A Diagnostic Chip for the Colorimetric Detection of Legionella pneumophila in Less than 3 h at the Point of Need.

Legionella pneumophila has been pinpointed by the World Health Organization as the highest health burden of all waterborne pathogens in the European Union and is responsible for many disease outbreaks around the globe. Today, standard analysis methods (based on bacteria culturing onto agar plates) need several days (~12) in specialized analytical laboratories to yield results, not allowing for timely actions to prevent outbreaks. Over the last decades, great efforts have been made to develop more efficient waterborne pathogen diagnostics and faster analysis methods, requiring further advancement of microfluidics and sensors for simple, rapid, accurate, inexpensive, real-time, and on-site methods. Herein, a lab-on-a-chip device integrating sample preparation by accommodating bacteria capture, lysis, and DNA isothermal amplification with fast (less than 3 h) and highly sensitive, colorimetric end-point detection of L. pneumophila in water samples is presented, for use at the point of need. The method is based on the selective capture of viable bacteria on on-chip-immobilized and -lyophilized antibodies, lysis, the loop-mediated amplification (LAMP) of DNA, and end-point detection by a color change, observable by the naked eye and semiquantified by computational image analysis. Competitive advantages are demonstrated, such as low reagent consumption, portability and disposability, color change, storage at RT, and compliance with current legislation.

Νovel Polylactic Acid/Tetraethyl Citrate Self-Healable Active Packaging Films Applied to Pork Fillets' Shelf-Life Extension.

Nowadays, increased food safety and decreased food waste are two of the major global interests. Self-healable active packaging materials are an attractive option to achieve such targets. This property is critical for the hygiene and the consumption appropriateness of the food. Polylactic acid is a very promising polymeric matrix that potentially could replace the widely used low-density polyethylene due to its biobased origin and its easy biodegradable nature. The main drawback of this polymeric matrix is its brittle, fragile nature. On the other hand, tetraethyl citrate is a biobased approved food additive which became an attractive option as a plasticizer for industries seeking alternative materials to replace the traditional petrochemically derived compounds. A novel biobased film exhibiting self-healing behavior suitable for food-active packaging was developed during this study. Polylactic acid's brittleness was reduced drastically by incorporating tetraethyl citrate, and a random cut on the original self-repairing film was fully healed after 120 s. The optimum concentration of tetraethyl citrate in the polylactic acid was around 15% v/w with a water/oxygen barrier close to the relevant of polylactic acid and low migration. According to the EC50 parameter, the antioxidant activity was 300% higher than the relevant of pure polylactic acid, while according to the thiobarbituric acid and heme iron parameters, the film resisted lipid oxidation and deterioration. Finally, the total viable count parameter indicates the strong antimicrobial activity of this sample.

Thymol@activated Carbon Nanohybrid for Low-Density Polyethylene-Based Active Packaging Films for Pork Fillets' Shelf-Life Extension.

Τhe replacement of food packaging additives and preservatives with bio-based antioxidant/antibacterial compounds has been a common practice in recent years following the trend of bioeconomy and nanotechnology. Such bio-additives are often enclosed in nanocarriers for a controlled release process. Following this trend in this work, a thymol (TO)-rich activated carbon (AC) nanohybrid was prepared and characterized physicochemically with various techniques. This TO@AC nanohybrid, along with the pure activated carbon, was extruded with low-density polyethylene (LDPE) to develop novel active packaging films. The codenames used in this paper were LDPE/xTO@AC and LDPE/xAC for the nanohybrid and the pure activated carbon, respectively. X-ray diffractometry, Fourier-transform infrared spectroscopy, and scanning electron microscopy measurements showed high dispersity of both the TO@AC nanohybrid and the pure AC in the LDPE matrix, resulting in enhanced mechanical properties. The active film with 15 wt.% of the TO@AC nanohybrid (LDPE/15TO@AC) exhibited a 230% higher water/vapor barrier and 1928% lower oxygen permeability than the pure LDPE film. For this active film, the highest antioxidant activity referred to the DPPH assay (44.4%), the lowest thymol release rate (k2 ≈ 1.5 s-1), and the highest antibacterial activity were recorded, resulting in a 2-day extension of fresh pork fillets' shelf-life.

Characterization and Antibiotic Resistance of Listeria monocytogenes Strains Isolated from Greek Myzithra Soft Whey Cheese and Related Food Processing Surfaces over Two-and-a-Half Years of Safety Monitoring in a Cheese Processing Facility.

Listeriosis is a serious infectious disease with one of the highest case fatality rates (ca. 20%) among the diseases manifested from bacterial foodborne pathogens in humans, while dairy products are often implicated as sources of human infection with Listeria monocytogenes. In this study, we characterized phenotypically and genetically by whole-genome sequencing (WGS) 54 L. monocytogenes strains isolated from Myzithra, a traditional Greek soft whey cheese (48 isolates), and swabs collected from surfaces of a cheese processing plant (six isolates) in the Epirus region of Greece. All but one strain of L. monocytogenes belonged to the polymerase chain reaction (PCR) serogroups IIa (16.7%) and IIb (81.5%), corresponding to serotypes 1/2a, 3a and 1/2b, 3b, 7, respectively. The latter was identified as a PCR-serogroup IVb strain (1.8%) of serotypes 4b, 4d, 4e. Bioinformatics analysis revealed the presence of five sequence types (STs) and clonal complexes (CCs); ST1, ST3, ST121, ST 155, ST398 and CC1, CC3, CC121, CC155, CC398 were thus detected in 1.9, 83.3, 11.0, 1.9, and 1.9% of the L. monocytogenes isolates, respectively. Antibiograms of the pathogen against a panel of seven selected antibiotics (erythromycin, tetracycline, benzylpenicillin, trimethoprim-sulfamethoxazole, ampicillin, ciprofloxacin, and meropenem) showed that 50 strains (92.6%), the six surface isolates also included, were intermediately resistant to ciprofloxacin and susceptible to the rest of the six antimicrobial agents tested, whereas strong resistance against the use of a single from three implicated antibiotics was recorded to four strains (7.4%) of the pathogen isolated from Myzithra cheese samples. Thence, the minimum inhibitory concentrations (MICs) were determined for erythromycin (MIC = 0.19 µg/mL), ciprofloxacin (MIC ≥ 0.19 µg/mL), and meropenem (MIC = 0.64 µg/mL), and finally, just one strain was deemed resistant to the latter antibiotic. The phylogenetic positions of the L. monocytogenes strains and their genetic variability were determined through WGS, whilst also stress response and virulence gene analysis for the isolates was conducted. Findings of this work should be useful as they could be utilized for epidemiological investigations of L. monocytogenes in the food processing environment, revealing possible contamination scenarios, and acquired antimicrobial resistance along the food production chain.

Investigating Transcriptomic Induction of Resistance and/or Virulence in Listeria monocytogenes Cells Surviving Sublethal Antimicrobial Exposure.

The potential transcriptomic induction of resistance and/or virulence in two L. monocytogenes strains belonging to the most frequent listeriosis-associated serovars (i.e., 1/2a and 4b), following their sublethal antimicrobial exposure, was studied through qPCR determination of the relative expression of 10 selected related genes (i.e., groEL, hly, iap, inlA, inlB, lisK, mdrD, mdrL, prfA, and sigB). To induce sublethal stress, three common antimicrobials (i.e., benzalkonium chloride, thymol, and ampicillin) were individually applied for 2 h at 37 °C against stationary phase cells of each strain, each at a sublethal concentration. In general, the expression of most of the studied genes remained either stable or was significantly downregulated following the antimicrobial exposure, with some strain-specific differences to be yet recorded. Thymol provoked downregulation of most of the studied genes, significantly limiting the expression of 6/10 and 4/10 genes in the strains of ser. 1/2a and ser. 4b, respectively, including those coding for the master regulators of stress response and virulence (SigB and PrfA, respectively), in both strains. At the same time, the two genes coding for the invasion internalin proteins (InlA and InlB), with crucial role in the onset of L. monocytogenes pathogenesis, were both importantly upregulated in ser. 4b strain. The results obtained increase our knowledge of the stress physiology of L. monocytogenes under certain sublethal antimicrobial conditions that could be encountered within the food chain and in clinical settings, and may assist in better and more effective mitigation strategies.

Listeria monocytogenes serotype prevalence and biodiversity in diverse food products.

The aim of this study was to assess serotype prevalence and biodiversity of Listeria monocytogenes strains isolated from diverse food products, i.e., minced pork, fruits, and vegetables. Three hundred twenty-six samples previously purchased from supermarkets and street markets within the Athens area were studied for L. monocytogenes prevalence. A total of 121 strains were isolated from the 36 samples that were positive for L. monocytogenes. Serotyping was performed with multiplex PCR, and biodiversity was assessed with random amplified polymorphic DNA (RAPD) PCR analysis using M13, UBC155, and HLWL85 as primers and with repetitive element palindromic (rep) PCR analysis using (GTG)5 as the primer. The majority (17 of 22) of the contaminated minced pork samples contained strains identified as serotype 1/2a, either alone or in combination with strains belonging to serotypes 1/2b, 4a, 4c, or 4ab. However, all L. monocytogenes isolates from fruits and vegetables belonged to serotype 4b. Rep-PCR provided better differentiation of the isolates than did RAPD PCR and resulted in discrimination of the isolates into a larger number of unique profiles. Complete differentiation was achieved only with the combination of these subtyping techniques.

Quantifying Listeria monocytogenes prevalence and concentration in minced pork meat and estimating performance of three culture media from presence/absence microbiological testing using a deterministic and stochastic approach.

Listeria monocytogenes poses a serious threat to public health, and the majority of cases of human listeriosis are associated with contaminated food. Reliable microbiological testing is needed for effective pathogen control by food industry and competent authorities. The aims of this work were to estimate the prevalence and concentration of L. monocytogenes in minced pork meat by the application of a Bayesian modeling approach, and also to determine the performance of three culture media commonly used for detecting L. monocytogenes in foods from a deterministic and stochastic perspective. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media according to ISO 11290-1:1996 and 11290-2:1998 methods. Presence of the pathogen was confirmed by conducting biochemical and molecular tests. Independent experiments (n = 10) for model validation purposes were performed. Performance attributes were calculated from the presence-absence microbiological test results by combining the results obtained from the culture media and confirmative tests. Dirichlet distribution, the multivariate expression of a Beta distribution, was used to analyze the performance data from a stochastic perspective. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. L. monocytogenes concentration was estimated at 14-17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = -2.17%). The results showed that all media were best at ruling in L. monocytogenes presence than ruling it out. Sensitivity and specificity varied depending on the culture-dependent method. None of the culture media was perfect in detecting L. monocytogenes in minced pork meat alone. The use of at least two culture media in parallel enhanced the efficiency of L. monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L. monocytogenes presence and the uncertainty of the results obtained. Furthermore, the problem of observing zero counts may be overcome by applying Bayesian analysis, making the determination of a test performance feasible.

Performance of three culture media commonly used for detecting Listeria monocytogenes.

Listeria monocytogenes poses a serious threat to public health, and the majority of cases of human listeriosis are associated with contaminated food. Reliable microbiological testing is needed for effective control of this pathogen by the food industry and competent authorities. The aim of this study was to determine the performance of three culture media commonly used for detecting L. monocytogenes in foods. Minced pork meat samples (n = 100) were subjected to microbiological testing for L. monocytogenes according to International Organization for Standardization methods 11290-1:1996 and 11290-2:1998 using PALCAM, ALOA, and RAPID'L. mono culture media in parallel. Presence of the pathogen was confirmed by conducting biochemical and molecular tests on the presumptive L. monocytogenes colonies. Performance attributes of sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, diagnostic odds ratios, error odds ratios, receiving operating characteristic (ROC) curve, and area under this curve were calculated from the presence-absence microbiological test results by combining the results obtained from the culture media and confirmative tests. PALCAM had the best performance in terms of positive predictive value (i.e., a positive result indicates high probability of L. monocytogenes presence) but not in terms of sensitivity (i.e., the ability of the medium to detect the pathogen when present). RAPID'L. mono was the most sensitive medium. None of the culture media were perfect for detecting L. monocytogenes in minced pork meat alone. The pathogen was detected in 16, 19, and 26% (apparent prevalence) of the samples by PALCAM, ALOA, and RAPID'L. mono, respectively, although the true prevalence of the pathogen was 22%. These findings indicate that the use of a single culture medium may lead to erroneous determination of the prevalence of L. monocytogenes.

Comparison of decontamination efficacy of antimicrobial treatments for beef trimmings against Escherichia coli O157:H7 and 6 non-O157 Shiga toxin-producing E. coli serogroups.

UNLABELLED: The decontamination efficacy of 6 chemical treatments for beef trimmings were evaluated against Escherichia coli O157:H7 and 6 non-O157 Shiga toxin-producing E. coli (nSTEC) serogroups. Rifampicin-resistant 4-strain mixtures of E. coli O157:H7 and nSTEC serogroups O26, O45, O103, O111, O121, and O145 were separately inoculated (3 to 4 log CFU/cm(2)) onto trimmings (10 × 5 × 1 cm; approximately 100 g) fabricated from beef chuck rolls, and were immersed for 30 s in solutions of acidified sodium chlorite (0.1%, pH 2.5), peroxyacetic acid (0.02%, pH 3.8), sodium metasilicate (4%, pH 12.5), Bromitize(®) Plus (0.0225% active bromine, pH 6.6), or AFTEC 3000 (pH 1.2), or for 5 s in SYNTRx 3300 (pH 1.0). Each antimicrobial was tested independently together with an untreated control. Results showed that all tested decontamination treatments were similarly effective against the 6 nSTEC serogroups as they were against E. coli O157:H7. Irrespective of pathogen inoculum, treatment of beef trimmings with acidified sodium chlorite, peroxyacetic acid, or sodium metasilicate effectively (P < 0.05) reduced initial pathogen counts (3.4 to 3.9 log CFU/cm(2)) by 0.7 to 1.0, 0.6 to 1.0, and 1.3 to 1.5 log CFU/cm(2), respectively. Reductions of pathogen counts (3.1 to 3.2 log CFU/cm(2)) by Bromitize Plus, AFTEC 3000, and SYNTRx 3300 were 0.1 to 0.4 log CFU/cm(2), depending on treatment. Findings of this study should be useful to regulatory authorities and the meat industry as they consider nSTEC contamination in beef trimmings. PRACTICAL APPLICATIONS: Findings of this study should be useful to: (i) meat processors as they design and conduct studies to validate the efficacy of antimicrobial treatments to control pathogen contamination on fresh beef products; and (ii) regulatory agencies as they consider approaches for better control of the studied pathogens.

Bayesian inference for quantifying Listeria monocytogenes prevalence and concentration in minced pork meat from presence/absence microbiological testing.

The purpose of this work was to estimate the prevalence and concentration of Listeria monocytogenes in minced pork meat by the application of a Bayesian modeling approach. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media. Presence of the pathogen was confirmed through biochemical and molecular tests. Independent experiments (n = 10) for validation purposes were performed. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. Sensitivity and specificity varied depending on the culture method. L. monocytogenes concentration was estimated at 14-17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = -2.17%). The use of at least two culture media in parallel enhanced the efficiency of L. monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L. monocytogenes presence and the uncertainty of the results obtained.

Estimating the diagnostic accuracy of three culture-dependent methods for the Listeria monocytogenes detection from a Bayesian perspective.

The objective of this study was to estimate the test accuracy measures (classification probabilities [CPs], predictive values [PVs], likelihood ratios [LRs] and area under receiving operating characteristic curve [AUC]) of three different culture-dependent methods, commonly used during routine analysis for the detection of the foodborne pathogen Listeria monocytogenes, from a Bayesian perspective. Data from a previous study by Andritsos et al. (2010) were used to define measures of accuracy for the diagnostic tests. Samples of minced pork meat obtained from local markets were tested for L. monocytogenes presence by parallel testing using selective media (PALCAM, ALOA and RAPID'L.mono). Dirichlet distribution, which is the multivariate expression of a Beta distribution, was used to analyze the data. Bayesian analysis determines characteristics of the posterior distribution from available prior information. Results showed that all methods were best at ruling in L. monocytogenes presence than ruling it out. PALCAM seemed to have better performance based on positive PV, positive LR and AUC, but it was not so sensitive as RAPID'L.mono was. Results also showed that none of the media were perfect in detecting L. monocytogenes, i.e. sensitivity and specificity equal to one. Besides, the problem of observing zero counts may be overcome by applying Bayesian analysis, making the determination of a test performance feasible.

The microbiological condition of minced pork prepared at retail stores in Athens, Greece.

Minced pork samples (n = 150) obtained from butchers' shops and supermarkets in Greece, during summer (n = 75) and winter (n = 75), were subjected to microbiological analysis. Microbial counts (log CFU/g) for the parameters tested were: total viable count (TVC), 6.8 ± 1.0; Pseudomonas spp., 6.4 ± 1.2; Brochothrix thermosphacta, 5.9 ± 1.1; lactic acid bacteria, 5.3 ± 1.0; yeasts and moulds, 4.6 ± 0.7; hydrogen sulfide (H(2)S)-producing bacteria, 4.3 ± 1.3; Enterobacteriaceae, 3.6 ± 1.2; total coliforms, 2.9 ± 1.1; Escherichia coli, 1.4 ± 0.7; Staphylococcus spp., 4.3 ± 1.0; S. aureus, 2.4 ± 0.9, and Listeria spp., 1.4 ± 0.6. The highest correlations were between TVC and pseudomonads, B. thermosphacta and H(2)S-producing bacteria, while the lowest were between total coliforms and all other groups of microorganisms except Enterobacteriaceae. The type of retail outlet and the seasonality of sampling did not have any significant effects (p>0.05) on minced pork meat quality. Interrelationships between (i) meat quality and shelf life, (ii) hygienic conditions during mince preparation and (iii) personnel hygiene were revealed.