Foodborne illnesses caused by the ingestion of foods contaminated with pathogens and/or their toxins arestill one of the major public health threats worldwide. Disposable devices, allowing the on-site, early andmultiplexed quantitative detection of pathogenic bacteria are therefore highly sought. Herein, we reportbiochips that are able to quantitatively detect two of the most common food-associated pathogens,namely Listeria monocytogenes and Staphylococcus aureus from the suspensions of bacteria stationaryphasebroth culture. With a detection limit as low as 5.00 CFU ml-1 for L. monocytogenes and 1.26 CFUml-1 for S. aureus, our platform may be a promising point-of-care device not only for clinical and fooddiagnostics but also for biosecurity purposes.

The correct identification and quantitative detection of Lactobacillus casei-group members continues tobe a key issue due to the extensive use of members of this group as (probiotic) adjuncts, corrective and/orreinforcement cultures. Numerous selective/differential media use selective agents to which injured orstressed cells are sensitive, resulting in an underestimation of the actual number of target bacteria. Wedeveloped a selective medium and applied it to detect members of the Lb. casei-group reliably, regardlessof their physiological condition, quantitatively detect and isolate strains of this group from probiotic milkand cheeses, and monitor the probiotic Lb. paracasei CRL 431 strain in Caciotta cheeses made either fromgoats' or cows' milk inoculated with this probiotic strain and a multiple strain culture. This approachcould be useful for control bodies, agro-food and pharmaceutical industries to isolate Lb. casei-groupstrains, develop probiotic food/supplements and demonstrate their compliance with the specieslabelling.

Food safety is embedded in food-related problems, and in proposed solutions. Despite continuous investment, the WHO estimates 23 million cases of foodborne illness and 5000 deaths in Europe every year and Europeans are not confident in the food system. Now, the circular economy aims to improve global food security through sustainable production, thus new ingredients, methods and food safety challenges. Food is unequivocally linked to non-communicable diseases, and changes are needed for nutritional food safety. Emerging and re-emerging foodborne pathogens are changing the epidemiology of foodborne diseases. Additionally, some chemicals are of concern, and food is a major source of human exposure. Finally, risk communication is required for management of consumer-based foodborne hazards, yet this foodborne illness is common. We ignore food safety challenges at our peril as potential consequences of a lapse are huge; keeping the food supply safe is a never-ending task.

Despite great advances in the diagnostics and better awareness for food safety and security worldwide, significant numbers of foodborne outbreaks have been traced back to the consumption of milk and dairy products contaminated with pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Campylobacter spp., and pathogenic Escherichia coli. Several culture-dependent and culture-independent nucleic acid-based methods have been proposed to identify, detect, and type milk- and dairyborne pathogenic bacteria. In our review, we will provide an overview on why it is of utmost importance to ascertain the presence of pathogenic microorganisms in milk and milk products; thereafter, we will describe the most commonly used culture-dependent and culture-independent methods, as well as the most attractive ones with regard to their future exploitation, providing the reader with new insights into how and when they can be exploited to ensure the enumeration, and accurate detection at both species and strain level of the most important milk- and dairyborne pathogenic bacteria, even if in a viable but nonculturable state.

A set of degenerate PCR primers was designed and used to amplify and sequence about 75% of the catalase(kat) gene from each of 49 staphylococcal strains. In some strains of Staphylococcus xylosus, S. saprophyticus, andS. equorum, two catalase genes, katA and katB, were found. A phylogenetic tree was generated and showeddiversities among 66 partial (about 900-bp) staphylococcal kat nucleotide sequences (including 17 sequencesfound in GenBank) representing 26 different species. The topology of this tree showed a distribution ofstaphylococcal species similar, but not identical, to those reported previously based on 16S rRNA, hsp60, sodA,rpoB, tuf, and gap genes. The kat gene sequences were less conserved than those of 16S rRNA, rpoB, hsp60, andtuf genes and slightly more conserved than those of the gap gene. Therefore, kat gene sequence analysis mayprovide an additional marker for inferring phylogenetic relationships of staphylococci. Moreover, the discretenucleotide polymorphism revealed in this gene could be exploited for rapid, low-cost identification of staphylococcalspecies through PCR-restriction fragment length polymorphism (RFLP) analysis. In this study, aPCR-RFLP assay performed by using only the TaqI restriction enzyme was successfully developed for rapidunequivocal identification/differentiation, at species and subspecies levels, of coagulase-positive staphylococci(CPS). The assay was validated by testing the DNA from 100 staphylococcal strains, including reference andwild CPS strains isolated from different environments. This reliable, rapid, and low-cost approach (requiringabout 6 h from DNA isolation to the achievement of results and <5 Euros for each strain tested) allowedunambiguous identification of all the strains assayed, including the newly described S. delphini and S. pseudintermediusCPS species.

We analyzed the microbiological quality and safety of raw milk from vending machine and assessed the evolution of its microbiota during the refrigeration at 7°C. We also monitored the survival of E. coli O157:H7 strain artificially added to the raw milk during the refrigeration and investigated the possibility to use the probiotic Lactobacillus rhamnosus GG strain to control pathogenic and spoilage bacteria in raw milk. Total aerobic mesophilic microbiota of raw milk exceeded the limit allowed by the current European legislation and increased of about two log units after 48 hr of refrigeration at 7°C. The spiked E. coli O157:H7 strain multiplied slowly in the refrigerated raw milk increasing of about 1 log unit, whereas the probiotic strain was able to slightly lower the populations of Pseudomonas spp., Listeria spp. and Staphylococcus aureus but was unable to inhibit the growth of E. coli O157:H7. Practical applications: This study allowed us to confirm the microbiological threat associated to the direct consumption of raw milk. Ad hoc strategies to improve the overall microbiological quality of raw milk from vending machines within the whole chain (from farm to consumption) are needed. A protective and probiotic adjunct able to control spoilage and pathogenic bacteria in raw milk with an adequate microbiological quality (at least compliant with the current legislation) may be a valuable mitigation strategy, but probiotic strains more effective than L. rhamnosus GG should be used.

Ethnic foods are becoming popular worldwide. Nevertheless, foodborne outbreaks and food recalls due to the contamination of these foods with pathogenic agents, toxins, undeclared allergens and hazardous chemical compounds are increasing in recent years together with their growing popularity. In this context, ad hoc prevention and control strategies are needed, to assess and face the emerging microbiological and toxicological risks associated with the consumption of ethnic foods.

A simple and specific method for the rapid detection and identification of Lactobacillus brevis was developed. A fAFLP (Fluorescent Amplified Fragment Length Polymorphisms) marker for L. brevis was used to design oligonucleotide primers for a species-specific PCR assay, targeting a 125 bp fragment of the gene encoding the aldo/keto reductase of the diketogulonate-reductase family of L. brevis. This assay resulted in 100% inclusivity and exclusivity of assignment of strains to the species L. brevis. The analytical specificity of this assay was successfully tested to identify L. brevis isolates from sourdoughs.

Matsoni, a traditional Georgian fermented milk, has variable quality and stability besides a short shelf-life (72-120 h at 6 °C) due to inadequate production and storage conditions. To individuate its typical traits as well as select and exploit autochthonous starter cultures to standardize its overall quality without altering its typicality, we carried out a thorough physico-chemical, sensorial and microbial characterization of traditional Matsoni. A polyphasic approach, including a culture-independent (PCR-DGGE) and two PCR culture-dependent methods, was employed to study the ecology of Matsoni. Overall, the microbial ecosystem of Matsoni resulted largely dominated by Streptococcus (S.) thermophilus and Lactobacillus (Lb.) delbrueckii subsp. bulgaricus. High loads of other lactic acid bacteria species, including Lb. helveticus, Lb. paracasei and Leuconostoc (Leuc.) lactis were found to occur as well. A selected autochthonous multiple strain culture (AMSC) composed of one Lb. bulgaricus, one Lb. paracasei and one S. thermophilus strain, applied for the pilot-scale production of traditional Matsoni, resulted the best in terms of enhanced shelf-life (one month), sensorial and nutritional quality without altering its overall typical quality. This AMSC is at disposal of SMEs who need to exploit and standardize the overall quality of this traditional fermented milk, preserving its typicality.

An extensive use of Weissella (W.) confusa is currently being made for the production of a variety of fermented foods and beverages although some strains of this species have emerged as opportunistic pathogens for humans and animals. Nevertheless, no rapid methods are available for the reliable identification of W. confusa.We developed a novel PCR using AFLP (Amplified Fragment Length Polymorphism)-derived primers for the rapid and unequivocal identification of W. confusa. Fluorescent AFLP of 30 strains of W. confusa, Leuconostoc citreum, Lactobacillus (Lb.) brevis, Lb. rossiae, Lb. plantarum and Lb. buchneri allowed us to detect, purify and sequence several W. confusa specific AFLP fragments. The homology search in BLAST of a 303 bp nucleotide sequence revealed a d77% identity of the purified fragment with the lepA gene of several lactic acid bacteria. A PCR assay targeting 225 bp of this fragment was developed and tested against the DNA of 109 strains, including 34 foodborne and clinical W. confusa and 75 strains of 47 phylogenetically closely and distantly related species, resulting in 100% specificity with a detection limit of 16 pg. Being the first species-specific PCR to date developed for the rapid and unambiguous identification of W. confusa, this novel assay could be a reliable and efficient tool for detecting W. confusa not only in food and beverages, but also in clinical specimens, thus contributing to clarify its real significance in human and animal infections.

We re-examined the sensitivity and specificity of 31 PCR assays (including four commercially available and developed in-house methods) for the identification of Campylobacter species, with particular reference to taxa described since 2004, which are closely related to C. jejuni and C. coli, the pathogenic species of most interest. Each of the assays was used by at least one of the participating nine laboratories in eight countries. The sensitivity and specificity of these PCR assays examined varied considerably and ranged from 100% to 0% for sensitivity and 100% to 55% for specificity. None of the three assays examined for C. lari were successful in detecting all strains of this species, possibly reflecting its complex taxonomy. A number of assays for C. jejuni, C. coli, and a subgroup of enteropathogenic campylobacters, were found to yield false positive results for Campylobacter species described since PCR tests were reported, including C. cuniculorum, C. subantarcticus, C. peloridis and C. volucris. Our study supports the need for attention to detail in initial PCR assay design and evaluation, and also for on-going revalidation of laboratory assays to ensure that diagnoses are correct. Recommendations to guide the revalidation process are presented.

A rich variety of indigenous fruits and vegetables grow in Africa, which contribute to thenutrition and health of Africa's populations. Fruits and vegetables have high moistureand are thus inherently prone to accelerated spoilage. Food fermentation still plays amajor role in combating food spoilage and foodborne diseases that are prevalent inmany of Africa's resource disadvantaged regions. Lactic acid fermentation is probablythe oldest and best-accepted food processing method among the African people,and is largely a home-based process. Fermentation of leafy vegetables and fruits is,however, underutilized in Africa, although such fermented products could contributetoward improving nutrition and food security in this continent, where many are stillmalnourished and suffer from hidden hunger. Fermentation of leafy vegetables and fruitsmay not only improve safety and prolong shelf life, but may also enhance the availabilityof some trace minerals, vitamins and anti-oxidants. Cassava, cow-peas, amaranth,African nightshade, and spider plant leaves have a potential for fermentation, as dovarious fruits for the production of vinegars or fruit beers and wines. What is needed toaccelerate efforts for production of fermented leaves and vegetables is the developmentof fermentation protocols, training of personnel and scale-up of production methods.Furthermore, suitable starter cultures need to be developed and produced to guaranteethe success of the fermentations.

A culture-independent and two culture-dependent real time (rt-) PCR approaches were developed toquantitatively identify Listeria monocytogenes in raw milk and soft cheeses. The optimised rt-PCRrevealed 100% inclusivity and exclusivity. DNA- and cell-based standard curves showed a good linearityof response (R2
0.987 and R2
0.998, respectively) for five orders of magnitude (39  105 e3  100 genome equivalents and 106e101 CFU equivalents, respectively) with about 100% relative accuracyand inter-assay variability 0.90%. Up to 1 genome equivalent/and 10 CFU/reaction were quantifiedin the DNA and cell standard curves, respectively. The rt-PCR was then combined with a liquid-(MPN technique) or a solid- (ALOA and PALCAM) based enumeration. The diagnostic sensitivity of thedifferent approaches was investigated in artificially contaminated raw milk and soft cheeses. The rt-PCRculture-independent approach performed well in raw milk and (with a lower sensitivity) in stracchinocheese-based standard curves. MPN/rt-PCR was the best approach to enumerate low levels ofL. monocytogenes in raw milk and stracchino cheese, while the ALOA-based rt-PCR quantification wasmore effective than the PALCAM-based. These performances were confirmed when 23 real samples ofraw milk and soft cheeses by both the rt-PCR approaches were assayed.

A TaqMan and a SYBR Green real time PCR (rt-PCR) were developed for the reliable identification and quantitative detection of Staphylococcus (S.) aureus strains harbouring the enterotoxin gene cluster (egc) regardless of its variants. Both approaches revealed 100% specificity against a panel of 70 reference strains, including 29 clinical and foodborne S. aureus strains harbouring all the egc variants to date known, 4 egc-S. aureus strains and 37 strains of phylogenetically closely and distantly related species. Standard curves made by 10 fold dilutions of either genomic DNA or cells from an egc+ S. aureus log-phase broth culture showed a good linearity of response (R2e0.993) for six orders of magnitude, with about 100% relative accuracy and a low inter-assay variability (CVd3.02). The overall limit of quantification (LOQ) for both rt-PCR assays (about 100% PCR efficiency; running time 30 min) was 10 cfu or 10 genome equivalents per reaction mixture although 1 cfu or 1 genome equivalent was detected with a 33.33% probability. These performances were confirmed in raw milk artificially contaminated with log-phase broth cultures of either a single egc+ S. aureus strain or a mixture of S. aureus strains harbouring all the egc variants to date known. Similar results were also obtained with a raw milk based standard curve of the S. aureus egc+ mixture in the presence of 106 cfu/mL of egc- S. aureus strains harbouring some of the commonest enterotoxin genes associated to the staphylococcal food poisoning. Nonetheless, the TaqMan based approach resulted in a lower sensitivity (LOQ=100 cfu equivalents per reaction mixture) than the SYBR Green based assay (LOQ=10 cfu equivalents per reaction mixture). When applied to real milk samples, both PCR assays provided a good response with 100% diagnostic specificity and 96–107% relative accuracy, as compared to conventional culture-based PCR approaches. Due to the high specificity, the wide dynamic range of detection and the high sensitivity demonstrated even in a complex and potentially highly contaminated raw milk matrix, the SYBR Green rt-PCR assay is a useful diagnostic tool for quick, high throughput and reliable routine screening of egc+ S. aureus isolates. Moreover, the SYBR Green based quantitative detection of these pathogens in raw milk could remarkably contribute to clarify their actual role in staphylococcal food poisoning and other clinical syndromes associated with the consumption of milk and milk-based products.

Despite the use of several Weissella (W.) strains for biotechnological and probioticpurposes, certain species of this genus were found to act as opportunistic pathogens,while strains of W. ceti were recognized to be pathogenic for farmed rainbow trout.Herein, we investigated the pathogenic potential of weissellas based on in silico analysesof the 13 whole genome sequences available to date in the NCBI database. Ourscreening allowed us to find several virulence determinants such as collagen adhesins,aggregation substances, mucus-binding proteins, and hemolysins in some species.Moreover, we detected several antibiotic resistance-encoding genes, whose presencecould increase the potential pathogenicity of some strains, but should not be regardedas an excluding trait for beneficial weissellas, as long as these genes are not present onmobile genetic elements. Thus, selection of weissellas intended to be used as startersor for biotechnological or probiotic purposes should be investigated regarding theirsafety aspects on a strain to strain basis, preferably also by genome sequencing, sincenucleotide sequence heterogeneity in virulence and antibiotic resistance genes makesPCR-based screening unreliable for safety assessments. In this sense, the applicationof W. confusa and W. cibaria strains as starter cultures or as probiotics should beapproached with caution, by carefully selecting strains that lack pathogenic potential.

The Institute of Sciences of Food Production (ISPA) is a centre of excellence acting in the fields of scientific research, innovation and technology transfer aimed to improve safety and quality of agro-food products. The Institute policy targets continuous innovation and socio-economic growth of territories by building and implementing strategic cooperative partnerships with world leader Institutions in the area of agro-food (FAO, EFSA, FSA, USDA, etc.) and with SMEs and large enterprises as well as local universities and Consortia of P.D.O. products within funding programs promoted by local, national and international bodies.

A mid-log phase broth culture of Escherichia (E.) coli O157:H7 381 (final concentration 104 cfu/mL) was monitored by conventional liquid- and solid-based enumeration techniques combined with PCR while it was subjected to thermal stress in gradually more complex systems (i.e., Tryptone Soya Broth, pasteurized milk and during lab-scale productions of a pasta filata fior di latte cheese obtained from raw or pasteurized milk). Our results highlighted: i) the incapability of the selective medium, ii) the effectiveness of the thin agar layer-PCR method, and iii) the effectiveness of the most probable number (MPN)-PCR method (in comparison with both plating-based methods) in recovering and selectively counting viable and stressed or injured E. coli O157:H7. Moreover, MPN-PCR was superior to both plating-based methods in terms of speed and easiness to get results. The thermal stresses herein applied (heating at 55 °C for 5 and 8 min) were less effective on the pasteurized milk than on the Tryptone Soya Broth and the pathogen was more protected in the raw milk-based matrices than in the pasteurized ones. Moreover, given the contamination level (104 cfu/mL of milk) of the strain, the temperature/time of stretching and the hardening and brining conditions herein used, the complete inactivation of the pathogen is not achievable.