A Review of Regulatory Standards and Advances in Essential Oils as Antimicrobials in Foods.

As essential oils (EOs) possess GRAS status, there is a strong interest in their application to food preservation. Trends in the food industry suggest consumers are drawn to environmentally friendly alternatives and less synthetic chemical preservatives. Although the use of EOs has increased over the years, adverse effects have limited their use. This review aims to address the regulatory standards for EO usage in food, techniques for delivery of EOs, essential oils commonly used to control pathogens and molds, and advances with new active compounds that overcome sensory effects for meat products, fresh fruits and vegetables, fruit and vegetable juices, seafood, dairy products, and other products. This review will show adverse sensory effects can be overcome in various products by the use of edible coatings containing encapsulated EOs to facilitate the controlled release of EOs. Depending on the method of cooking, the food product has been shown to mask flavors associated with EOs. In addition, using active packaging materials can decrease the diffusion rate of the EOs, thus controlling undesirable flavor characteristics while still preserving or prolonging the shelf life of food. The use of encapsulation in packaging film can control the release of volatile or active ingredients. Further, use of EOs in the vapor phase allows for contact indirectly, and use of nanoemulsion, coating, and film wrap allows for the controlled release of the EOs. Research has also shown that combining EOs can prevent adverse sensory effects. Essential oils continue to serve as a very beneficial way of controlling undesirable microorganisms in food systems.

Peptide 1018 inhibits swarming and influences Anr-regulated gene expression downstream of the stringent stress response in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that causes considerable human morbidity and mortality, particularly in nosocomial infections and individuals with cystic fibrosis. P. aeruginosa can adapt to surface growth by undergoing swarming motility, a rapid multicellular movement that occurs on viscous soft surfaces with amino acids as a nitrogen source. Here we tested the small synthetic host defense peptide, innate defense regulator 1018, and found that it inhibited swarming motility at concentrations as low as 0.75 µg/ml, well below the MIC for strain PA14 planktonic cells (64 µg/ml). A screen of the PA14 transposon insertion mutant library revealed 29 mutants that were more tolerant to peptide 1018 during swarming, five of which demonstrated significantly greater swarming than the WT in the presence of peptide. Transcriptional analysis (RNA-Seq) of cells that were inoculated on swarming plates containing 1.0 µg/ml peptide revealed differential expression of 1,190 genes compared to cells swarming on plates without peptide. Furthermore, 1018 treatment distinctly altered the gene expression profile of cells when compared to that untreated cells in the centre of the swarm colonies. Peptide-treated cells exhibited changes in the expression of genes implicated in the stringent stress response including those regulated by anr, which is involved in anaerobic adaptation, indicative of a mechanism by which 1018 might inhibit swarming motility. Overall, this study illustrates potential mechanisms by which peptide 1018 inhibits swarming surface motility, an important bacterial adaptation associated with antibiotic resistance, virulence, and dissemination of P. aeruginosa.

Contribution of Swarming Motility to Dissemination in a Pseudomonas aeruginosa Murine Skin Abscess Infection Model.

Swarming motility in Pseudomonas aeruginosa is a multicellular adaptation induced by semisolid medium with amino acids as a nitrogen source. By phenotypic screening, we differentiated swarming from other complex adaptive phenotypes, such as biofilm formation, swimming and twitching, by identifying a swarming-specific mutant in ptsP, a metabolic regulator. This swarming-deficient mutant was tested in an acute murine skin abscess infection model. Bacteria were recovered at significantly lower numbers from organs of mice infected with the ∆ptsP mutant. We also tested the synthetic peptide 1018 for activity against different motilities and efficacy in vivo. Treatment with peptide 1018 mimicked the phenotype of the ∆ptsP mutant in vitro, as swarming was inhibited at low concentrations (<2 µg/mL) but not swimming or twitching, and in vivo, as mice had a reduced bacterial load recovered from organs. Therefore, PtsP functions as a regulator of swarming, which in turn contributes to dissemination and colonization in vivo.

The Small RNAs PA2952.1 and PrrH as Regulators of Virulence, Motility, and Iron Metabolism in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that undergoes swarming motility in response to semisolid conditions with amino acids as a nitrogen source. With a genome encoding hundreds of potential intergenic small RNAs (sRNAs), P. aeruginosa can easily adapt to different conditions and stresses. We previously identified 20 sRNAs that were differentially expressed (DE) under swarming conditions. Here, these sRNAs were overexpressed in strain PAO1 and were subjected to an array of phenotypic screens. Overexpression of the PrrH sRNA resulted in decreased swimming motility, whereas a ΔprrH mutant had decreased cytotoxicity and increased pyoverdine production. Overexpression of the previously uncharacterized PA2952.1 sRNA resulted in decreased swarming and swimming motilities, increased gentamicin and tobramycin resistance under swarming conditions, and increased trimethoprim susceptibility. Transcriptome sequencing (RNA-Seq) and proteomic analysis were performed on the wild type (WT) overexpressing PA2952.1 compared to the empty vector control under swarming conditions, and these revealed the differential expression (absolute fold change [FC] ≥ 1.5) of 784 genes and the differential abundance (absolute FC ≥ 1.25) of 59 proteins. Among these were found 73 transcriptional regulators, two-component systems, and sigma and anti-sigma factors. Downstream effectors included downregulated pilus and flagellar genes, the upregulated efflux pump MexGHI-OpmD, and the upregulated arn operon. Genes involved in iron and zinc uptake were generally upregulated, and certain pyoverdine genes were upregulated. Overall, the sRNAs PA2952.1 and PrrH appeared to be involved in regulating virulence-related programs in P. aeruginosa, including iron acquisition and motility.IMPORTANCE Due to the rising incidence of multidrug-resistant (MDR) strains and the difficulty of eliminating P. aeruginosa infections, it is important to understand the regulatory mechanisms that allow this bacterium to adapt to and thrive under a variety of conditions. Small RNAs (sRNAs) are one regulatory mechanism that allows bacteria to change the amount of protein synthesized. In this study, we overexpressed 20 different sRNAs in order to investigate how this might affect different bacterial behaviors. We found that one of the sRNAs, PrrH, played a role in swimming motility and virulence phenotypes, indicating a potentially important role in clinical infections. Another sRNA, PA2952.1, affected other clinically relevant phenotypes, including motility and antibiotic resistance. RNA-Seq and proteomics of the strain overexpressing PA2952.1 revealed the differential expression of 784 genes and 59 proteins, with a total of 73 regulatory factors. This substantial dysregulation indicates an important role for the sRNA PA2952.1.

Unique inducible filamentous motility identified in pathogenic Bacillus cereus group species.

Active migration across semi-solid surfaces is important for bacterial success by facilitating colonization of unoccupied niches and is often associated with altered virulence and antibiotic resistance profiles. We isolated an atmospheric contaminant, subsequently identified as a new strain of Bacillus mobilis, which showed a unique, robust, rapid, and inducible filamentous surface motility. This flagella-independent migration was characterized by formation of elongated cells at the expanding edge and was induced when cells were inoculated onto lawns of metabolically inactive Campylobacter jejuni cells, autoclaved bacterial biomass, adsorbed milk, and adsorbed blood atop hard agar plates. Phosphatidylcholine (PC), bacterial membrane components, and sterile human fecal extracts were also sufficient to induce filamentous expansion. Screening of eight other Bacillus spp. showed that filamentous motility was conserved amongst B. cereus group species to varying degrees. RNA-Seq of elongated expanding cells collected from adsorbed milk and PC lawns versus control rod-shaped cells revealed dysregulation of genes involved in metabolism and membrane transport, sporulation, quorum sensing, antibiotic synthesis, and virulence (e.g., hblA/B/C/D and plcR). These findings characterize the robustness and ecological significance of filamentous surface motility in B. cereus group species and lay the foundation for understanding the biological role it may play during environment and host colonization.

Author Correction: The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Overexpression of the Small RNA PA0805.1 in Pseudomonas aeruginosa Modulates the Expression of a Large Set of Genes and Proteins, Resulting in Altered Motility, Cytotoxicity, and Tobramycin Resistance.

Pseudomonas aeruginosa is a motile species that initiates swarming motility in response to specific environmental cues, i.e., a semisolid surface with amino acids as a nitrogen source (relevant to the human lung). Swarming is an intricately regulated process, but to date posttranscriptional regulation has not been extensively investigated. Small noncoding RNAs (sRNAs) are hypothesized to play posttranscriptional regulatory roles, largely through suppression of translation, and we previously demonstrated 20 sRNA species that were dysregulated under swarming conditions. One of these, sRNA PA0805.1 (which was 5-fold upregulated under swarming conditions), when cloned, transformed into wild-type (WT) PAO1, and overexpressed, led to broad phenotypic changes, including reduced swarming, swimming, and twitching motilities, as well as increased adherence, cytotoxicity, and tobramycin resistance. A ΔPA0805.1 deletion mutant was more susceptible to tobramycin than the WT under swarming conditions. The strain overexpressing PA0805.1 was compared to the empty-vector strain by transcriptome sequencing (RNA-Seq) and proteomics under swarming conditions to determine sRNA targets. Broad transcriptional and proteomic profiles showed 1,121 differentially expressed genes and 258 proteins with significantly different abundance. Importantly, these included 106 transcriptional regulators, two-component regulatory systems, and sigma and anti-sigma factors. Downstream of these regulators were found downregulated type IV pilus genes, many upregulated adherence and virulence factors, and two multidrug efflux systems, mexXY and mexGHI-opmD Therefore, the sRNA PA0805.1 appears to be a global regulator that influences diverse bacterial lifestyles, most likely through a regulatory cascade.IMPORTANCE P. aeruginosa is an opportunistic pathogen of humans. With roughly 10% of its genes encoding transcriptional regulators, and hundreds of small noncoding RNAs (sRNAs) interspersed throughout the genome, P. aeruginosa is able to fine-tune its response to adapt and survive in the host and resist antimicrobial agents. Understanding mechanisms of genetic regulation is therefore crucial to combat pathogenesis. The previously uncharacterized sRNA PA0805.1 was overexpressed in P. aeruginosa strain PAO1, resulting in decreased motility, increased adherence, cytotoxicity, and tobramycin resistance. In contrast, a ΔPA0805.1 deletion mutant had increased susceptibility to tobramycin under swarming conditions. Omic approaches uncovered 1,121 transcriptomic and 258 proteomic changes in the overexpression strain compared with the empty-vector strain, which included 106 regulatory factors. Downstream of these regulators were upregulated adherence factors, multidrug efflux systems, and virulence factors in both transcriptomics and proteomics. This study provides insights into the role of the sRNA PA0805.1 in modulating bacterial adaptations.

The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations.

Antimicrobial use in livestock production is a driver for the development and proliferation of antimicrobial resistance (AMR). Wildlife interactions with livestock, acquiring associated AMR bacteria and genes, and wildlife's subsequent dispersal across the landscape are hypothesized to play an important role in the ecology of AMR. Here, we examined priority AMR phenotypes and genotypes of Escherichia coli isolated from the gastrointestinal tracts of European starlings (Sturnus vulgaris) found on concentrated animal feeding operations (CAFOs). European starlings may be present in high numbers on CAFOs (>100,000 birds), interact with urban environments, and can migrate distances exceeding 1,500 km in North America. In this study, 1,477 European starlings from 31 feedlots in five U.S. states were sampled for E. coli resistant to third generation cephalosporins (3G-C) and fluoroquinolones. The prevalence of 3G-C and fluoroquinolone-resistant E. coli was 4% and 10%, respectively. Multidrug resistance in the E. coli isolates collected (n = 236) was common, with the majority of isolates displaying resistance to six or more classes of antibiotics. Genetic analyses of a subset of these isolates identified 94 genes putatively contributing to AMR, including seven class A and C ß-lactamases as well as mutations in gyrA and parC recognized to confer resistance to quinolones. Phylogenetic and subtyping assessments showed that highly similar isolates (≥99.4% shared core genome, ≥99.6% shared coding sequence) with priority AMR were found in birds on feedlots separated by distances exceeding 150 km, suggesting that European starlings could be involved in the interstate dissemination of priority AMR bacteria.

Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells.

Swarming surface motility is a complex adaptation leading to multidrug antibiotic resistance and virulence factor production in Pseudomonas aeruginosa Here, we expanded previous studies to demonstrate that under swarming conditions, P. aeruginosa PA14 is more resistant to multiple antibiotics, including aminoglycosides, ß-lactams, chloramphenicol, ciprofloxacin, tetracycline, trimethoprim, and macrolides, than swimming cells, but is not more resistant to polymyxin B. We investigated the mechanism(s) of swarming-mediated antibiotic resistance by examining the transcriptomes of swarming cells and swarming cells treated with tobramycin by transcriptomics (RNA-Seq) and reverse transcriptase quantitative PCR (qRT-PCR). RNA-Seq of swarming cells (versus swimming) revealed 1,581 dysregulated genes, including 104 transcriptional regulators, two-component systems, and sigma factors, numerous upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Strain PA14 mutants in resistome genes that were dysregulated under swarming conditions were tested for their ability to swarm in the presence of tobramycin. In total, 41 mutants in genes dysregulated under swarming conditions were shown to be more resistant to tobramycin under swarming conditions, indicating that swarming-mediated tobramycin resistance was multideterminant. Focusing on two genes downregulated under swarming conditions, both prtN and wbpW mutants were more resistant to tobramycin, while the prtN mutant was additionally resistant to trimethoprim under swarming conditions; complementation of these mutants restored susceptibility. RNA-Seq of swarming cells treated with subinhibitory concentrations of tobramycin revealed the upregulation of the multidrug efflux pump MexXY and downregulation of virulence factors.

Community-led comparative genomic and phenotypic analysis of the aquaculture pathogen Pseudomonas baetica a390T sequenced by Ion semiconductor and Nanopore technologies.

Pseudomonas baetica strain a390T is the type strain of this recently described species and here we present its high-contiguity draft genome. To celebrate the 16th International Conference on Pseudomonas, the genome of P. baetica strain a390T was sequenced using a unique combination of Ion Torrent semiconductor and Oxford Nanopore methods as part of a collaborative community-led project. The use of high-quality Ion Torrent sequences with long Nanopore reads gave rapid, high-contiguity and -quality, 16-contig genome sequence. Whole genome phylogenetic analysis places P. baetica within the P. koreensis clade of the P. fluorescens group. Comparison of the main genomic features of P. baetica with a variety of other Pseudomonas spp. suggests that it is a highly adaptable organism, typical of the genus. This strain was originally isolated from the liver of a diseased wedge sole fish, and genotypic and phenotypic analyses show that it is tolerant to osmotic stress and to oxytetracycline.

Anti-biofilm peptides as a new weapon in antimicrobial warfare.

Microorganisms growing in a biofilm state are very resilient in the face of treatment by many antimicrobial agents. Biofilm infections are a significant problem in chronic and long-term infections, including those colonizing medical devices and implants. Anti-biofilm peptides represent a very promising approach to treat biofilm-related infections and have an extraordinary ability to interfere with various stages of the biofilm growth mode. Anti-biofilm peptides possess promising broad-spectrum activity in killing both Gram-positive and Gram-negative bacteria in biofilms, show strong synergy with conventional antibiotics, and act by targeting a universal stringent stress response. Understanding downstream processes at the molecular level will help to develop and design peptides with increased activity. Anti-biofilm peptides represent a novel, exciting approach to treating recalcitrant bacterial infections.

Colorimetric paper-based detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes of agricultural water.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (µPADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the µPAD. Alternatively, digital images of the reacted µPADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24 hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Content and correlates of checking related to symptoms of obsessive compulsive disorder and generalized anxiety disorder.

In addition to the central role of compulsive behaviors in obsessive compulsive disorder (OCD), recent data have documented the presence of compulsive behaviors in individuals with generalized anxiety disorder (GAD). However, there is a lack of information about potential similarities and differences with regard to the quality, or content, of checking associated with worry and obsessions. The two studies presented herein are an initial step towards gathering this information. Findings of Study 1, from a large unselected undergraduate sample, showed that symptoms of OCD and GAD were both significantly associated with checking behaviors. However, while OCD symptoms were associated with checking related to both objects and interpersonal situations, GAD symptoms were only significantly associated with interpersonal checking. Findings of Study 2, using a separate sample, suggest links between interpersonal checking and features characteristic of GAD, namely emotion regulation difficulties, and between object checking and a cognitive feature of OCD, namely thought-action fusion. In summary, the current studies add to a growing body of literature suggesting that checking may be important in numerous forms of psychopathology, while also suggesting that the nature and function of checking may differ for various symptom profiles.

Oxidative stability of gelatin coated pork at refrigerated storage.

As consumers are chiefly concerned with odor and appearance, refrigerated meats have a greater possibility of rejection due to their perishability. A treatment was designed to preserve the quality parameters such as color and oxidation rate. The treatment encompassed coating pork loin samples with aqueous porcine gelatin solutions (175 bloom) at three concentrations (0%, 10% and 20%) and studying its effect on lipid oxidation, protein oxidation, oxidation pre-cursors, color and purge of meat. Vacuum packed Longissimus dorsi muscle was cut into 10 x 5 x 1 cm samples and randomly dipped in one of three solutions. Samples were stored at 4 degrees C and analyzed for thio-barbituric acid reactive substances, metmyoglobin content, protein carbonyls, heme iron content and color change. There was a significant difference (p<0.05) for TBARS values, protein carbonyls, heme iron content, total color change, metmyoglobin content with treatments maintaining values more similar to a fresher product when compared to control on days 3, 5 and 7. There was no significant difference (p>0.05) between 10% and 20% gelatin coating on any of the parameters between storage days.

Barriers to treatment seeking for anxiety disorders: initial data on the role of mental health literacy.

BACKGROUND: Anxiety disorders represent the single largest mental health problem in the United States [Greenberg et al., 1999. J Clin Psychiatry 60:427-435; Rice and Miller, 1998. Br J Psychiatry 173:4-9]. However most individuals with anxiety disorders never seek treatment [Henderson et al., 2002. Can J Psychiatry 47:819-824; Mojtabai et al., 2002. Arch Gen Psychiatry 59:77-84; Roness et al., 2005. Acta Psychiatr Scand 111:51-58]. Deficits in the ability to recognize anxiety disorders and beliefs about them, (i.e., "mental health literacy") may contribute to low levels of help seeking. METHODS: Survey data assessing mental health literacy for multiple anxiety disorders and for depression were collected from 284 undergraduate students enrolled in psychology courses at a public university in the United States. Specifically, respondents were presented with vignettes portraying individuals experiencing various forms of mental illness and were asked to label the disorder, its cause and whether or not they would recommend treatment. RESULTS: Findings showed that social phobia and obsessive compulsive disorder (OCD) were associated with recognition rates that were generally high and similar to depression (approximately 80%). In contrast, less than half of the respondents labeled panic disorder or generalized anxiety disorder (GAD) correctly. Symptoms of OCD were attributed to mental illness by approximately 50% of respondents, but such attributions were rare for the other anxiety disorders studied (<12%). Finally, data on help-seeking recommendations suggested that such recommendations are far from universal and varied between different anxiety disorders and according to perceptions of the causes of symptoms. CONCLUSIONS: Given that the current sample was well-educated young adults, mental health literacy of the general public may be even lower.

Extension of the sexual abuse questionnaire to other abuse categories: the initial psychometric validation of the Binghamton Childhood Abuse Screen.

Study I represents a follow-up to a recently published study dealing with the initial development of the Sexual Abuse Questionnaire (SAQ). The SAQwas designed to be used to identify individuals experiencing psychological distress resulting from a history of childhood sexual abuse. Using a new data set (N = 2,806), this study utilized item analyses, exploratory factor analyses, and Receiver Operating Characteristics Curves to replicate and expand upon the initial psychometric properties of the SAQ, and to examine the sensitivity of the SAQ to other abuse categories. Findings from Study I indicated that the SAQ was sensitive to other abuse categories and in need of revision. Study II, using a new data set (N = 1,454), provided the initial psychometric evaluation of the newly constructed Binghamton Childhood Abuse Screen (BCAS).