Application of Sample6 DETECT™ HT/L Kit for the Detection of Listeria in Mixed Leafy Greens.

BACKGROUND: Early and accurate detection of Listeria in foods is vital. Current methods require 24 h enrichment for detection. OBJECTIVE: This study aimed to demonstrate enhanced early detection of Listeria in mixed leafy greens using Sample6 DETECT™ HT/L, a phage-based detection system. METHODS: A method comparison between the reference method (U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 10) for the detection of Listeria spp. and the Sample6 DETECT HT/L using a new proprietary R2 Medium was performed in mixed leafy greens. RESULTS: Using the R2 Medium enrichment with Sample6 DETECT HT/L, detection of L. innocua was possible at 12 h (with a centrifugation step), and L. monocytogenes was detected by 18 h, with equivalent performance as the 24 h enrichments using the reference method detection. The Sample6 DETECT HT/L gave an equivalent performance for L. innocua at 15 h as the reference method at 24 h. Detection was accomplished by the addition of reagents in the kit, following the package insert, and reading results in a detection chamber using a 96-well plate reader that detects a fluorescent signal. CONCLUSIONS: Results indicate the new R2 Medium and Sample6 DETECT HT/L allowed for earlier detection of Listeria spp. in mixed leafy greens. HIGHLIGHTS: Sample6 DETECT HT/L with the new R2 Medium allowed the early detection of Listeria spp. and may be applied in other food matrices and environmental samples.

Occurrence and Levels of Salmonella, Enterohemorrhagic Escherichia coli, and Listeria in Raw Wheat.

HIGHLIGHTS: Prevalence of Salmonella and E. coli in raw wheat emphasizes the need to cook wheat products. 3,891 grain samples were tested for E. coli and Salmonella; 1,285 were tested for Listeria. Of wheat berries sampled, 0.44% were positive for E. coli and 1.23% were positive for Salmonella. Salmonella diversity was high, indicating various animal sources that are difficult to prevent. Cooking wheat products is the best preventative measure against foodborne illness from wheat.

Application of an Environmental Phage-Based Assay (Sample6 Detect HT/L) for the Detection of Listeria spp. in Ice Cream.

Background: Dairy products are common sources of Listeria outbreaks, and early detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses for dairy producers. Objective: This study aimed to evaluate Sample6 Detect HT/L for effective detection of Listeria monocytogenes and L. innocua in ice cream. Methods: Performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 method for detection of Listeria spp. in ice cream using an unpaired study design. Results: R2-enriched samples tested with Sample6 Detect HT/L performed as well as the reference method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the test kit that helps with early detection. All the dPODC values (Sample6 Detect HT/L presumptive and confirmed results) equaled zero, indicating 100% concordance between the methods. Both Sample6 Detect HT/L and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 Detect HT/L and reference method results. Conclusions: Sample6 Detect HT/L is suitable to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect HT/L demonstrated equivalent detection of L. monocytogenes and L. innocua from R2-enriched samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA BAM method for L. monocytogenes. Highlights: These results indicate that Sample6 Detect HT/L, primarily developed for environmental samples, can be used to detect Listeria spp. in ice cream with less incubation time, resulting in faster detection.

Earthworm symbiont Verminephrobacter eiseniae mediates natural transformation within host egg capsules using type IV pili.

The dense microbial communities commonly associated with plants and animals should offer many opportunities for horizontal gene transfer through described mechanisms of DNA exchange including natural transformation (NT). However, studies of the significance of NT have focused primarily on pathogens. The study presented here demonstrates highly efficient DNA exchange by NT in a common symbiont of earthworms. The obligate bacterial symbiont Verminephrobacter eiseniae is a member of a microbial consortium of the earthworm Eisenia fetida that is transmitted into the egg capsules to colonize the embryonic worms. In the study presented here, by testing for transformants under different conditions in culture, we demonstrate that V. eiseniae can incorporate free DNA from the environment, that competency is regulated by environmental factors, and that it is sequence specific. Mutations in the type IV pili of V. eiseniae resulted in loss of DNA uptake, implicating the type IV pilus (TFP) apparatus in DNA uptake. Furthermore, injection of DNA carrying antibiotic-resistance genes into egg capsules resulted in transformants within the capsule, demonstrating the relevance of DNA uptake within the earthworm system. The ability to take up species-specific DNA from the environment may explain the maintenance of the relatively large, intact genome of this long-associated obligate symbiont, and provides a mechanism for acquisition of foreign genes within the earthworm system.

A global survey of the bacteria within earthworm nephridia.

Earthworms comprise 16 described families in the Crassiclitellata plus a few other minor groups. Microscopy studies of the early 20th century detected bacteria within the excretory organs, the nephridia, of species within a few of these families. More recent evidence for the consistent and specific association of bacteria with nephridia within the Lumbricidae has been well documented, but the presence and identity of nephridial bacteria among the rest of the Crassiclitellata families had not been explored. The study presented here aimed to identify members of Crassiclitellata families that harbor bacteria in their nephridia, and identify these bacteria based on 16S rRNA gene sequences. Eleven earthworm families were surveyed from countries of six continents, and two island nations. The results revealed members of four bacterial orders commonly occurred within nephridia of genera within nine Crassiclitellata families. Members of the bacterial phyla Bacteroidetes (order Sphingobacteriales), Betaproteobacteria (order Burkholderiales; family Comamonadaceae), and Alphaproteobacteria (orders Rhodospirillales and Rhizobiales) were detected in the nephridia of basal Crassiclitellata, as well as in derived families. Earthworm genera with meronephridia, multiple small nephridia per segment, lacked bacteria, whereas bacteria were often detected in holonephridia, single pairs of large nephridia with a distinct morphology and external excretory pore. The Acanthodrilidae members, a large derived family of earthworms, did not appear to possess nephridial bacteria regardless of nephridial form. Although earthworms from a variety of habitat types were sampled, there were no clear correlations of lifestyle with symbiont types, with the exception of the aquatic earthworms that contained bacteria unrelated to those in any other earthworms. The findings support an evolutionarily long association of bacteria within the Crassiclitellata, and suggest a contribution to nitrogen conservation for the earthworms.

Verminephrobacter eiseniae type IV pili and flagella are required to colonize earthworm nephridia.

The bacterial symbiont Verminephrobacter eiseniae colonizes nephridia, the excretory organs, of the lumbricid earthworm Eisenia fetida. E. fetida transfers V. eisenia into the egg capsule albumin during capsule formation and V. eiseniae cells migrate into the earthworm nephridia during embryogenesis, where they bind and persist. In order to characterize the mechanistic basis of selective tissue colonization, methods for site-directed mutagenesis and colonization competence were developed and used to evaluate the consequences of individual gene disruptions. Using these newly developed tools, two distinct modes of bacterial motility were shown to be required for V. eiseniae colonization of nascent earthworm nephridia. Flagella and type IV pili mutants lacked motility in culture and were not able to colonize embryonic earthworms, indicating that both twitching and flagellar motility are required for entrance into the nephridia.

Diversity and host specificity of the Verminephrobacter-earthworm symbiosis.

Symbiotic bacteria of the genus Verminephrobacter (Betaproteobacteria) were detected in the nephridia of 19 out of 23 investigated earthworm species (Oligochaeta: Lumbricidae) by 16S rRNA gene sequence analysis and fluorescence in situ hybridization (FISH). While all four Lumbricus species and three out of five Aporrectodea species were densely colonized by a mono-species culture of Verminephrobacter, other earthworm species contained mixed bacterial populations with varying proportions of Verminephrobacter; four species did not contain Verminephrobacter at all. The Verminephrobacter symbionts could be grouped into earthworm species-specific sequence clusters based on their 16S rRNA and RNA polymerase subunit B (rpoB) genes. Closely related host species harboured more closely related symbionts than did distantly related hosts. Co-diversification of the symbiotic partners could not be demonstrated unambiguously due to the poor resolution of the host phylogeny [based on histone H3 and cytochrome c oxidase subunit I (COI) gene sequence analyses]. However, there was a pattern of symbiont diversification within four groups of closely related hosts. The mean rate of symbiont 16S rRNA gene evolution was determined using a relaxed clock model, and the rate was calibrated with paleogeographical estimates of the time of origin of Lumbricid earthworms. The calibrated rates of symbiont 16S rRNA gene evolution are 0.012-0.026 substitutions per site per 50 million years and thus similar to rates reported from other symbiotic bacteria.

Transmission of a bacterial consortium in Eisenia fetida egg capsules.

The earthworm Eisenia fetida harbours Verminephrobacter eiseniae within their excretory nephridia. This symbiont is transferred from the parent into the egg capsules where the cells are acquired by the developing earthworm in a series of recruitment steps. Previous studies defined V. eiseniae as the most abundant cell type in the egg capsules, leaving approximately 30% of the bacteria unidentified and of unknown origin. The study presented here used terminal restriction fragment length polymorphism analysis together with cloning and sequencing of 16S rRNA genes to define the composition of the bacterial consortium in E. fetida egg capsules from early to late development. Newly formed capsules of E. fetida contained three bacterial types, a novel Microbacteriaceae member, a Flexibacteriaceae member and the previously described V. eiseniae. Fluorescent in situ hybridization (FISH) using specific and general rRNA probes demonstrated that the bacteria are abundant during early development, colonize the embryo and appear in the adult nephridia. As the capsules mature, Herbaspirillum spp. become abundant although they were not detected within the adult worm. These divergent taxa could serve distinct functions in both the adult earthworm and in the egg capsule to influence the competitive ability of earthworms within the soil community.

Dietary protein and cellulose effects on chemical and microbial characteristics of Swine feces and stored manure.

The objectives of this study were to investigate the effects of dietary crude protein (14.5 or 12.0%) and cellulose (8.7 or 2.5%) levels on composition of feces and manure after 8 wk of diet feeding and storage. Pigs were fed twice daily; after each feeding, urine and feces were collected and added to manure storage containers. On weeks 2 and 8 after initiation of the experiment, fresh fecal and manure samples were obtained. On Week 8, increased dietary cellulose resulted in significantly higher levels of volatile fatty acids (VFA) and phenols in feces compare to other diets. In contrast, dietary protein had the greatest effect on manure chemical composition; lower protein decreased sulfur content, ammonia, and phenolic compound concentrations. High levels of either dietary cellulose or protein tended to increase microbial community similarity in fecal samples, but only high protein increased similarity among manure sample microbial communities. Fecal and manure samples from Week 8 differed from samples taken in Week 2 both in chemical and microbiological composition. Week 2 samples had lower concentrations of many of chemical compounds and microbial diversity than samples from Week 8. The fecal results indicate that after 2 wk of feeding experimental diets the animals were not fully adapted to the diets. More importantly, after only 2 wk of urine and fecal collection, manure was not representative of stored manure, limiting its usefulness in developing standards and recommendations for on-farm management practices.

Verminephrobacter eiseniae gen. nov., sp. nov., a nephridial symbiont of the earthworm Eisenia foetida (Savigny).

A Gram-negative, flagellated, heterotrophic, catalase-negative, rod-shaped bacterium previously identified as an earthworm symbiont was isolated from nephridia of the earthworm Eisenia foetida. Comparisons of 16S rRNA gene sequences indicated its relatedness to the betaproteobacterial genus Acidovorax and the novel isolates shared 92-94% sequence similarity with recognized species of this genus. Gene sequence phylogenies revealed that the group of earthworm symbionts formed a cohesive and independent clade. The DNA G+C content was 67.0+/-0.2 mol%. Major fatty acids were C(16:0), C(16:1)omega7c and C(17:0) cyclo. While capable of growing in fully aerated media, all isolates favoured low oxygen concentrations and all required biotin or a mix of amino acids in order to grow on defined mineral media. Based on phylogenies inferred from three housekeeping gene sequences (gap, recA and rpoC), DNA-DNA hybridization values, the unique ecology and the distinct physiology of the novel strains, the new genus Verminephrobacter gen. nov. is proposed for the earthworm nephridial symbionts. The name Verminephrobacter eiseniae sp. nov. is proposed for the type species with strain EF01-2(T) (=ATCC BAA-1489(T)=DSM 19286(T)) as the type strain of the type species.

Selective recruitment of bacteria during embryogenesis of an earthworm.

Earthworms of the family Lumbricidae harbor specific and stable populations of Acidovorax-like bacteria within their excretory organs, the nephridia. The symbionts of Eisenia foetida are deposited into the egg capsules during mating and the nephridia of the juveniles are colonized before they hatch. The timing and mechanisms governing bacterial recruitment and colonization are unknown for the earthworm-Acidovorax association. This study examined the process of colonization of the symbiotic organ during development of the embryos within the egg capsules. Bacteria associated with the developing embryos were visualized using in situ hybridization to bacterial cells and laser scanning confocal microscopy. Bacterial cells were associated with earthworm embryos during the earliest stages of development-the ova through to hatching. Three-dimensional examination of stages of development revealed an embryonic duct that recruits the Acidovorax-like symbiont cells. As each segment matures, Acidovorax-like symbiotic bacteria are recruited into this duct, excluding most other bacterial types, and remain there for a period of days prior to migration into the nephridium. After colonization of the nephridial ampulla, the canal remains bacteria-free. In addition to the known Acidovorax-like bacteria, multiple types of bacteria interact with the embryos externally and internally during the full course of development, and ultimately fill the gut lumen near the end of development prior to hatching. Colonization of the correct tissues by specific bacteria during differentiation and maturation of the organs must involve selective host defenses and signaling between the two partners to prevent over growth of nascent tissues.

Localization of 'Candidatus Endobugula sertula' and the bryostatins throughout the life cycle of the bryozoan Bugula neritina.

'Candidatus Endobugula sertula,' the uncultivated gamma-proteobacterial symbiont of the marine bryozoan Bugula neritina, synthesizes bryostatins, complex polyketides that render B. neritina larvae unpalatable to predators. Although the symbiosis is well described, little is known about the locations of 'E. sertula' or the bryostatins throughout larval settlement, metamorphosis and early development. In this study, we simultaneously localized 'E. sertula' and the bryostatins in multiple stages of the B. neritina life cycle, using a novel bryostatin detection method based on its known ability to bind mammalian protein kinase C. Our results suggest that the bryostatins are deposited onto the exterior of B. neritina larvae during embryonic development, persist on the larval surface throughout metamorphosis and are shed prior to cuticle formation. During metamorphosis, 'E. sertula' remains adhered to the larval pallial epithelium and is incorporated into the preancestrula cystid tissue layer, which ultimately develops into a bud and gives rise to the next zooid in the colony. Colocalization of bryostatin signal with aggregates of 'E. sertula' in buds of ancestrulae suggested new synthesis of bryostatins in ancestrulae. In adult B. neritina colonies, symbiont microcolonies were observed in the funicular cords of rhizoids, which likely result in asexual transmission of 'E. sertula' to regenerated colonies. Furthermore, bryostatin signal was detected on the surface of the rhizoids of adult B. neritina colonies. Through simultaneous localization of the bryostatins and the 'E. sertula,' we determined how 'E. sertula' is transmitted, and identified shifts in bryostatin localization throughout the life cycle of the host B. neritina.

Transmission of nephridial bacteria of the earthworm Eisenia fetida.

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.

NO means 'yes' in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association.

During colonization of the Euprymna scolopes light organ, symbiotic Vibrio fischeri cells aggregate in mucus secreted by a superficial ciliated host epithelium near the sites of eventual inoculation. Once aggregated, symbiont cells migrate through ducts into epithelium-lined crypts, where they form a persistent association with the host. In this study, we provide evidence that nitric oxide synthase (NOS) and its product nitric oxide (NO) are active during the colonization of host tissues by V. fischeri. NADPH-diaphorase staining and immunocytochemistry detected NOS, and the fluorochrome diaminofluorescein (DAF) detected its product NO in high concentrations in the epithelia of the superficial ciliated fields, ducts, and crypt antechambers. In addition, both NOS and NO were detected in vesicles within the secreted mucus where the symbionts aggregate. In the presence of NO scavengers, cells of a non-symbiotic Vibrio species formed unusually large aggregates outside of the light organ, but these bacteria did not colonize host tissues. In contrast, V. fischeri effectively colonized the crypts and irreversibly attenuated the NOS and NO signals in the ducts and crypt antechambers. These data provide evidence that NO production, a defense response of animal cells to bacterial pathogens, plays a role in the interactions between a host and its beneficial bacterial partner during the initiation of symbiotic colonization.

Acidovorax-like symbionts in the nephridia of earthworms.

Dense accumulations of bacteria in the excretory organs, nephridia, were first described more than 75 years ago in members of the annelid family Lumbricidae (earthworms). These nephridial symbionts were assumed to play a role in the degradation of proteins in the excretory fluid for nitrogen recycling. In the present study, the phylogenetic affiliation of the nephridial bacteria of the earthworms Lumbricus terrestris, Aporrectodea tuberculata, Octolasion lacteum and Eisenia foetida was resolved. The 16S rRNA gene sequences of the symbionts formed a monophyletic cluster within the genus Acidovorax. Similarity between symbiont sequences from different host species was 95.5-97.6%, whereas similarity was> 99% between symbiont sequences from individuals of the same species. Densely packed bacteria were detected in the ampulla of the nephridia by fluorescence in situ hybridization (FISH) using Acidovorax-specific oligonucleotide probes. No other bacterial cells could be found by FISH, although a few sequences other than Acidovorax had been found by PCR and cloning. These results suggest that the Acidovorax-earthworm symbiosis is a stable, host-specific association that has evolved from a common bacterial ancestor. Given the close phylogenetic relationship of the symbionts to proteolytic, free-living Acidovorax species, they may indeed play a role in protein degradation during nitrogen excretion by earthworms.