O157:H7 and the bacterial count of the curd at time zero was 2.31 log10 cfu g-1. During the first day of ripening, the bacterial load has increased to 5.73 log10 cfu g-1 to more than 6.20 log10 cfu g-1 during the first week. The bacterial load remained constant up to 28 days and then slightly decreased until the end of ripening, with values of aw and pH of 0.88 and 5.41 respectively. The results of this study highlighted that E. coli O157:H7 is able to survive the manufacturing process and they suggest that the 90-day period of ripening alone is insufficient to remove E. coli O157:H7 in contaminated Cacioricotta goat cheese. Moreover, these results support the assumption that the presence of a low contamination of milk with E. coli O157:H7 could represent a potential source of infection and a threat to consumers.

Prevalence and characterization of methicillin-resistant Staphylococcus aureus isolates in food industry workers.

BACKGROUND: Methicillin-resistant S. aureus (MRSA) is a pathogen spread not only in the hospital environment but also in the community and amongst livestock (LA-MRSA). LA-MRSA can be transmitted to humans that live in close contact with MRSA-colonized animals, and human colonization and/or infection has been reported worldwide, particularly among those involved with livestock farming. In this study the authors evaluated the prevalence of S. aureus and MRSA among healthy carriers who worked in the food industry in Apulia, Southern Italy. METHODS: Nasal swabs were taken from pasta and pork industry workers. All swab samples were subjected to tests for the isolation, identification and typing of S. aureus and MRSA strains. The identification of the strains was confirmed by molecular assessment using multiplex-PCR for the amplification of the nuc and mecA genes. The strains identified as MRSA were then subjected to a PCR protocol for the characterization of sequence type ST398. RESULTS: In total 26.3% of examined nasal swabs were positive for S. aureus, 8.2% of them were methicillin resistant strains and 28.5% of MRSA isolates were characterized as ST398. The MRSA prevalence among pork factory workers was 3% , whereas among the pasta operators the prevalence was 11.5. CONCLUSION: The presence of S. aureus and MRSA among food workers represents a public health risk. Further, considering the dissemination of S. aureus and MRSA among non-nosocomial environments, including communities and livestock, careful surveillance and continuous monitoring of the emergence of MRSA is fundamental for safeguarding public health.

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.

High occurrence of Helicobacter pylori in raw goat, sheep and cow milk inferred by glmM gene: a risk of food-borne infection?

Helicobacter pylori is an organism widespread in humans and sometimes responsible for serious illnesses, such as gastric and duodenal ulcers, MALToma and even gastric cancer. It has been hypothesized that the infection route by H. pylori involves multiple pathways including food-borne transmission, as the microorganism has been detected from foods such as sheep and cow milk. This work reports the results of a survey conducted in order to investigate the presence of H. pylori in raw goat, sheep and cow milk produced in Southern Italy, employing a Nested Polymerase Chain Reaction (Nested-PCR) assay for the detection of the phosphoglucosamine mutase gene (glmM), as screening method followed by conventional bacteriological isolation. Out of the 400 raw milk samples examined, 139 (34.7%) resulted positive for the presence of glmM gene, but no strains were isolated. In this work H. pylori DNA has been firstly detected from 41 (25.6%) raw goat milk samples. The results deserve further investigations on the contamination source/s of the milk samples and on the major impact that it may have on consumers.

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products.

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.

Escherichia coli O26 in minced beef: prevalence, characterization and antimicrobial resistance pattern.

Verocytotoxin-producing Escherichia coli (VTEC) non-O157 serogroups are among the most important emerging food-borne pathogen groups. In particular, the O26 serogroup is able to cause a large spectrum of illnesses in humans which have a significant public health impact as they may range from haemorrhagic colitis (HC) to haemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). It is known that VTEC organisms are associated with animal reservoirs, i.e. ruminants, and foods of animal origin, especially undercooked meat and raw milk, are often involved in outbreaks. In this study, 250 minced beef samples collected at retail outlets in southern Italy were tested for the presence of E. coli O26 and the isolates were characterized and studied for their antimicrobial resistance properties. Three minced beef samples (1.2%) tested positive for E. coli O26; one isolate per positive sample was characterized. One isolate harboured the genes encoding for virulence factors intimin (eaeA) and enterohaemolysin (hlyA), while none presented verocytotoxin-encoding genes (stx1 and stx2) and all were negative at the verotoxicity assay. All the isolates showed resistance properties to at least four antimicrobial agents tested and two were multi-drug resistant (MDR). Although no verocytotoxin-encoding genes were found in the isolates, the presence of potentially pathogenic E. coli O26 strains in minced beef points to the need for proper hygiene during meat production to reduce the risk of food-borne illnesses and transmission of MDR organisms via foods to humans. This paper is the first report on the presence and characterization of E. coli O26 in minced beef marketed in Italy.

Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a global health concern. The present study regarded 160 S. aureus strains that had been isolated from 1634 foodstuff samples of animal origin in a previous survey conducted in Italy during 2003-2005. The strains were characterized by detecting the mecA gene, the production of type A to D staphylococcal enterotoxins (SEs), and studying their resistance properties against several antibiotics; their ecological origin was determined by biotyping. Of the 160 analyzed S. aureus strains six (3.75%) were mecA positive and derived from six different samples; four isolates were from bovine milk and two from dairy products (pecorino cheese and mozzarella cheese). Two strains isolated from milk belonged to the non-host-specific biovar while the others to the ovine biovar. The strain isolated from mozzarella cheese belonged to the non-host-specific biovar and the strain isolated from pecorino cheese to the ovine biovar. All the MRSA strains isolated were enterotoxigenic; two strains synthesized SEA/SED two SED and one SEC. All the strains showed resistance to at least one of the antibiotics tested but none was resistant to glycopeptides.

Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products.

Staphylococcus aureus is considered to be one of the leading causes of food-borne illnesses. Milk, dairy products and meats are often contaminated with enterotoxigenic strains of this bacterium. Foodstuff contamination may occur directly from infected food-producing animals or may result from poor hygiene during production processes, or the retail and storage of foods, since humans may carry the microorganism. The number of S. aureus strains that exhibits antimicrobial-resistance properties has increased, together with the potential risk of transmitting the same properties to the human microflora via foods or inducing infections hard to be treated. This paper reports the results of a 3-year survey (2003-2005) on the occurrence of S. aureus in meat and dairy products. Of 1634 samples examined, 209 (12.8%) were contaminated with S. aureus. A total of 125 enterotoxigenic S. aureus strains were biotyped and their antimicrobial resistance pattern tested. Most of the isolated strains produced SED (33.6%), followed by SEA (18.4%), SEC (15.2%), SEB (6.4%) and belonged mainly to the Human ecovar (50.4%), followed by Ovine (23.2%), Non-Host-Specific (17.6%), Bovine (7.2%) and Poultry-like (1.6%) ecovars. Finally, the 68.8% analysed strains showed antimicrobial resistance properties at least at one of antibiotics tested. Human biotype showed antimicrobial resistance at more than one antibiotic than the other biotypes (p<0.05). The results provided evidence that the presence of enterotoxigenic and antimicrobial resistant strains of S. aureus has become remarkably widespread in foods. This calls for better control of sources of food contamination and of the spread of antimicrobial-resistance organisms.

Survival of Helicobacter pylori in artificially contaminated ultrahigh temperature and pasteurized milk.

Helicobacter pylori (H. pylori) is a very important bacterial pathogen of humans which may cause gastrointestinal illnesses ranging from gastric and duodenal ulcers to neoplastic diseases such as MALToma and gastric cancer. Transmission via contaminated food is still uncertain but several authors believe this can realistically occur and milk may act as a vehicle of infection. This paper reports the results of H. pylori survival trials in pasteurized and ultrahigh temperature (UHT) milks artificially contaminated and aerobically stored at 4 degrees C. The results obtained showed that the four strains used in this study (H. pylori nat 18-19-20 and H. pylori ATCC 43504), had a progressive reduction in bacterial load with a median survival of 9 days in pasteurized milk and 12 days in UHT milk, with approximate average of initial inoculum of 10(5) and 10(6)cfu/ml, respectively. These findings are very important to clarify the route of transmission of H. pylori to humans via food and for implementation of a correct risk analysis for food safety purposes.

Prevalence, molecular characterization and antimicrobial resistance of thermophilic campylobacter isolates from cattle, hens, broilers and broiler meat in south-eastern Italy.

Eleven cattle farms, 8 layer farms, 7 broiler farms and 30 broiler meat samples were investigated in south-eastern Italy throughout 2003 to evaluate the prevalence, the molecular type and antimicrobial resistance of thermophilic Campylobacters. A total of 398 samples were analysed. One Campylobacter isolate for each positive faecal swab and three isolates per positive broiler meat sample were selected for further analysis. Multiplex PCR was performed for species-level identification and PCR-RFLP of the flagellin A gene for genotyping. Resistance to 14 antimicrobials was studied in 188 Campylobacter isolates. Prevalence of campylobacters was high both on farms (100%) and in food samples (73%). On 4/11 cattle farms and on 10/15 poultry farms more than one species was isolated. The presence of more than one genotype was found on 8/11 cattle farms, on 10/15 poultry farms and in 8/22 Campylobacter-positive food samples. High rates of resistance to quinolone were observed: 9/31 (29%) C. jejuni bovine isolates, 4/22 (18%) C. jejuni poultry isolates, and 14/26 (54%) C. coli poultry isolates. Resistance to sulphamethoxazole-trimethoprim was also observed frequently: 18/26 (69%) of the avian C. coli strains, 25/31 (80%) of the C. jejuni strains isolated from poultry and 15/22 (68%) of those isolated from cattle were resistant. There was a significant difference between the rate of resistance to macrolides of C. coli and C. jejuni isolated in poultry, which amounted to 23% and 3%, respectively. This study provided data on the prevalence and antimicrobial resistance of thermophilic campylobacters in south-eastern Italy and confirmed that flaA-typing is an efficient tool to study the epidemiology of Campylobacter strains in short-term investigations.

Vibrio parahaemolyticus, Vibrio vulnificus and microorganisms of fecal origin in mussels (Mytilus galloprovincialis) sold in the Puglia region (Italy).

Mytilus galloprovincialis is one of the most commonly consumed of all bivalve molluscs. The consumption of raw bivalve molluscs has caused outbreaks of food poisoning due to Vibrio parahaemolyticus and Vibrio vulnificus. This paper reports the results of a survey on the presence of V. parahaemolyticus, V. vulnificus fecal coliform bacteria, Escherichia coli and Salmonella spp. in 600 M. galloprovincialis samples collected from retail outlets in the Puglia region. V. parahaemolyticus and V. vulnificus were found in 47 (7.83%) and 17 (2.83%) of the samples, respectively. One sample (0.16%) was contaminated with Salmonella spp. but no relationship was observed between vibrios and fecal coliforms and E. coli. There were no significant differences among vibrios present in bivalve molluscs during the 3-year survey.

Multiplex-touchdown PCR assay for the detection and genotyping of Helicobacter pylori from artificially contaminated sheep milk.

Helicobacter pylori (Hp) is an organism commonly present worldwide in the human population, sometimes causing serious illnesses such as duodenal and gastric ulcers, adenocarcinoma of the stomach, and low-grade B-cell mucosa-associated lymphoid tissue lymphoma of the stomach. This article describes a multiplex-touchdown PCR method for the identification and genotyping (vacA-s1/m1, sl/m2, and s2/m2-and cagA genes) of Hp directly from sheep milk artificially contaminated with Hp strains from human gastric biopsies and with Hp ATCC 43504. The strains from humans carried sl/m2 cagA+ and s2/m2 cagA allelic combinations, while the ATCC strains carried an sl/ml cagA+ allelic combination. The technique showed a sensitivity of 15 CFU/ml for species identification and of 1,500 CFU/ml for the detection of genes encoding for VacA and CagA. It has proven to be specific and rapid, and the authors suggest that it be used as a rapid screening method to ensure that sheep milk is uncontaminated with this organism.

Coagulase-positive Staphylococci and Staphylococcus aureus in food products marketed in Italy.

Staphylococcus aureus is a very common organism capable of producing several enterotoxins (SEs) that cause intoxication symptoms of varying intensity in humans when ingested through contaminated food. This paper reports the results of an investigation on the presence of Coagulase-Positive Staphylococci (CPS) and S. aureus in several food products marketed in Italy and on food contact surface swabs sampled from the food industry. A total of 11,384 samples were examined and 1971 of them (17.3%) were found to contain CPS. The assays performed on 541 CPS strains led to the identification of 537 S. aureus strains on which characterization of type A, B, C and D staphylococcal enterotoxins (SEA, SEB, SEC and SED) was performed. A total of 298 S. aureus strains (55.5%) produced one or more SEs: 33.9% of the strains produced SEC, 26.5% SEA, 20.5% SEA+SED, 13.4% SED, 2.7% SEB, 1.7% SEA+SEB, 0.7% SEC+SED and 0.3% produced SEA+SEC and SEB+SEC. The investigation highlighted that these organisms are very common and constitute a potential risk for consumers' health.

Market survey of Vibrio spp. and other microrganisms in Italian shellfish.

A survey was conducted of Vibrio spp., Escherichia coli, fecal coliforms, and Salmonella in 644 molluscan shellfish samples marketed in the Apulia region of southern Italy. Vibrios were found in 278 samples (43%), and levels of E. coli and fecal coliforms were above the Italian legal limit in 27 and 34 samples (4 and 5%), respectively. Salmonella was not detected in any of the samples. Because the majority of the vibrio isolates were found in samples that were compliant with Italian regulations, there appears to be no relationship between the presence of microorganisms of fecal origin and the presence of vibrios potentially harmful to human health.

Enterotoxins of Staphylococcus aureus induce a cytopathic effect in cell lines.

We describe the use of cell cultures for the detection of Staphylococcus aureus enterotoxins. Madin Darby Bovine Kidney (MDBK), Bovine Embryo lung cells (PEB) and Dog Carcinoma Cell line (A-72) cell monolayers were tested. MDBK, A-72 and PEB cell lines proved to be susceptible to the cytotoxic effect induced by staphylococcal enterotoxins. A cytopathic effect was observed in some cases after 2 hours of incubation. The PEB cell line was the most susceptible and may be used as a useful and cheap method for screening enterotoxigenic S. aureus strains.

Left main coronary artery aneurysm: a case report and review of the literature.

Left main coronary artery aneurysm is an uncommon feature of coronary artery disease in adults. We describe the case of a large aneurysm in a 58-year-old patient undergoing cardiac catheterization for effort angina and inducible myocardial ischemia. Specific considerations about the underlying causes and therapeutic options are discussed.

[Immunologic abortion and its treatment by immunotherapy]. / La maladie abortive immunologique et son traitement par immunothérapie.

Recurrent spontaneous abortions in most cases, can be explained by classical abnormalities; but for some cases without etiology and consequently without appropriate therapy until a few years ago, there is hope a successful treatment, thanks to recent advances. Contrary to what was suspected in the past, during pregnancy, the mother is immunologically competent against the paternal antigens of the fetus; this competence is necessary for her to respond to the trophoblast paternal antigen stimulations and develop her immune tolerance. If, because of insufficient stimulation, the woman does not succeed in producing this tolerance, it is now possible to help her by vaccination with paternal lymphocytes, before she becomes pregnant. Our results confirm these data: Again, we observe a greater frequency of HLA antigen sharing in couples with recurrent spontaneous abortions (RSAs), especially at the DR locus, Women with three or more RSAs, produce fewer antibodies against their husbands HLA antigens than regular normal fertile women (none out of the 50 cases studied), Anti-paternal antibodies the specificity of which cannot be determined at the moment, are shown by means of the microlymphocytotoxicity test at 37 degrees C carried out on the paternal B lymphocytes. They appear with the cure of the abortive illness after treatment by paternal lymphocyte injections. In the control women who did not receive any immunotherapy, those who developed anti-paternal antibodies spontaneously had a new normal pregnancy; 57.2 of those who did not produce any anti-paternal antibodies aborted once more.