Universal Preenrichment Research Articles

A flow cytometric assay to detect viability and persistence of Salmonella enterica subsp. enterica serotypes in nuclease-free water at 4 and 25°C.

Salmonella spp. is one of the most isolated microorganisms reported to be responsible for human foodborne diseases and death. Water constitutes a major reservoir where the Salmonella spp. can persist and go undetected when present in low numbers. In this study, we assessed the viability of 12 serotypes of Salmonella enterica subsp. enterica for 160 days in nuclease-free water at 4 and 25°C using flow cytometry and Tryptic Soy Agar (TSA) plate counts. The results show that all 12 serotypes remain viable after 160 days in distilled water using flow cytometry, whereas traditional plate counts failed to detect ten serotypes incubated at 25°C. Moreover, the findings demonstrate that 4°C constitutes a more favorable environment where Salmonella can remain viable for prolonged periods without nutrients. Under such conditions, however, Salmonella exhibits a higher susceptibility to all tested antibiotics and benzalkonium chloride (BZK). The pre-enrichment with Universal Pre-enrichment Broth (UP) and 1/10 × Tryptic Soy broth (1/10 × TSB) resuscitated all tested serotypes on TSA plates, nevertheless cell size decreased after 160 days. Furthermore, phenotype microarray (PM) analysis of S. Inverness and S. Enteritidis combined with principal component analysis (PCA) revealed an inter-individual variability in serotypes with their phenotype characteristics, and the impact of long-term storage at 4 and 25°C for 160 days in nuclease-free water. This study provides an insight to Salmonella spp. long-term survivability at different temperatures and highlights the need for powerful tools to detect this microorganism to reduce the risk of disease transmission of foodborne pathogens via nuclease-free water.

Evaluation of universal preenrichment broth and comparison of rapid molecular methods for the detection of Salmonella from spent sprout irrigation water (SSIW)

Sprouts and spent sprout irrigation water (SSIW) present unique challenges for the development of a Salmonella detection method in food matrices. This study aimed to compare universal preenrichment broth (UPB) and lactose broth (LB) as preenrichment media for cultural and rapid screening methods and to compare their abilities to recover Salmonella in SSIW samples from different sprout varieties (i.e., alfalfa, broccoli, and mung bean sprouts). The associated co-enriched microbiota with different sprout varieties using different preenrichment media were also examined using a quasi-metagenomic approach. The performance of media and detection methods was compared using the relative level of detection (RLOD) value, as recommended by ISO 16140-2:2016. The level of detection (LOD) for Salmonella culture method with UPB was similar to that with LB in low aerobic plate count (APC) background samples (the relative LOD, i.e., RLOD, was nearly 1 after adjusting for the effects of SSIW variety and serovar), but significantly lower than that with LB in high APC background samples (RLOD = 0.32). The LOD for Salmonella with selected rapid methods was comparable to each other (RLOD from 0.97 to 1.50) and to the culture method (RLOD from 0.69 to 1.03), and no significant difference was detected between preenrichment broths in low APC background samples with RLOD values between 0.76 and 1.04. In samples with a high APC background, however, a drastic difference in LOD was observed between methods and between preenrichment broths for each method. The RLOD ranged from 0.03 to 0.32 when UPB was compared to LB as preenrichment broth. The composition and relative abundance (RA) of co-enriched microbiota was affected by multiple factors including food matrices, preenrichment media and Salmonella contamination. Altogether, this study validated UPB as a better preenrichment broth than LB for the detection of Salmonella enterica from SSIW. This study also suggested UPB may also be an optimal preenrichment medium for rapid screening methods when APC level is high. The observation of potential exclusion of Salmonella in preenrichment through the overgrowth of competitive microflora from the quasi-metagenomic study provided novel information that may be used to further optimize preenrichment formulations.

Effectiveness of selected pre-enrichment broths for the detection of Salmonella spp. in meat analogs

With the increasing variety of meat analog products, it is important to verify that the methods used for Salmonella detection are effective. The objective of this study was to compare the efficacy of three pre-enrichment broths for the detection of Salmonella in meat analog burgers: the currently used Bacteriological Analytical Manual (BAM) broth (lactose broth with Triton X-100, LB-T), universal pre-enrichment (UP) broth, and buffered peptone water (BPW). Five different meat analog products (coded A-E) were inoculated with Salmonella serotypes (S. Enteritidis or S. Agona) following the Food and Drug Administration Method Modification and Method Extension Criteria for Existing Validated Microbiology Methods. Seven replicates were tested per product with each broth, for a total of 105 inoculated samples and 105 uninoculated samples. Salmonella was isolated as described in the BAM, and two isolated colonies from each sample were tested with real-time PCR. The total number of positive real-time PCR detections associated with each pre-enrichment broth was compared across all products (70 PCR tests per broth) and within each brand (14 PCR tests per broth). Overall, LB-T showed the greatest detection rate of 65/70 (92.9%), while BPW and UP showed rates of 63/70 (90.0%) and 62/70 (88.6%), respectively. However, these differences were not statistically significant (p-value > 0.05, Test for Equality of Proportions). When the broths were compared separately for each product, LB-T showed a significantly greater detection rate of 13/14 (92.9%) in Brand C samples compared to UP at 7/14 (50.0%). These findings demonstrate that LB-T is an effective pre-enrichment for detecting Salmonella in various market-available meat analogs.

Mixed Salmonella cultures reveal competitive advantages between strains during pre‐enrichment and selective enrichment

AbstractCulture‐based Salmonella isolation consists of nonselective pre‐enrichment, followed by selective enrichment in Rappaport–Vassiliadis (RV) or tetrathionate (TT) broths, and subsequent plating on selective indicator agar. This study aimed to assess the recovery of two strains belonging to serovars Montevideo (strain ATCC‐8387) and Typhimurium (strain ATCC‐14028) when grown together in different media. The two strains were co‐inoculated 1:1, 10:1, 100:1, and the reciprocal, and CRISPR‐SeroSeq was used to assess the relative frequency of both strains after each culture step. Individually, there was no growth difference between both strains in universal pre‐enrichment (UP) or RV broths, though both strains had a higher growth in the former. In TT, both strains had reduced growth, especially ser. Montevideo‐8387. When the strains were combined, the growth of ser. Typhimurium‐14028 strain was higher in UP and TT broths, while the ser. Montevideo strain was higher in RV broth. The ser. Typhimurium strain also grew better on xylose lysine tergitol‐4 (XLT‐4) agar. Using media that are commonly used for Salmonella isolation, this work reveals strain‐to‐strain differences in growth and that in three conditions (UP, RV, XLT‐4), these differences were only manifested when the strains were in competition with each other. These findings illustrate the importance of dual selective enrichments to be able to capture all Salmonella present in a sample.

Screening of crows and waterfowls for Salmonella and Listeria monocytogenes infection

Background and Aim: Wild birds can carry a number of potential human and animal pathogens. These birds can intrude into human habitats giving the opportunity to transmission of such infection. Therefore, the current study was designed to investigate the role of crows and waterfowls as vectors of Salmonella and Listeria monocytogenes and to compare cultivation methods with direct polymerase chain reaction (PCR) for the detection of infection. Materials and Methods: A total of 200 fecal dropping samples were collected. Salmonella was cultivated by three step method (Universal Pre-enrichment Broth [UPB], Rappaport-Vassiliadis Broth, and Xylose Lysine Desoxycholate agar). The recovered isolates were characterized by biotyping, serotyping, and PCR detection of enterotoxin (stn) gene. The antibiogram pattern of isolates against a panel of 8 antibiotics was recorded. L. monocytogenes was cultivated on UPB, then on Listeria Oxford Agar and Listeria CHROMagar. The recovered isolates were characterized by biotyping and PCR detection of listeriolysin (hylA) gene. Results: The percentages of Salmonella infection in crows and waterfowls were 10 and 20, respectively. The most frequently recovered serovars were Typhimurium, Potengi, Enteritidis, and Kentucky. Antimicrobial susceptibility analysis of Salmonella isolates showed that the resistance rates for gentamicin were the highest (92%), followed by amoxicillin (88%) and cefixime (60%). Resistance to 8 antibiotics was recorded in 60% (15/25) of Salmonella isolates. The percentages of L. monocytogenes infection in crows and waterfowls were 1.3 and 2, respectively. Direct PCR applied to UPB revealed that 12% and 3% of samples were positive for Salmonella and L. monocytogenes, respectively, and there was no significant difference between direct PCR and cultivation method for the detection of infection. Conclusion: The present findings indicate that wild birds can harbor zoonotic enteric pathogens and this necessitates monitoring the epidemiologic status of these pathogens among wild birds and humans and applying the appropriate intervention measures to prevent the spread of infection.

Prevalence of Shiga-toxigenic and atypical enteropathogenic Escherichia coli in untreated surface water and reclaimed water in the Mid-Atlantic U.S

The microbial quality of irrigation water has increasingly become a concern as a source of contamination for fruits and vegetables. Non-traditional sources of water are being used by more and more growers in smaller, highly diversified farms in the Mid-Atlantic region of the U.S. Shiga-toxigenic E. coli (STEC) have been responsible for several outbreaks of infections associated with the consumption of leafy greens. Our study evaluated the prevalence of the “big seven” STEC serogroups and the associated enterohemorrhagic E. coli (EHEC) virulence factors (VF) genes in conventional and nontraditional irrigation waters in the Mid-Atlantic region of the U.S. Water samples (n = 510) from 170 sampling events were collected from eight untreated surface water sites, two wastewater reclamation facilities, and one vegetable processing plant, over a 12-month period. Ten liters of water were filtered through Modified Moore swabs (MMS); swabs were then enriched into Universal Pre-enrichment Broth (UPB), followed by enrichment into non-O157 STEC R&F broth and isolation on R & F non-O157 STEC chromogenic plating medium. Isolates (n = 2489) from enriched MMS from water samples were screened for frequently reported STEC serogroups that cause foodborne illness: O26, O45, O103, O111, O121, O145, and O157, along with VF genes stx1, stx2, eae, and ehxA. Through this screening process, STEC isolates were found in 2.35% (12/510) of water samples, while 9.0% (46/510) contained an atypical enteropathogenic E. coli (aEPEC) isolate. The eae gene (n = 88 isolates) was the most frequently detected EHEC VF of the isolates screened. The majority of STEC isolates (stx1 or stx2) genes mainly came from either a pond or reclamation pond water site on two specific dates, potentially indicating that these isolates were not spatially or temporally distributed among the sampling sites. STEC isolates at reclaimed water sites may have been introduced after wastewater treatment. None of the isolates containing eae were determined to be Escherichia albertii. Our work showed that STEC prevalence in Mid-Atlantic untreated surface waters over a 12-month period was lower than the prevalence of atypical EPEC.

Effects of Different Broth Enrichment upon Phage Magnetoelastic Biosensor for Fast Detecting Low <i>Salmonella</i> Counts on Problematic Produce

According to the FDA Bacteriological Analytical Manual (BAM) for Salmonella identification in produce, two pre-enrichment steps with 48 hours of incubation are the golden procedures. Lactose broth is recommended for the first pre-enrichment step medium for leafy greens, and the universal pre-enrichment (UP) broth is for tomatoes. However, the suggested broths were evaluated to have the maximum performance using the culture-dependent methods, and may not be applied to other methods, such as biosensor detection platform. A wireless bacteriophage magnetoelastic (ME) biosensor has been recently developed for real-time or rapid detection of food-borne pathogens in various foods. This affinity-based biosensor utilizes a phage oligonucleotide as the probe to capture target bacteria. In this study, the efficiencies of different pre-enrichment media for early detection of low Salmonella on spinach leaves and tomatoes use ME biosensors to shorten detection time. Four broths of modified peptone water, Lennox broth (LB), lactose broth, and UP broth were selected in this study. Various pre-enrichment times for ME biosensor detection were investigated. After spiking 4 cfu/g Salmonella on the tomatoes surfaces, the phage biosensor was able to detect Salmonella within 5 hours of pre-enrichment comparing to 24 hours in the FDA procedures. For Salmonella spiked spinach leaves, the same medium showed Salmonella positive within 7 hours. This study demonstrated that LB broth is the best medium to shorten pre-enrichment time to pass Salmonella number detection thresholds for ME biosensor detection in spinach and tomatoes when comparing to FDA procedures.

Incidence of fecal indicator and pathogenic bacteria in reclaimed and return flow waters in Arizona, USA

The quality of irrigation water used to cultivate produce that is consumed raw is an important issue with regard to food safety. In this study, the microbiological quality of potential irrigation water sources in Arizona was evaluated by testing for the presence of indicator and pathogenic bacteria. Reclaimed water samples were collected from two wastewater treatment plants and return flow samples were collected from two drainage canals and one return flow pond. Standard membrane filtration methods were used for detection of indicator bacteria. Water samples (n = 28) were filtered through cellulose ester membrane filters and bacterial populations were enumerated by placing the filters on selective agar. For detection of pathogens (Salmonella enterica, Listeria monocytogenes and Shiga toxin-producing E. coli (STEC)), water samples were filtered through Modified Moore swabs and enriched in Universal Pre-enrichment Broth, followed by selective enrichment broth for each pathogen. The enriched broth was streaked onto agar media selective for each pathogen. Presumptive colonies were confirmed by PCR/real-time PCR. Among the 14 reclaimed water samples from two sites, the ranges of recovered populations of E. coli, total coliforms, and enterococci were 0–1.3, 0.5–8.3 × 103, and 0–5.5 CFU/100 mL, respectively. No L. monocytogenes, Salmonella or STEC were found. In the 13 return flow water samples from 3 sites, the ranges of recovered populations of E. coli, total coliforms and enterococci were 1.9–5.3 × 102, 6.5 × 102−9.1 × 104, and 2.9–3.7× 103 CFU/100 mL, respectively. All samples were negative for L. monocytogenes. One (7.1%) of the return flow samples was positive for E. coli O145. Nine (64.3%) of the samples were positive for Salmonella. Both real-time PCR and culture-based methods were used for the detection of Salmonella and L. monocytogenes, and the results from the two methods were comparable. The findings of this study provide evidence that irrigation waters in Arizona, including reclaimed water and return flows, could be potential sources of bacterial contamination of produce. Additional work is needed to evaluate whether bacteria present in irrigation water sources transfer to the edible portion of irrigated plants and are capable of persisting through post-harvest activities.

The effect of environmental poultry samples on the pH of typical Salmonella pre-enrichment and enrichment media following incubation

The first step to detect Salmonella in feed and other dry contaminated samples is a pre-enrichment broth that can assist in the recovery of small numbers of stressed Salmonella cells. A previous study demonstrated that incubation of feed and feed ingredients in commonly used pre-enrichment media resulted in a low pH that injured or killed the Salmonella. The objective of this study was to determine which environmental samples and pre-enrichment and selective broths could interfere with the accuracy of Salmonella detection by allowing pH to drop. Samples were collected from commercial poultry operations. Triplicate 10-g subunits were dispensed into sterile 18-oz Whirl Pak bags and 90 mL of each of the media [lactose broth (LB), buffered peptone water (BPW), Universal Pre-enrichment (UP), minimal salts (M-9), tetrathionate broth (TT) and Rappaport-Vassiliadis broth (RV)] were added to the bags and incubated 24 h at 37°C (or 42°C for RV and TT). The pH was measured after incubation. With turkey litter, fluff, and eggshells, regardless of media, the pH never went below 5.7. Regardless of sample type, the lowest pH was 6.1, 6.2, and 6.4 for UP, M-9, and BPW, respectively. For the pre-enrichment medium commonly used in the US, (LB) the pH dropped to 4.7 to 4.9 with broiler litter and 4.2 for boot covers used in turkey or broiler houses. Many researchers testing these sample types are unaware of this potential for pH change during pre-enrichment and may be underestimating the presence of Salmonella.

Simultaneous and individual quantitative estimation of Salmonella, Shigella and Listeria monocytogenes on inoculated Roma tomatoes (Lycopersicon esculentum var. Pyriforme) and Serrano peppers (Capsicum annuum) using an MPN technique

Simultaneous and individual enumeration of Salmonella, Shigella and Listeria monocytogenes was compared on inoculated Roma tomatoes and Serrano peppers using an Most Probable Number (MPN) technique. Samples consisting of tomatoes (4 units) or peppers (8 units) were individually inoculated with a cocktail of three strains of Salmonella, Shigella or L. monocytogenes, or by simultaneous inoculation of three strains of each pathogen, at low (1.2–1.7 log CFU/sample) and high (2.2–2.7 log CFU/sample) inocula. Samples were analyzed by an MPN technique using universal pre-enrichment (UP) broth at 35 °C for 24 ± 2 h. The UP tubes from each MPN series were transferred to enrichment and plating media following adequate conventional methods for isolating each pathogen. Data were analyzed using multifactorial analysis of variance (p < 0.05) and LSD multiple rang test. There were differences (p < 0.05) in recovery of simultaneous and individual bacteria inoculated (individual > simultaneous), type of bacteria (Salmonella > Shigella and L. monocytogenes), type of sample (UP broth > pepper and tomato), and inoculum level (high > low). The MPN technique was effective for Salmonella on both commodities. Shigella counts were higher on tomatoes compared to peppers, (p < 0.05), and for L. monocytogenes on peppers (p < 0.05).

Evaluation of TA10 Broth for Recovery of Listeria monocytogenes from Ground Beef.

In 2009, the enrichment broth TA10 was released for simultaneous recovery of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. This medium was compared with other Salmonella enrichment broths [lactose (LAC) broth, buffered peptone water (BPW), and universal pre-enrichment (UP) broth] for the recovery of heat- and freeze-injured Salmonella spp. in beef by the conventional culture method. There was a significant difference between TA10 and LAC enrichment broths for detecting injured Salmonella spp. In this study, the International Organization for Standardization Listeria pre-enrichment broth (Half-Fraser/Fraser) was compared with TA10 broth for the recovery of L. monocytogenes from ground beef. Ground beef samples were contaminated with single Listeria serovars at levels of 0.096 to 0.001 most probable number/g. Twenty 25 g test portions of the contaminated ground beef were pre-enriched in each broth, and the ISO-11290-1 Listeria official isolation protocol was used thereafter. There was a significant difference between TA10 broth (48 h) and Half-Fraser/Fraser broth (72 h) in the recovery of L. monocytogenes. In addition, the incubation time for TA10 broth was shorter than for Half-Fraser/Fraser broth. The results indicate that TA10 broth should be used instead of Half-Fraser/Fraser broth for analysis of beef that may be contaminated with very low levels of L. monocytogenes.

Early Recovery of Salmonella from Food Using a 6-Hour Non-selective Pre-enrichment and Reformulation of Tetrathionate Broth.

Culture based methods are commonly employed to detect pathogens in food and environmental samples. These methods are time consuming and complex, requiring multiple non-selective and selective enrichment broths, and usually take at least 1 week to recover and identify pathogens. Improving pathogen detection in foods is a primary goal for regulatory agencies and industry. Salmonella detection in food relies on a series of culture steps in broth formulations optimized to resuscitate Salmonella and reduce the abundance of competitive bacteria. Examples of non-selective pre-enrichment broths used to isolate Salmonella from food include Lactose, Universal Pre-enrichment, BPW, and Trypticase Soy broths. Tetrathionate (TT) and Rappaport–Vassiliadis (RV) broths are employed after a 24-h non-selective enrichment to select for Salmonella and hamper the growth of competitive bacteria. In this study, we tested a new formulation of TT broth that lacks brilliant green dye and has lower levels of TT . We employed this TT broth formulation in conjunction with a 6-h non-selective pre-enrichment period and determined that Salmonella recovery was possible one day earlier than standard food culture methods. We tested the shortened culture method in different non-selective enrichment broths, enumerated Salmonella in the non-selective enrichments, and used 16S rRNA gene sequencing to determine the proportional abundances of Salmonella in the TT and RV selective enrichments. Together these data revealed that a 6-h non-selective pre-enrichment reduces the levels of competitive bacteria inoculated into the selective TT and RV broths, enabling the recovery of Salmonella 1 day earlier than standard culture enrichment methods.

Relative effectiveness of selected preenrichment media for the detection of Salmonella from leafy green produce and herbs

Four buffered preenrichment media (BAX® System MP Media (BAX)), Universal Preenrichment Broth (UPB), modified Buffered Peptone Water (mBPW), and Buffered Peptone Water (BPW)) were compared with lactose broth (LB) in the Bacteriological Analytical Manual’s (BAM) Salmonella culture method for the analysis of 9 leafy green produce and herb types. Artificially contaminated test portions were pre-enriched in each medium and the results were analyzed statistically using Fisher’s Exact 2-tailed F test (p < 0.05) with pairwise comparisons. There was no difference in recovery of Salmonella from curly parsley and basil among the five media (p > 0.05). UPB was consistently among the most effective media for recovery of Salmonella from the nine produce types; however, S. Typhimurium and S. Newport were isolated from cabbage more frequently with mBPW than with UPB (p < 0.05). Comparisons of the results among the preenrichment media from all experimental trials, with leafy green produce and herbs, demonstrate that Salmonella is more effectively detected and isolated using buffered enrichments than with the currently recommended LB (p < 0.05). There were no significant differences among the buffered preenrichments for the detection of Salmonella-positive test portions of the produce tested (BAX (160 Salmonella-positive test portions/480 test portions), UPB (176/480), mBPW (184/480), BPW (169/480), LB (128/480))(p > 0.05).

Validation of a high-throughput immunobead array technique for multiplex detection of three foodborne pathogens in chicken products

This study rigorously evaluated a previously developed immunobead array method to simultaneously detect three important foodborne pathogens, Campylobacter jejuni, Listeria monocytogenes, and Salmonella spp., for its actual application in routine food testing. Due to the limitation of the detection limit of the developed method, an enrichment step was included in this study by using Campylobacter Enrichment Broth for C. jejuni and Universal Pre-enrichment Broth for L. monocytogenes and Salmonella spp.. The findings showed that the immunobead array method was capable of detecting as low as 1CFU of the pathogens spiked in the culture media after being cultured for 24h for all three pathogens. The immunobead array method was further evaluated for its pathogen detection capabilities in ready-to-eat (RTE) and ready-to-cook (RTC) chicken samples and proven to be able to detect as low as 1CFU of the pathogens spiked in the food samples after being cultured for 24h in the case of Salmonella spp., and L. monocytogenes and 48h in the case of C. jejuni. The method was subsequently validated with three types of chicken products (RTE, n=30; RTC, n=20; raw chicken, n=20) and was found to give the same results as the conventional plating method. Our findings demonstrated that the previously developed immunobead array method could be used for actual food testing with minimal enrichment period of only 52h, whereas the conventional ISO protocols for the same pathogens take 90–144h. The immunobead array was therefore an inexpensive, rapid and simple method for the food testing.

A duplex qPCR for the simultaneous detection of Escherichia coli O157:H7 and Listeria monocytogenes using LNA probes.

In this study, a duplex qPCR assay was developed for the needs of the Irish fish industry to screen for the two major food-borne pathogens of fish, Listeria monocytogenes and Escherichia coli O157:H7. The assay can claim positive or negative results for two pathogens in one go in only 20 h including 16 h universal pre-enrichment and compared to traditional ISO approved plate culture methods the labour and the cost involved in testing of one sample is reduced to minimum. The highly specific genomic areas targeted for PCR amplification in the assay are the hly gene for listeriolysin O (LLO) of L. monocytogenes and the stx gene for Shiga-like toxin expressed by E. coli O157:H7. The detection limit of the assay is consistent with the consumer protection limits of 1 pg genomic DNA or 1 CFU 25 g(-1) fish meat (with enrichment) allowing the test to be considered as a substitute to standard plate culture methods. The study highlights a novel duplex qPCR for Listeria monocytogenes and Escherichia coli O157:H7 that could be used as an alternative to plate-based ISO or singleplex PCR methods while minimizing the costs. The assay uses rapid DNA extraction methods and locked nucleic acid probes. Sensitivity and specificity are 100 and 98·95% respectively. The potential for quantitative rage of the assay is 10(8) -10(1) CFU ml(-1) .

Evaluation and comparison of rapid methods for the detection of Salmonella in naturally contaminated pine nuts using different pre enrichment media

Foodborne outbreaks, involving pine nuts and peanut butter, illustrate the need to rapidly detect Salmonella in low moisture foods. However, the current Bacteriological Analytical Manual (BAM) culture method for Salmonella, using lactose broth (LB) as a pre enrichment medium, has not reliably supported real-time quantitative PCR (qPCR) assays for certain foods. We evaluated two qPCR assays in LB and four other pre enrichment media: buffered peptone water (BPW), modified BPW (mBPW), Universal Pre enrichment broth (UPB), and BAX® MP media to detect Salmonella in naturally-contaminated pine nuts (2011 outbreak). A four-way comparison among culture method, Pathatrix® Auto, VIDAS® Easy SLM, and qPCR was conducted. Automated DNA extraction techniques were compared with manual extraction methods (boiling or InstaGene™). There were no significant differences (P > 0.05) among the five pre enrichment media for pine nuts using the culture method. While both qPCR assays produced significantly (P ≤ 0.05) higher false negatives in 24 h pre enriched LB than in the other four media, they were as sensitive as the culture method in BPW, mBPW, UPB, and BAX media. The VIDAS Easy and qPCR were equivalent; Pathatrix was the least effective method. The Automatic PrepSEQ™ DNA extraction, using 1000 μL of pre enrichment, was as effective as manual extraction methods.

Casamino Acids and Oxyrase enhance growth of Listeria monocytogenes in multi-pathogen enrichments

Rapid methods have been developed as relatively faster alternatives to plate culture for the detection of pathogenic bacteria in foods. However, since most rapid methods are subject to logistical limitations (e.g., sample volume size, analysis time, matrix effects) and/or a detection scheme with insufficient sensitivity needed to detect very low levels of bacteria in foods, culture enrichment is often employed to increase the concentration of targeted pathogens prior to detection. Multiplexed rapid detection platforms, capable of simultaneous detection of different bacteria in a single sample, necessitate co-enrichment (or mixed culture enrichment) of as many different targeted microorganisms as possible in a timely manner. This investigation compares the growth of four major foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, and Yersinia enterocolitica) inoculated into pristine media or ground pork and enriched in various culture media. Initial results revealed that, after 24 h incubation, the growth of L. monocytogenes (the slowest-growing pathogen examined) was increased by approximately 1-log by the supplementation of Universal Preenrichment Broth with Casamino Acids and/or Oxyrase. Overnight (24 h) growth of L. monocytogenes in ground pork enrichment cultures was enhanced up to ca. 2-log by the addition of either Casamino Acids or Casamino Acids and Oxyrase for each of the tested growth media. Ultimately, an overnight culture of the inoculated pathogens in any of the selected media containing both Casamino Acids and Oxyrase was observed to yield target bacterial concentrations that were at sufficient levels (between 10e5 and 10e6 CFU/mL) for detection by most rapid methods.

Comparison of Enrichment Broths for the Recovery of Healthy and Heat-Injured Salmonella Typhimurium on Raw Duck Wings

This study was performed to optimiz eSalmonella Typhimurium recovery from raw duck wings with five nonselective broths (buffered peptone water, tryptic soy broth, lactose broth, universal preenrichment broth, nutrient broth) and four selective broths (selenite broth, BAX System MP media [MP], Salmonella AD media [AD], ONE broth-Salmonella [OB]). Healthy or heat-injured (50 and 85% injury) cells were inoculated at a level of 10(2), 10(1), or 10(0) CFU/25 g on raw duck wings. Growth was modeled using DMfit with four growth parameters: lag-phase duration, maximum growth rate, doubling time, and maximum population density. Most enrichments were able to recover Salmonella Typhimurium to greater than 6 log CFU/ml. AD, MP, and OB had significantly (P < 0.05) higher maximum growth rate (0.9 to 1.0/h) and lower doubling time (0.7 to 0.8 h). Buffered peptone water, AD, MP, and OB recovered healthy and 50%-injured cells at low inoculum levels to more than 6.0 log CFU/ml; OB achieved the greatest recovery (7.6 and 7.9 log CFU/ml), following 24 h of incubation. The 85%-injured cells at 10(0) and 10(1) CFU/25 g, however, were only recovered in OB, reaching 7.3 and 7.5 log CFU/ml, respectively. These results suggest that OB may be an appropriate enrichment broth for the recovery of Salmonella Typhimurium from raw duck wings in standard diagnostic tests or other rapid detection methods, to avoid false-negative results.

Comparison of different preenrichment broths, egg:preenrichment broth ratios, and surface disinfection for the detection of Salmonella enterica ssp. enterica serovar Enteritidis in shell eggs

Salmonella enterica ssp. enterica serovar Enteritidis is the leading reported cause of Salmonella infections. Most Salmonella Enteritidis infections are associated with whole shell eggs and egg products. This project attempted to lay the foundation for improving the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella Enteritidis in shell eggs. Two Salmonella Enteritidis isolates were used for comparisons among different preenrichment and enrichment media and for the evaluation of egg:preenrichment broth ratios for the detection of Salmonella Enteritidis in shell eggs. The effect of surface disinfection on the detection of Salmonella Enteritidis in shell eggs was also investigated. The results indicated that tryptic soy broth (TSB) was similar to TSB plus ferrous sulfate, but significantly (α = 0.05) better than nutrient broth, Universal Preenrichment broth, and buffered peptone water when used for preenrichment of Salmonella in shell eggs. Salmonella Enteritidis populations after enrichment with Rappaport-Vassiliadis broth were 0.40 to 1.11 log cfu/mL of culture lower than those in preenrichment cultures. The reduction was statistically significant (α = 0.05). Egg:broth ratios at 1:9 and 1:2 produced significantly (α = 0.05) higher Salmonella Enteritidis populations after preenrichment with TSB with inoculum levels at 4 cfu/100 g of eggs and 40 cfu/1,000 g of eggs than the ratio at 1:1. Salmonella Enteritidis populations in TSB preenrichment cultures of shell eggs surface-disinfected with 70% alcohol:iodine/potassium iodide solution and untreated control were 9.11 ± 0.11 and 9.18 ± 0.05 log cfu/mL, respectively, for SE 13–2, and 9.20 ± 0.04 and 9.16 ± 0.05 log cfu/mL, respectively, for SE CDC_2010K_1543. Surface disinfection of eggs did not reduce the sensitivity of detection of Salmonella Enteritidis in liquid eggs. These results could improve the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella in shell eggs by simplifying the preenrichment medium and changing the sample handling before enrichment.

Co-enriching microflora associated with culture based methods to detect Salmonella from tomato phyllosphere.

The ability to detect a specific organism from a complex environment is vitally important to many fields of public health, including food safety. For example, tomatoes have been implicated numerous times as vehicles of foodborne outbreaks due to strains of Salmonella but few studies have ever recovered Salmonella from a tomato phyllosphere environment. Precision of culturing techniques that target agents associated with outbreaks depend on numerous factors. One important factor to better understand is which species co-enrich during enrichment procedures and how microbial dynamics may impede or enhance detection of target pathogens. We used a shotgun sequence approach to describe taxa associated with samples pre-enrichment and throughout the enrichment steps of the Bacteriological Analytical Manual's (BAM) protocol for detection of Salmonella from environmental tomato samples. Recent work has shown that during efforts to enrich Salmonella (Proteobacteria) from tomato field samples, Firmicute genera are also co-enriched and at least one co-enriching Firmicute genus (Paenibacillus sp.) can inhibit and even kills strains of Salmonella. Here we provide a baseline description of microflora that co-culture during detection efforts and the utility of a bioinformatic approach to detect specific taxa from metagenomic sequence data. We observed that uncultured samples clustered together with distinct taxonomic profiles relative to the three cultured treatments (Universal Pre-enrichment broth (UPB), Tetrathionate (TT), and Rappaport-Vassiliadis (RV)). There was little consistency among samples exposed to the same culturing medias, suggesting significant microbial differences in starting matrices or stochasticity associated with enrichment processes. Interestingly, Paenibacillus sp. (Salmonella inhibitor) was significantly enriched from uncultured to cultured (UPB) samples. Also of interest was the sequence based identification of a number of sequences as Salmonella despite indication by all media, that samples were culture negative for Salmonella. Our results substantiate the nascent utility of metagenomic methods to improve both biological and bioinformatic pathogen detection methods.