Flow cytometry for rapid detection of Salmonella spp. in seed sprouts

Seed sprouts (alfalfa, mung bean, radish, etc.) have been implicated in several recent national and international outbreaks of salmonellosis. Conditions used for sprouting are also conducive to the growth of Salmonella. As a result, this pathogen can quickly grow to very high cell densities during sprouting without any detectable organoleptic impact. Seed sprouts typically also support heavy growth (~108 CFU g−1) of a heterogeneous microbiota consisting of various bacterial, yeast, and mold species, often dominated by non-pathogenic members of the family Enterobacteriaceae. This heavy background may present challenges to the detection of Salmonella, especially if this pathogen is present in relatively low numbers. We combined DNA-based fluorescence in situ hybridization (FISH) with flow cytometry (FCM) for the rapid molecular detection of Salmonella enterica ser. Typhimurium in artificially contaminated alfalfa and other seed sprouts. Components of the assay included a set of cooperatively binding probes, a chemical blocking treatment intended to reduce non-specific background, and sample concentration via tangential flow filtration (TFF). We were able to detect S. Typhimurium in sprout wash at levels as low as 103 CFU ml−1 sprout wash (104 CFU g−1 sprouts) against high microbial backgrounds (~108 CFU g−1 sprouts). Hybridization times were typically 30 min, with additional washing, but we ultimately found that S. Typhimurium could be readily detected using hybridization times as short as 2 min, without a wash step. These results clearly demonstrate the potential of combined DNA-FISH and FCM for rapid detection of Salmonella in this challenging food matrix and provide industry with a useful tool for compliance with sprout production standards proposed in the Food Safety Modernization Act (FSMA)

A model for the prediction of antimicrobial resistance in Escherichia coli based on a comparative evaluation of fatty acid profiles

Antimicrobial resistance is a threat to agricultural production and public health. In this proof-of-concept study, we investigated predicting antimicrobial sensitive/resistant (S/R) phenotypes and host sources of Escherichia coli (n = 128) based on differential fatty acid abundance. Myristic (14:0), pentadecanoic acid (15:0), palmitic (16:0), elaidic (18:19) and steric acid (18:0) were significantly different (α = 0.05) using a two-way ANOVA for predicting nalidixic acid, ciprofloxacin, aztreonam, cefatoxime, and ceftazidime S/R phenotypes. Additionally, analyses of palmitoleic (16:1), palmitic acid (16:0), methyl palmitate (i-17:0), and cis-9,10-methyleneoctadecanoic acid (19:0Δ) showed these markers were significantly different (α = 0.05) between isolates obtained from cattle and raccoons. S/R phenotype prediction for the above antibiotics or host source, based on linear regression models of fatty acid abundance, were made using a replicated-randomized subsampling and modeling approach. This model predicted S/R phenotype with 79% and 81% accuracy for nalidixic acid and ciprofloxacin, respectively. The isolate host source was predicted with 63% accuracy

Advances in Foodborne Pathogen Analysis

As the world population has grown, new demands on the production of foods have been met by increased efficiencies in production, from planting and harvesting to processing, packaging and distribution to retail locations. These efficiencies enable rapid intranational and global dissemination of foods, providing longer “face time” for products on retail shelves and allowing consumers to make healthy dietary choices year-round. However, our food production capabilities have outpaced the capacity of traditional detection methods to ensure our foods are safe. Traditional methods for culture-based detection and characterization of microorganisms are time-, labor- and, in some instances, space- and infrastructure-intensive, and are therefore not compatible with current (or future) production and processing realities. New and versatile detection methods requiring fewer overall resources (time, labor, space, equipment, cost, etc.) are needed to transform the throughput and safety dimensions of the food industry. Access to new, user-friendly, and point-of-care testing technologies may help expand the use and ease of testing, allowing stakeholders to leverage the data obtained to reduce their operating risk and health risks to the public. The papers in this Special Issue on “Advances in Foodborne Pathogen Analysis” address critical issues in rapid pathogen analysis, including preanalytical sample preparation, portable and field-capable test methods, the prevalence of antibiotic resistance in zoonotic pathogens and non-bacterial pathogens, such as viruses and protozoa

Relationships among intramammary health, udder and teat characteristics, and productivity of extensively managed ewes

Mastitis is an economically important disease and its subclinical state is difficult to diagnose, which makes mitigation more challenging. The objectives of this study were to screen clinically healthy ewes in order to 1) identify cultivable microbial species in milk, 2) evaluate somatic cell count (SCC) thresholds associated with intramammary infection, and 3) estimate relationships between udder and teat morphometric traits, SCC, and ewe productivity. Milk was collected from two flocks in early (\u3c5 \u3ed) and peak (30 to 45 d) lactation to quantify SCC (n = 530) and numerate cultivable microbial species by culture-based isolation followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; n = 243) identification. Within flock and lactation stage, 11% to 74% (mean = 36%) of samples were culture positive. More than 50 unique identifications were classified by MALDI-TOF MS analysis, and Bacillus licheniformis (18% to 27%), Micrococcus flavus (25%), Bacillus amyloliquefaciens (7% to 18%), and Staphylococcus epidermidis (26%) were among the most common within flock and across lactation stage. Optimum SCC thresholds to identify culture-positive samples ranged from 175 × 103 to 1,675 × 103 cells/mL. Ewe productivity was assessed as total 120-d adjusted litter weight (LW120) and analyzed within flock with breed, parity, year, and the linear covariate of log10 SCC (LSCC) at early or peak lactation. Although dependent on lactation stage and year, each 1-unit increase in LSCC (e.g., an increase in SCC from 100 × 103 to 1,000 × 103 cells/mL) was predicted to decrease LW120 between 9.5 and 16.1 kg when significant. Udder and teat traits included udder circumference, teat length, teat placement, and degree of separation of the udder halves. Correlations between traits were generally low to moderate within and across lactation stage and most were not consistently predictive of ewe LSCC. Overall, the frequencies of bacteria-positive milk samples indicated that subclinical mastitis (SCM) is common in these flocks and can impact ewe productivity. Therefore, future research is warranted to investigate pathways and timing of microbial invasion, genomic regions associated with susceptibility, and husbandry to mitigate the impact of SCM in extensively managed ewes

Validation of a screening method for the detection of colistin-resistant \u3ci\u3eE. coli\u3c/i\u3e containing mcr-1 in feral swine feces

A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 μg/mL) and vancomycin (8 μg/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 100 and 104 CFU/g), for a total of 1110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inocula of 0.1–9.99 CFU/g (n=340), 10–49.99 CFU/g (n=170), 50–99 CFU/g (n=255), 100–149 CFU/g (n=60), and 200–2200 CFU/g (n=175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger

Prophage induction reduces Shiga toxin producing \u3ci\u3eEscherichia coli\u3c/i\u3e (STEC) and Salmonella enterica on tomatoes and spinach: A model study

Fresh produce is increasingly implicated in foodborne outbreaks and most fresh produce is consumed raw, emphasizing the need to develop non-thermal methods to control foodborne pathogens. This study investigates bacterial cell lysis through induction of prophages as a novel approach to control foodborne bacterial pathogens on fresh produce. Shiga toxin producing Escherichia coli (STEC) and Salmonella enterica isolates were exposed to different prophage inducers (i.e. mitomycin C or streptonigrin) and growth of the cells was monitored by measuring the optical density (OD600) during incubation at 37C. Beginning at three hours after addition of the inducer, all concentrations (0.5, 1, 2 mg/mL) of mitomycin C, or 2 mg/mL streptonigrin significantly reduced the OD600 in broth cultures, in a concentration dependent manner, relative to cultures where no inducer was added. PCR confirmed bacterial release of induced bacteriophages and demonstrated that a single compound could successfully induce multiple types of prophages. The ability of mitomycin C to induce prophages in STEC O157:H7 and in S. enterica (serovars Typhimurium and Newport) on fresh produce was evaluated by inoculating red greenhouse tomatoes or spinach leaves with 5 x 107 and 5 x 108 colony forming units, respectively. After allowing time for the inoculum to dry on the fresh produce samples, 6 mg/mL mitomycin C was sprayed onto each sample, while control samples were sprayed with water. Following overnight incubation at 4C, the bacterial cells were recovered and plate counts were performed. A 3 log reduction in STEC O157:H7 cells was observed on tomatoes sprayed with mitomycin C compared to those sprayed with water, while a 1 log reduction was obtained on spinach. Similarly, spraying mitomycin C on tomatoes and spinach inoculated with S. enterica isolates resulted in a 1-1.5 log and 2 log reduction, respectively. These findings serve as a proof of concept that prophage induction can effectively control bacterial foodborne pathogens on fresh produce

Control of Listeria monocytogenes in ready-to-eat (RTE) vacuum packaged turkey roll via single or combined use of organic acid salts, ALTA 2341, and electron-beam irradiation

The effectiveness of electron beam irradiation for controlling LIsteria monocytogenes in ready-to-eat (RTE) turkey roll, formulated with ALTA 2341 (6,000 or 12,000 AU) alone or combined with sodium lactate (SL, 2%) + sodium diacetate (SDA, 0.25%), was investigated. Slices of turkey roll were inoculated with a five-strain mixture of L. monocytogenes to give [Difference]106 CFU/cm2. Inoculated product without ALTA 2341 or SL + SDA served as controls. Meat samples were vacuum-packaged, then irradiated at 0, 1.0, 1.5, 2.0, and 2.5 kGy. Samples irradiated at 0, 1.5, and 2.5 kGy were stored at 40C (42 days) or 100C (30 days). L. monocytogenes were enumerated by plating serial dilutions of meat homogenate on Modified Oxford (MOX) agar and counting bacterial colonies on agar plates after incubation (350C, 48h). Irradiation at 1.5 and 2.5 kGy reduced initial populations of L. monocytogenes by [Difference]2.4 and [Difference]4.4 log, respectively, irrespective of product formulation. Irradiation D-values were not significantly affected by differences in formulation (p\u3e0.05). Growth of survivors in RTE turkey roll with ALTA (6,000 or 12,000 AU) alone was not inhibited (p\u3e0.05). Growth of survivors was completely inhibited in samples with SL + SDA (P\u3c0.05). For example, L. monocytogenes in irradiated (2.5 kGy) turkey roll with SL +SDA + ALTA 2341 were less than 102 CFU/cm2 throughout storage at 40C or 100C. In contrast, survivors in 2.5 kGy-treated samples without SL + SDA reached [Difference]108 CFU/cm2 at 20 days (100C) and 35 days (40C). Irradiation (2.5 kGy) combined with SL (2.0%) + SDA (0.25%) with or without ALTA (6,000 or 12,000 AU) is effective in reducing initial populations of L. monocytogenes in RTE turkey roll and preventing growth of survivors during refrigeration (40C) and temperature abuse (100C)

Control of Listeria monocytogenes in ready-to-eat (RTE) vacuum packaged turkey roll via single or combined use of organic acid salts, ALTA 2341, and electron-beam irradiation

The effectiveness of electron beam irradiation for controlling LIsteria monocytogenes in ready-to-eat (RTE) turkey roll, formulated with ALTA 2341 (6,000 or 12,000 AU) alone or combined with sodium lactate (SL, 2%) + sodium diacetate (SDA, 0.25%), was investigated. Slices of turkey roll were inoculated with a five-strain mixture of L. monocytogenes to give [Difference]106 CFU/cm2. Inoculated product without ALTA 2341 or SL + SDA served as controls. Meat samples were vacuum-packaged, then irradiated at 0, 1.0, 1.5, 2.0, and 2.5 kGy. Samples irradiated at 0, 1.5, and 2.5 kGy were stored at 40C (42 days) or 100C (30 days). L. monocytogenes were enumerated by plating serial dilutions of meat homogenate on Modified Oxford (MOX) agar and counting bacterial colonies on agar plates after incubation (350C, 48h). Irradiation at 1.5 and 2.5 kGy reduced initial populations of L. monocytogenes by [Difference]2.4 and [Difference]4.4 log, respectively, irrespective of product formulation. Irradiation D-values were not significantly affected by differences in formulation (p>0.05). Growth of survivors in RTE turkey roll with ALTA (6,000 or 12,000 AU) alone was not inhibited (p>0.05). Growth of survivors was completely inhibited in samples with SL + SDA (P<0.05). For example, L. monocytogenes in irradiated (2.5 kGy) turkey roll with SL +SDA + ALTA 2341 were less than 102 CFU/cm2 throughout storage at 40C or 100C. In contrast, survivors in 2.5 kGy-treated samples without SL + SDA reached [Difference]108 CFU/cm2 at 20 days (100C) and 35 days (40C). Irradiation (2.5 kGy) combined with SL (2.0%) + SDA (0.25%) with or without ALTA (6,000 or 12,000 AU) is effective in reducing initial populations of L. monocytogenes in RTE turkey roll and preventing growth of survivors during refrigeration (40C) and temperature abuse (100C).</p