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.

Antimicrobial peptides (AMPs) are effective components of the host immune response and are widely distributed throughout nature. Recently, nontoxic antimicrobial polymers that mimic the structures of naturally occurring AMPs have been designed and are under development commercially as novel therapeutics. These compounds have several potential advantages over natural AMPs, including greater stability and reduced immunogenicity compared to natural peptides, relatively simple and scalable syntheses and the ability to tailor or “fine tune” their activities through combinatorial approaches. In previous work, we demonstrated the utility of certain generally regarded as safe (GRAS) flavorant and aroma compounds as enhancers of uptake and activity of clinically important antibiotics (Brehm-Stecher & Johnson, 2003). Here, we have extended this approach to include enhancement of biomimetic antimicrobial polymers. Three low molecular weight (<1000 D), broad-spectrum arylamide polymers (PolyMedix, Inc., Radnor, PA) were examined for their antimicrobial activities against gram-negative bacteria, gram-positive bacteria, yeast and filamentous fungi, both alone and when co-administered with sesquiterpenoid enhancers. Assay formats included disk diffusion, automated turbidimetry, time course (kinetic) plating of antimicrobial-treated cell suspensions, outer membrane assays with 1-N-phenylnaphthylamine (NPN) and transmission electron microscopy (TEM). Although results differed according to the polymer and test organism used, treatments containing sesquiterpenoids were marked by either increased ZOIs, decreased MICs or more rapid inactivation when compared with polymer-only treatments. Antimicrobial activity, expressed as decimal reduction times (D-value), showed that after 5 min, the combination of sesquiterpenoid and polymer was significantly different from the controls (p < 0.05) with a D-value of 3.92 min when incubated with Escherichia coli ATCC 25922. Collectively, our results indicate that the combination of sesquiterpenoid-enhancing agents with biomimetic antimicrobial polymers shows promise for the development of new, faster-acting and more broadly effective antimicrobial therapies.

Ready-to-Eat (RTE) meat and poultry products manufactured with natural or organic methods may be at greater risk for Listeria monocytogenes growth, if contaminated, than their conventional counterparts due to the required absence of preservatives and antimicrobials. Thus, the objective of this study was to investigate the use of commercially available natural antimicrobials in combination with post-lethality interventions for the control of L. monocytogenes growth and recovery on alternatively-cured RTE ham. Antimicrobials evaluated were cranberry powder (90 MX), vinegar (DV), and vinegar and lemon juice concentrate (LV1 X). Post-lethality interventions studied included high hydrostatic pressure at 400 MPa (HHP), lauricarginate (LAE), octanoic acid (OA), and post-packaging thermal treatment (PPTT). Viable L. monocytogenes on modified Oxford (MOX) and thin agar layer (TAL) media were monitored through 98 days of product storage at 4 ± 1°C. The post-lethality treatments of HHP, OA, and LAE significantly reduced initial viable L. monocytogenes numbers compared to the control, regardless of the antimicrobial ingredient used in the formulation while PPTT did not. Only when used in combination with DV and LV1 X did HHP, OA, and LAE exhibit sustained suppression, of L. monocytogenes recovery and growth throughout refrigerated storage. As a result, the use of natural antimicrobial ingredients such as DV and LV1 X in combination with post-lethality interventions such as HHP, LAE, and OA represents an effective multi-hurdle approach that could be instituted by manufacturers of organic and natural processed meat and poultry products for L. monocytogenes control.

Ready-to-eat (RTE) meat and poultry products manufactured with natural or organic methods are at greater risk for Listeria monocytogenes growth, if contaminated, than their conventional counterparts due to the required absence of preservatives and antimicrobials. Thus, the objective of this study was to investigate the use of commercially available natural antimicrobials and postlethality interventions in the control of L. monocytogenesgrowth and recovery on a RTE ham product. Antimicrobials evaluated were cranberry powder (90MX), vinegar (DV), and vinegar/lemon juice concentrate (LV1X). Postlethality interventions studied were high hydrostatic pressure at 400 (HHP400) or 600 (HHP600) MPa, lauric arginate (LAE), octanoic acid (OA), and postpackaging thermal treatment (PPTT). Parameters evaluated through 98 days of storage at 4±1°C were residual nitrite concentrations, pH, a_w, and viable L. monocytogenes on modified Oxford (MOX) media. On day 1, OA, 90MX, DV, and LV1X yielded lower residual nitrite concentrations than the control, whereas HHP400, HHP600, and LAE did not. LAE, HHP400, and OA reduced L. monocytogenes population compared to the control after 1 day of storage by 2.38, 2.21, and 1.73 log₁₀ colony-forming units per gram, respectively. PPTT did not achieve a significant reduction in L. monocytogenes populations. L. monocytogenes recovered and grew in all postlethality intervention treatments except HHP600. 90MX did not inhibit the growth of L. monocytogenes, while DV and LV1X did. Results of this study demonstrate the bactericidal properties of HHP, OA, and LAE and the bacteriostatic potential of natural antimicrobial ingredients such as DV and LV1X against L. monocytogenes.

Previously, we demonstrated capture and concentration of Salmonella enterica subspecies enterica ser. Typhimurium using magnetic ionic liquids (MILs), followed by rapid isothermal detection of captured cells via recombinase polymerase amplification (RPA). Here, we report work intended to explore the broader potential of MILs as novel pre-analytical capture reagents in food safety and related applications. Specifically, we evaluated the capacity of the ([P66614+][Ni(hfacac)3−]) (“Ni(II)”) MIL to bind a wider range of human pathogens using a panel of Salmonella and Escherichia coli O157:H7 isolates, including a “deep rough” strain of S. Minnesota. We extended this exploration further to include other members of the family Enterobacteriaceae of food safety and clinical or agricultural significance. Both the Ni(II) MIL and the ([P66614+][Dy(hfacac)4−]) (“Dy(III)”) MIL were evaluated for their effects on cell viability and structure-function relationships behind observed antimicrobial activities of the Dy(III) MIL were determined. Next, we used flow imaging microscopy (FIM) of Ni(II) MIL dispersions made in model liquid media to examine the impact of increasing ionic complexity on MIL droplet properties as a first step towards understanding the impact of suspension medium properties on MIL dispersion behavior. Finally, we used FIM to examine interactions between the Ni(II) MIL and Serratia marcescens, providing insights into how the MIL may act to capture and concentrate Gram-negative bacteria in aqueous samples, including food suspensions. Together, our results provide further characterization of bacteria-MIL interactions and support the broader utility of the Ni(II) MIL as a cell-friendly capture reagent for sample preparation prior to cultural or molecular analyses.

Plant extracts have been used for millennia for treatment of disease, with much recent interest focusing on the antimicrobial activities of plant essential oils (EOs). Although EOs are active against common microbial pathogens, their effective use as topical, environmental, or food antimicrobials will require EO-based formulations with enhanced antimicrobial activities. In this study, two polyionic compounds, sodium polyphosphate (polyP, a polyanion) and polyethylenimine (PEI, a polycation), were evaluated for their abilities to enhance the antimicrobial activities of six EOs against the human pathogens Escherichia coli O157:H7, Salmonella enterica subsp. enterica ser. Minnesota, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and Candida albicans. EOs tested were cinnamon, clove, regular and redistilled oregano, and two types of thyme oil. EOs were examined via disk diffusion and broth microdilution, either alone or in the presence of subinhibitory levels of polyP or PEI. Both polyP and PEI were found to be effective enhancers of EO activity against all strains examined, and calculation of fractional inhibitory indices for select EO/organism pairings demonstrated that true synergy was possible with this en‐ hancement approach. Experiments with a deep-rough strain of S. Minnesota probed the role of the outer membrane in both intrinsic resistance to EOs and enhancement by polyions. The use of polyP and PEI for boosting the antimicrobial activities of EOs may eventually facilitate the development of more effective EO-based antimicrobial treatments for use in applications such as wound treatment, surface disinfection, or as generally recognized as safe antimicrobials for use in foods or on food contact surfaces.

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).

We have previously investigated the extraction and concentration of bacteria from model systems using magnetic ionic liquid (MIL) solvents, while retaining their viability. Here, we combine MIL-based sample preparation with isothermal amplification and detection of Salmonella-specific DNA using Recombinase Polymerase Amplification (RPA). After initial developmental work with Serratia marcescens in water, Salmonella Typhimurium ATCC 14028 was inoculated in water, 2% milk, almond milk or liquid egg samples and extracted using one of two MILs, including: trihexyl(tetradecyl)phosphonium cobalt(II) hexafluoroacetylacetonate ([P66614+][Co(hfacac)3–]) and trihexyl(tetradecyl)phosphonium nickel(II) hexafluoroacetylacetonate ([P66614+][Ni(hfacac)3–]). Viable cells were recovered from the MIL extraction phase after the addition of modified LB broth, followed by a 20 min isothermal RPA assay. Amplification was carried out using supersaturated sodium acetate heat packs and results compared to those using a conventional laboratory thermocycler set to a single temperature. Results were visualized using either gel electrophoresis or nucleic acid lateral flow immunoassay (NALFIA). The combined MIL-RPA approach enabled detection of Salmonella at levels as low as 103 CFU mL-1. MIL-based sample preparation required less than 5 min to capture and concentrate sufficient cells for detection using RPA, which (including NALFIA or gel-based analysis) required approximately 30 - 45 min. Our results suggest the utility of MILs for the rapid extraction and concentration of pathogenic microorganisms in food samples, providing a means for physical enrichment that is compatible with downstream analysis using RPA.

Enterococci are important inhabitants of the animal intestine and are widely used in probiotic products. A probiotic strain is expected to possess several desirable properties in order to exert beneficial effects. Therefore, the objective of this study was to isolate, characterize and identify Enterococcus sp. from chicken cecal and fecal samples to determine potential probiotic properties. Enterococci were isolated from chicken ceca and feces of thirty three clinically healthy chickens from a local farm. In vitro studies were performed to assess antibacterial activity of the isolated LAB (using agar well diffusion and cell free supernatant broth technique against Salmonella enterica serotype Enteritidis), survival in acidic conditions, resistance to bile salts, and their survival during simulated gastric juice conditions at pH 2.5. Isolates were identified by biochemical carbohydrate fermentation patterns using an API 50 CHL kit and API ZYM kits and by sequenced 16S rDNA. An isolate belonging to E. faecium species exhibited inhibitory effect against S. enteritidis. This isolate producing a clear zone as large as 10.30 mm or greater and was able to grow in the coculture medium and at the same time, inhibited the growth S. enteritidis. In addition, E. faecium exhibited significant resistance under highly acidic conditions at pH 2.5 for 8 h and survived well in bile salt at 0.2% for 24 h and showing ability to survive in the presence of simulated gastric juice at pH 2.5. Based on these results, E. faecium isolate fulfills some of the criteria to be considered as a probiotic strain and therefore, could be used as a feed additive with good potential for controlling S. Enteritidis in chickens. However, in vivo studies are needed to determine the safety of the strain.

Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM.