Validation Using Diverse, Difficult-to-Detect Salmonella Strains and a Dark Chocolate Matrix Highlights the Critical Role of Strain Selection for Evaluation of Simplified, Rapid PCR-Based Methods Offering Next-Day Time to Results.

ABSTRACT: Modifications to pathogen detection kits to accomplish simplified protocols with reduced time to results may impact method performance, particularly when combining shortened enrichment times and simplified enrichment procedures. We used Salmonella detection in dark chocolate as a model to test the impact of different enrichment times (minimum and maximum validated times) and procedures on detection of low levels of difficult-to-detect Salmonella strains, for three PCR kits that were AOAC International Performance Tested Method certified for detection of Salmonella spp. in dark chocolate. Initial inclusivity studies with pure cultures showed that all three kits detected 70 of 70 Salmonella spp. strains at 1 log above the theoretical limit of detection, with some strains yielding later cycle threshold values or having variable detection among technical replicates, indicating reduced assay performance for these strains. Based on these data, we selected a S. enterica subsp. enterica serovar Poona strain as well as three non-subsp. enterica strains to test the ability of the three kits to detect Salmonella in dark chocolate inoculated at low levels (0.06 to 1.18 most probable number per 25 g). With primary enrichment in skim milk at 35°C, detection frequency for all assays did not significantly differ from the reference method for both the minimum and maximum validated enrichment times. However, a pilot study that used primary enrichment in buffered peptone water at 42°C yielded significantly fewer positive samples (13 of 80) than were obtained with the U.S. Food and Drug Administration Bacteriological Analytical Manual method using enrichment in skim milk at 35°C (40 of 80 positive samples); strains representing subsp. houtenae and salamae were detected in significantly fewer chocolate samples than enrichment with skim milk. Our data indicate that continued efforts to simplify rapid pathogen detection kits may reduce kit performance in a way that can only be detected with stringent evaluation protocols that are designed to identify kit failure modes.

Evaluation of invA Diversity among Salmonella Species Suggests Why Some Commercially Available Rapid Detection Kits May Fail To Detect Multiple Salmonella Subspecies and Species.

HIGHLIGHTS: Salmonella exhibits tremendous diversity, with 2,659 documented serovars. invA is a common gene target for detecting Salmonella spp. Detection methods should be validated with a sufficiently diverse strain set.

Dietary exposure to pesticide residues from foods of plant origin and drinks in Lebanon.

This study assesses the dietary exposure of Lebanese adults to 47 pesticide residues from both foods of plant origin and drinks. The study was conducted using the Total Diet Study protocol in two different areas of Lebanon: Greater Beirut (urban) and Keserwan (semi-rural). A total of 1860 individual foods were collected, prepared, and cooked prior to analysis. Composite samples of similar foods were analyzed, following the QuEChERS Multiresidue method. Eighteen residues were detected/quantified on at least one composite sample, with 66.7 % of the results being quantifiable and 33.3 % detectable. Quantifiable levels ranged between 10.3 and 208 µg/kg. For the composite samples where residues were detected, 55 % had one residue, while 45 % had 2-4 residues. The most frequently detected/quantified pesticide residues included Chlorpyrifos, Procymidone, Primiphos methyl, Dimethoate, and Dieldrin. The dietary exposure assessment was conducted using the deterministic approach with two scenarios: (1) the lower bound (LB) approach and (2) the upper bound (UB) approach. Using the LB approach, mean estimated daily exposures were far below the acceptable daily intakes (ADIs) for all investigated residues. Using the UB approach, which tends to overestimate exposure, mean estimated daily exposures were below the ADIs for all residues except for Dieldrin (semi-rural: 128.7 % ADI; urban: 100.7 % ADI). Estimates of mean exposure to Diazinon reached 50.3 % of ADI in the urban diet and 61.9 % in the semi-rural diet. Findings of this study identify specific pesticide residues as monitoring priorities for which more comprehensive and sensitive analyses are needed in order to refine exposure assessment.

Green synthesis of curcumin conjugated nanosilver for the applications in nucleic acid sensing and anti-bacterial activity.

Silver nanoparticles (Ag NPs) are often synthesized by chemical and physical methods. Natural and non-toxic molecules are recently being replaced for nanoparticles preparation. In this paper we have used curcumin, which interacts with Ag+ and subsequently synthesizes silver nanoparticles. Further continuation of the reaction often makes aggregation and forms dark brown/black silver oxide. Presence of glycerol in the reaction mixture gives mono-disperse curcumin conjugated Ag NPs, which can be made stable by capping with polyvinylpyrolidone (PVP). XRD data confirm that curcumin conjugated Ag NPs are crystalline in nature with a mean crystalline size of 13.27 nm. The Ag NPs are spherical and in the range of 10-50 nm though their hydrodynamic radius is found to be higher, ∼294 nm, due to polyvinylpyrolidone capping and aggregation of nanoparticles in solution. The production of curcumin conjugated Ag NPs follows first order kinetics and the effect of curcumin concentration during formation of Ag NPs indicates a linear enhancement in the production of Ag NPs with an increase in concentration of curcumin. These curcumin conjugated silver nanoparticles show anti-bacterial activity and can successfully determine nucleic acid (DNA and RNA) in the concentration range 100-1000 ng/mL with a linear regression coefficient >0.997 using Resonance Rayleigh Scattering spectra.

Standardisation of a new model of H9N2/Escherichia coli challenge in broilers in the Lebanon.

Primary infection by low pathogenic avian influenza (LPAI) predisposes for secondary infection by Escherichia coli in poultry, leading to significant economic losses. Future research in control of this ailment requires the establishment of a successful controlled challenge by avian influenza virus (AIV)/E. coli. Six groups of broilers (6 birds/group) were included for the standardisation of the controlled challenge by AIV/E. coli. Birds in groups 1, 2, 3, 4 and 5 received an intra-tracheal challenge of 0.5 ml of two haemagglutinating units of H9N2 virus at 20 days of age. At the age of 23 days, birds in group 1 received an intra-thoracic (right air sac)-E. coli challenge equivalent to 1.6 x 10 colony-forming units (cfu)/0.5 ml/bird, while birds in groups 2, 3, 4 and 5 received E. coli by the same route and in the following respective decreasing order of viable cells: 1.6 x 10(6), 1.6 x 10(5), 1.6 x 10(4) and 1.6 x 10(3); cfu. Birds in control group 6 were deprived of H9N2 and E. coli challenge. Results showed significant early mortality in group 1 that was challenged with the highest number of E. coli, in comparison to groups 2-6 (p<0.05); however, the average weight at 28 days of age was similar in surviving birds of groups 2-6 (p>0.05). The frequencies of four signs at 2 days and at 5 days post E. coli challenge (conjunctivitis, diarrhoea, ocular exudates and rales) in the surviving birds of groups 2-5 were most often higher than those observed in control group 6 (p<0.05). These four signs and five gross lesions (abdominal airsacculitis, left thoracic airsacculitis, pericarditis, right thoracic airsacculitis and tracheitis) had a decreasing pattern of frequency related to a decrease in the E. coli count used in the challenge.

Preliminary study on the efficacy and safety of eight individual and blended disinfectants against poultry and dairy indicator organisms.

Eight individual and blended chemical disinfectants were screened for preliminary evaluation of safety, bactericidal and virucidal effectiveness against poultry and dairy organisms. The test organisms were Escherichia coli, Salmonella Enteritidis, Staphylococcus aureus, Streptococcus spp. and Clostridium perfringens, in addition to avian influenza virus (AIV) and Newcastle disease virus (NDV). Viable counts of surviving bacteria were determined after 30 min contact with each disinfectant and in the presence or absence of skimmed milk, to simulate the interference of organic matter. The haemagglutination test was used to assess the survival of the test viruses in the presence of the different disinfectants after propagation in 10-day-old chick embryos. In the presence of skimmed milk, a higher concentration of most of the disinfectants examined was required to exert antimicrobial effectiveness. When used individually, quaternary ammonium showed no virucidal activity against NDV and AIV; peracetic acid was not effective against Streptococcus spp., S. Enteritidis and NDV, while iodophors showed low bactericidal and inconsistent virucidal activity. The single and blended disinfectants with high microbicidal activities included phenols (high bactericidal and virucidal activity), blends of quaternary ammonium compounds (high bactericidal activity) and blends of cresols and organic acids (high virucidal activity). This suggests the use of blends of compatible compounds for disinfection operations in poultry and dairy industries since they will target a wider range of micro-organisms. None of the disinfectants had a negative effect on the development of the different organs of chicken embryos and the iodine-based disinfectant, developed for dairy-teat dipping, also showed no adverse reactions in experimental cows.

Identification of antiadhesive fraction(s) in nonimmunized egg yolk powder: in vitro study.

The presence of antiadhesive component(s) in the hen egg yolk against foodborne pathogens was anticipated from results of a previous animal study conducted by the authors. The previous work showed egg yolk powder without specific antibodies is effective in controlling Salmonella enteritidis,Salmonella typhimurium, and Escherichia coli O157:H7 colonization in laying hens. Therefore, this study was necessary to locate the activity and identify the effective component(s). In vitro experiments were conducted using confluent Caco-2 cell monolayers. S. enteritidis, S. typhimurium, and E. coli O157:H7 were investigated against the various extracted granule and plasma fractions in three different assays: adhesion elimination, adhesion prevention, and antimicrobial. This study revealed original findings and identified the protective yolk fraction against the foodborne pathogens as the granule component, high-density lipoproteins (HDL). The protective activity conveyed by HDL was confirmed to remain intact despite peptic and tryptic enzymatic digestion and to have antiadhesive but not antimicrobial effect.

Eggshell matrix proteins as defense mechanism of avian eggs.

This study focused on the role of eggshell matrix proteins as a function of potential natural antimicrobial defenses of avian eggs. The electrophoretic profile of SDS-PAGE showed that the soluble eggshell matrix proteins had three major bands of 15 000, 36 000, and 66 000 and several minor bands comprising 17 000, 25 000, 30 000, and 75 000, while insoluble matrix proteins were consisting of various bands comprising at least 16 distinct migration bands between 10 000 and 200 000. Three bacteria species, Pseudomonas aureginosa, Bacillus cereus, and Staphylococcus aureus, were found to be inhibited in the presence of soluble eggshell matrix proteins (100 microg/mL). On the other hand, Escherichia coli and Salmonella enteritidis were weakly inhibited at only an early stage of incubation time (up to 4 h). Scanning electron microscopy revealed that eggshell matrix proteins might interact and disrupt the membrane integrity of bacteria. The present study clearly indicated that avian eggshell matrix proteins possess a potential of novel antimicrobial defensin mechanism.