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Listeria monocytogenes is a foodborne pathogen responsible for about 1600 illnesses each year in the United States (US) and about 2500 confirmed invasive human cases in European Union (EU) countries. Several technologies and antimicrobials are applied to control the presence of L. monocytogenes in food. Among these, the use of natural antimicrobials is preferred by consumers. This is due to their ability to inhibit the growth of foodborne pathogens but not prompt negative safety concerns. Among natural antimicrobials, plant extracts are used to inactivate L. monocytogenes. However, there is a large amount of these types of extracts, and their active compounds remain unexplored. The aim of this study was to evaluate the antibacterial activity against L. monocytogenes of about 800 plant extracts derived from plants native to different countries worldwide. The minimal inhibitory concentrations (MICs) were determined, and scanning electron microscopy (SEM) was used to verify how the plant extracts affected L. monocytogenes at the microscopic level. Results showed that 12 of the plant extracts had inhibitory activity against L. monocytogenes. Future applications of this study could include the use of these plant extracts as new preservatives to reduce the risk of growth of pathogens and contamination in the food industry from L. monocytogenes.

Listeria monocytogenes is a pathogenic bacterium responsible for foodborne illness worldwide. Antimicrobial peptides, or bacteriocins, produced by food-grade lactic acid bacteria can serve as preservatives to prevent Listeria's growth in various foods, including dairy products. This study investigated the anti-listerial activities of bacteriocin-producing lactic acid bacteria, Streptococcus thermophilus B59671, and Lactobacillus plantarum 076. In vitro studies showed that the concentration of pediocin produced by L. plantarum 076 (2560 AU/mL) inhibited the growth of a six-strain cocktail of L. monocytogenes. However, the concentration of thermophilin 110 produced by S. thermophilus B59671 (320 AU/mL) only delayed the growth by ~2 h. Higher concentrations of thermophilin 110 (≥640 AU/mL) suppressed Listeria growth for up to 22 h. Pasteurized skim milk fermented with a co-culture of S. thermophilus B59671 and L. plantarum 076 reduced the number of L. monocytogenes cells by > 4 Log CFU/mL due mainly to the activity of pediocin. The anti-listerial activity was not observed in whey samples collected from pasteurized skim milk fermented with this co-culture but was detected when raw milk was the substrate. Two additional whey preparations, the by-products from commercial bovine and goat raw-milk cheeses, also inhibited Listeria growth and reduced the number of cells following storage at 4 °C for one week. This study showed that a concentrated preparation of thermophilin 110 has potential as an anti-listerial compound. It demonstrated the prospect of using a co-culture of S. thermophilus B59671 and L. plantarum 076 to prevent Listeria contamination in dairy foods. Additionally, results showed that metabolites with antimicrobial activities may be generated during the fermentation of raw milk due to indigenous microflora.

Aims To evaluate the performance of the IQ-Check kits and the USDA Microbiology Laboratory Guidebook (MLG) methods for detection of the top seven Shiga toxin-producing Escherichia coli (STEC) (O157:H7, O26, O45, O103, O111, O121 and O145) in ground beef and both STEC and Salmonella in co-inoculated samples. Methods and Results Ground beef samples inoculated with ~10 CFU of STEC or both STEC and Salmonella Typhimurium were stored at 4°C for 72 h, followed by screening with the IQ-Check and BAX System kit (MLG) methods that employ different enrichment media. STEC and S. Typhimurium were detected after 12 and 18 h and their presence was confirmed by colony isolation. Conclusions Both methods were able to detect STEC in ground beef after 12 h of enrichment in samples inoculated with low levels of the pathogen. STEC and S. Typhimurium can be detected and isolated in co-inoculated ground beef samples. Significance and Impact of the Study The IQ-Check methods are comparable to the MLG methods for detection of STEC and simultaneous detection of STEC and S. Typhimurium in seeded ground beef after a short enrichment time, thus the IQ-Check method can be useful for the food industry for rapid detection of these pathogens.

Inclusion of novobiocin as a selective agent for enrichment media and selective agars inhibits the growth of some Shiga toxin-producing Escherichia coli (STEC) strains, particularly non-O157 STEC, which can yield false-negative detection results. Here, we report the draft genomic sequences of seven STEC O111 isolates with different sensitivities to novobiocin.

Bivalve molluscs are a well documented source of viral infection. Further data on shellfish viral contamination are needed to implement European Regulations with sanitary measures more effective against viral pathogens. To this aim, 336 samples of bivalve molluscs (185 mussels, 66 clams, 23 oysters and 62 samples from other species) collected in harvesting areas of class A and B of four Italian Regions were analyzed for qualitative and quantitative determination of hepatitis A virus (HAV) and Norovirus (NoV) GI and GII, using real time RT-PCR. The results showed a wide diffusion of viral contamination in the shellfish production areas considered. HAV prevalence was low (0.9%) with contamination levels that varied from 5 to 7 × 10(2)copies/g. On the contrary, NoV showed a high prevalence (51.5%), with a large variability according to the group considered (e.g. 47.8% for Crassostrea in Veneto, 79.7% for Mytilus in Campania, 84.6% for Tapes in Sardinia). NoV contamination affected class A and class B production areas to a different extent, with a statistically significant difference in both contamination prevalence (22.1% vs. 66.3%; p

The aim of this study was to measure the persistence of residues of the pyrethroid insecticide α-cypermethrin (ACYP) in the milk of lactating donkeys following pour-on treatment. Milk was collected from animals (n = 7) before the treatment and at 12, 24, 36, 48, 60, 72 and 84 h post-treatment. The last sampling was taken 7 days post-treatment (168 h). Milk samples were analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The analytical method was validated following requirements of Commission Decision 2002/657/EC. All samples showed levels of ACYP below the maximum residue limit (MRL) of 20 μg kg(-1) established for bovine milk (Commission Regulation (EU) No. 37/2010). The results demonstrate that there is minimal partitioning of ACYP into milk in lactating donkeys from pour-on treatment.

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015&ndash, 2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV, 8.9%), norovirus GI (NoVGI, 10.8%) and GII (NoVGII, 39.7%), rotavirus (RV, 9.0%), astrovirus (AsV, 20.8%), sapovirus (SaV, 18.8%), aichivirus-1 (AiV-1, 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.

Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. LMOf2365_0442, 0443, and 0444 encode for phosphotransferase transport system (PTS) permease (fructose-specific IIABC components) that is responsible for sugar transport. LMOf2365_0445 encodes for glycosyl hydrolase. These genes were induced by high pressure and inhibited under salt treatments; therefore, we hypothesized that genes encoding these PTS proteins may be involved in general stress responses. To study the function of these genes, deletion mutants of the PTS genes (LMOf2365_0442, LMOf2365_0443, and LMOf2365_0444) and the downstream gene LMOf2365_0445 were created in L. monocytogenes strain F2365. These deletion mutants were tested under different stress conditions. The growth of ∆LMOf2365_0445 was increased under nisin (125 μg/mL) treatments compared to the wild-type (P < 0.01). The growth of ∆LMOf2365_0442 in salt (brain-heart infusion medium with 5% NaCl) was significantly increased (P < 0.01), and ∆LMOf2365_0442 showed increased growth under acidic conditions (pH 5.0) compared to the wild-type (P < 0.01). The results from phenotypic arrays demonstrated that some of these mutants showed slightly slower growth under different carbon sources and basic conditions. The results indicate that deletion mutants ∆LMOf2365_0442 and ∆LMOf2365_0445 were more resistant to multiple stress conditions compared to the wild-type, suggesting that they may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions may assist in the development of intervention strategies to control L. monocytogenes in food.

Background/Aims: We studied the association of low birth weight with ultrasound-assessed nonalcoholic fatty liver disease (NAFLD) to test the hypothesis that fetal growth retardation followed by a rapid weight catch-up growth might be an additional factor responsible for liver steatosis via insulin resistance (IR) and/or intra-abdominal fat. Methods: We enrolled 23 children born small for gestational age (SGA) with a rapid catch-up growth within the first 6-12 months, and 24 appropriate for gestational age (AGA) children as controls. All children underwent anthropometric, body composition measurements and evaluation of liver function tests, lipid profile, plasma glucose and insulin levels. Abdominal ultrasonography was performed in order to asses liver steatosis and thickness of subcutaneous and visceral adipose tissue. Results: NAFLD were observed in 8 out of the 23 SGA children (34.8%). IR and visceral fat were significantly increased in children with hepatic steatosis compared to those without. IR index was significantly related to liver steatosis, independently of body mass index standard deviation score and visceral fat. Conclusions: NAFLD should be recognized as an emerging problem in SGA prepubertal children who presented a rapid weight gain in postnatal life, and IR plays the key role. An appropriate diet during pregnancy and in the first year of life might prevent metabolic syndrome and NAFLD in these subjects.

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx 2e (81%) was the most common Stx variant, followed by stx 1a (14%), stx 2d (3%), and stx 1c (1%). The STEC serogroups that carried stx 2d were O15:H27, O159:H16 and O159:H-. Similar to stx 2a and stx 2c, the stx 2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili (pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.

Shellfish are recognized as a potential vehicle of viral diseases. However, according to European Regulations, only bacteriological parameters (Salmonella and E. coli) are used to establish their microbiological quality. Because of the lack of a standard reference method for the detection of viruses, data on the incidence of enteric viruses in European production areas are still limited. Aim of the present study was the evaluation of the Norovirus (NoV) contamination in shellfish from different harvesting areas (both class A and B) and from registered and not- registered retails of the Campania region (Tyrrhenian Sea, Italy). Seventy shellfish (Mytilus galloprovincialis) were collected from 15 harvesting areas, one authorized and two not authorized retails and were examined for virus presence (NoV genogroups I and II) by Real Time PCR. Ten harvesting areas (66%) showed presence of viral contamination, with three of them belonging to A areas and seven to B areas. The shellfish collected from the registered retail tested positive for Norovirus presence, whereas the shellfish collected from not-authorized retails didn’t show viral contamination. Twenty-nine samples were positive for genogroup II, ten of which tested positive also for NoV genogroup I.

Listeria monocytogenes (Lm) is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC) transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877) were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that DLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

The incidence, the number and the behavior of L. monocytogenes in three lots of naturally-contaminated vacuum-packed sliced smoked salmon, processed in different plants, were investigated during storage at 2 and 10 degrees C. L. monocytogenes was isolated from 20 of the 100 packages stored at 2 degrees C (16 from lot 1, 1 from lot 2 and 3 from lot 3) and from 12 of the 65 packages stored at 10 degrees C (all from lot 1). The levels detected were 15, 20, 290, 1100 and1100 cfu/g in 5 packages, all belonging to lot 1, and10 cfu/g in the remaining samples. The high incidence of L. monocytogenes in lot 1 was assumed to be due mainly to the use of causal workers for the slicing and packing operations.

Functional genomics approaches enable us to investigate the biochemical, cellular, and physiological properties of each gene product and are nowadays applied to enhance food safety by understanding microbial stress responses in food and host-pathogen interactions. Listeria monocytogenes is a food-borne pathogen that causes listeriosis and is difficult to eliminate this pathogen since it can survive under multiple stress conditions such as low pH and low temperature. Detailed studies are needed to determine its mode of action and to understand the mechanisms that protect the pathogen when it is subjected to stress. In this study, deletion mutants of phosphotransferase transport system genes (PTS) and adenosine triphosphate(ATP)-binding cassette transporters (ABC) of Listeria monocytogenes F2365 were created using molecular techniques. These mutants and the wild-type were tested under different stress conditions, such as in solutions with different NaCl concentration, pH value and for nisin resistance. Results demonstrate that the behaviour of these deletion mutants is different from the wild type. In particular, deleted genes may be involved in L. monocytogenes resistance to nisin and to acid and salt concentrations. Functional genomics research on L. monocytogenes allows a better understanding of the genes related to stress responses and this knowledge may help in intervention strategies to control this food-borne pathogen. Furthermore, specific gene markers can be used to identify and subtype L. monocytogenes. Thus, future development of this study will focus on additional functional analyses of important stress response-related genes, as well as on methods for rapid and sensitive detection of L. monocytogenes such as using DNA microarrays.

Bivalve molluscan shellfish such as oysters may be contaminated by human pathogens. Currently, the primary pathogens associated with shellfish-related outbreaks are noroviruses. This study was conducted to improve understanding of oyster bioaccumulation when oysters were exposed to daily contamination or one accidental contamination event, i.e., different modes of contamination. Oysters were contaminated with two representative strains of norovirus (GI.1 and GII.3) and then analyzed with real-time reverse transcription PCR. Exposure to a repeated virus dose for 9 days (mimicking a growing area subjected to frequent sewage contamination) led to an additive accumulation that was not significantly different from that obtained when the same total dose of virus was added all at once (as may happen after accidental sewage discharge). Similarly, bioaccumulation tests performed with mixed strains revealed additive accumulation of both viruses. Depuration may not be efficient for eliminating viruses; therefore, to prevent contaminated shellfish from being put onto the market, continuous sanitary monitoring must be considered. All climatic events or sewage failures occurring in production areas must be recorded, because repeated low-dose exposure or abrupt events may lead to similar levels of accumulation. This study contributes to an understanding of norovirus accumulation in oysters and provides suggestions for risk management strategies.

Norovirus (NoV) is the main cause of human nonbacterial gastroenteritis throughout the world. NoVs are classified into five genogroups: GI, GII, GIII, GIV, and GV. NoVs from GI and GII are the most commonly reported NoVs associated with human infections, and raw or undercooked shellfish have been identified as the main potential infection vehicle. European Commission Regulation 2073/2005 defines only bacteriological parameters for use as safety criteria for shellfish because reference methods for detection of viruses are lacking. From July 2007 to April 2010, 163 shellfish samples were collected in southern Italy from harvesting areas, authorized or nonauthorized retailers, and a restaurant after an outbreak of human gastroenteritis. The shellfish were analyzed for the presence of NoVs from GI and GII using the one-step real-time reverse transcription PCR protocol. A total of 94 shellfish samples (57.7%) were positive for the presence of NoV, and GII was the most frequently identified genogroup.

Campylobacter is a major foodborne pathogen responsible for acute gastroenteritis characterized by diarrhea that is sometimes bloody, fever, cramps, and vomiting. Campylobacter species are carried in the intestinal tracts of mammals and birds, and sources of human infection include raw milk, contaminated water, direct contact with pets, and foods, particularly poultry. Campylobacter jejuni and C. coli are the species that account for the majority of human infections. The aim of this work was to determine the prevalence of Campylobacter in 190 poultry carcasses sampled at slaughter and to use a multiplex PCR assay to determine if the isolates were C. jejuni or C. coli. C. coli was not isolated, while C. jejuni was recovered from 52 (37.1%) of 140 carcasses for which pools of four sampling sites (neck, cloaca, breast, and back) were examined. In the remaining 50 carcasses, the four sites were analyzed separately, and C. jejuni was recovered from the samples in the following order: neck (n = 20), cloaca (n = 16), breast (n = 14), and back (n = 11). The results are in agreement with those of other studies, which showed that C. jejuni is more commonly associated with poultry than is C. coli. Control strategies for Campylobacter should include interventions to eliminate C. jejuni in poultry at various stages of production and processing, including at slaughter.

A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly 933 ), and chromosomal flagella (fliC h7 ; flagellar structural gene of H7 serogroup), Shiga toxins (stx 1 , stx 2 ), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E. coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was ≤ 1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.