Genomic Analysis Points to Multiple Genetic Mechanisms for Non-Transformable Campylobacter jejuni ST-50.

Campylobacter jejuni and Campylobacter coli are well known for their natural competence, i.e., their capacity for the uptake of naked DNA with subsequent transformation. This study identifies non-transformable C. jejuni and C. coli strains from domestic animals and employs genomic analysis to investigate the strain genotypes and their associated genetic mechanisms. The results reveal genetic associations leading to a non-transformable state, including functional DNase genes from bacteriophages and mutations within the cts-encoded DNA-uptake system, which impact the initial steps of the DNA uptake during natural transformation. Interestingly, all 38 tested C. jejuni ST-50 strains from the United States exhibit a high prevalence of non-transformability, and the strains harbor a variety of these genetic markers. This research emphasizes the role of these genetic markers in hindering the transfer of antimicrobial resistance (AMR) determinants, providing valuable insights into the genetic diversity of Campylobacter. As ST-50 is a major clone of C. jejuni globally, we additionally determined the prevalence of the genetic markers for non-transformability among C. jejuni ST-50 from different regions of the world, revealing distinct patterns of evolution and a strong selective pressure on the loss of competence in ST-50 strains, particularly in the agricultural environment in the United States. Our findings contribute to a comprehensive understanding of genetic exchange mechanisms within Campylobacter strains, and their implications for antimicrobial resistance dissemination and evolutionary pathways within specific lineages.

Complete Genome Sequence of the Arcobacter canalis Type Strain LMG 29148.

Arcobacter canalis was originally recovered from shellfish and from a sewage-contaminated canal. Arcobacter canalis is closely related to the marine bacterium Arcobacter marinus This study describes the complete whole-genome sequence of the A. canalis type strain LMG 29148 (=F138-33T; =CECT 8984T), which was recovered from oysters.

Draft Genome Sequences of Nine Campylobacter hyointestinalis subsp. lawsonii Strains.

With increasing reports of Campylobacter hyointestinalis species associated with human diseases, more genome sequences are required to understand the virulence mechanisms of this emerging pathogen. Here, we describe the genome sequences of nine C. hyointestinalis subsp. lawsonii strains.

Survival of foodborne pathogens on commercially packed table grapes under simulated refrigerated transit conditions.

We examined the survival of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica Thompson inoculated on commercially packed table grapes under simulated refrigerated transit conditions (1.1 ± 0.5 °C; 90% RH). Grapes were placed in perforated polyethylene cluster bags, within a commercial expanded polystyrene box equipped with either a SO2-generating pad; a perforated polyethylene box liner; a SO2-generating pad and a box liner; or none of them. L. monocytogenes was most sensitive to SO2-generating pad. SO2-generating pad or SO2-generating pad with box liner inactivated this pathogen completely on day 12 following the inoculation. S. enterica Thompson displayed a similar cold sensitivity as L. monocytogenes, but was more resistant to SO2-generating pad than L. monocytogenes. While SO2-generating pad eliminated S. enterica Thompson on day 20, a combination of box liner with SO2-generating pad inactivated this pathogen completely on day 13. E. coli O157:H7 had the highest tolerance to transit temperature and to SO2-generating pad; SO2-generating pad inactivated this pathogen completely on Day 20. Our data suggest that use of SO2-generating pad combined with box liner is effective in reducing foodborne pathogens L. monocytogenes and S. enterica Thompson, while the use of SO2-generating pad alone was more effective on E. coli O157:H7.

Complete Genome Sequence of the Hippuricase-Positive Campylobacter avium Type Strain LMG 24591.

Campylobacter avium is a thermotolerant Campylobacter species that has been isolated from poultry. C. avium was also the second hippuricase-positive species to be identified within Campylobacter Here, we present the genome sequence of the C. avium type strain LMG 24591 (=CCUG 56292T), isolated in 2006 from a broiler chicken in Italy.

Comparative Genomic Analysis Identifies a Campylobacter Clade Deficient in Selenium Metabolism.

The nonthermotolerant Campylobacter species C. fetus, C. hyointestinalis, C. iguaniorum, and C. lanienae form a distinct phylogenetic cluster within the genus. These species are primarily isolated from foraging (swine) or grazing (e.g., cattle, sheep) animals and cause sporadic and infrequent human illness. Previous typing studies identified three putative novel C. lanienae-related taxa, based on either MLST or atpA sequence data. To further characterize these putative novel taxa and the C. fetus group as a whole, 76 genomes were sequenced, either to completion or to draft level. These genomes represent 26 C. lanienae strains and 50 strains of the three novel taxa. C. fetus, C. hyointestinalis and C. iguaniorum genomes were previously sequenced to completion; therefore, a comparative genomic analysis across the entire C. fetus group was conducted (including average nucleotide identity analysis) that supports the initial identification of these three novel Campylobacter species. Furthermore, C. lanienae and the three putative novel species form a discrete clade within the C. fetus group, which we have termed the C. lanienae clade. This clade is distinguished from other members of the C. fetus group by a reduced genome size and distinct CRISPR/Cas systems. Moreover, there are two signature characteristics of the C. lanienae clade. C. lanienae clade genomes carry four to ten unlinked and similar, but nonidentical, flagellin genes. Additionally, all 76 C. lanienae clade genomes sequenced demonstrate a complete absence of genes related to selenium metabolism, including genes encoding the selenocysteine insertion machinery, selenoproteins, and the selenocysteinyl tRNA.

Campylobacter pinnipediorum sp. nov., isolated from pinnipeds, comprising Campylobacter pinnipediorum subsp. pinnipediorum subsp. nov. and Campylobacter pinnipediorum subsp. caledonicus subsp. nov.

During independent diagnostic screenings of otariid seals in California (USA) and phocid seals in Scotland (UK), Campylobacter-like isolates, which differed from the established taxa of the genus Campylobacter, were cultured from abscesses and internal organs of different seal species. A polyphasic study was undertaken to determine the taxonomic position of these six isolates. The isolates were characterized by 16S rRNA gene and AtpA sequence analysis and by conventional phenotypic testing. The whole-genome sequences were determined for all isolates, and the average nucleotide identity (ANI) was determined. The isolates formed a separate phylogenetic clade, divergent from all other taxa of the genus Campylobacter and most closely related to Campylobactermucosalis. Although all isolates showed 100 % 16S rRNA gene sequence homology, AtpA and ANI analyses indicated divergence between the otariid isolates from California and the phocid isolates from Scotland, which warrants subspecies status for each clade. The two subspecies could also be distinguished phenotypically on the basis of catalase activity. This study shows clearly that the isolates obtained from pinnipeds represent a novel species within the genus Campylobacter, for which the name Campylobacter pinnipediorum sp. nov. is proposed. Within this novel species, the Californian isolates represent a separate subspecies, for which the name C. pinnipediorum subsp. pinnipediorum subsp. nov. is proposed. The type strain for both this novel species and subspecies is RM17260T (=LMG 29472T=CCUG 69570T). The Scottish isolates represent another subspecies, for which the name C. pinnipediorum subsp. caledonicus subsp. nov. is proposed. The type strain of this subspecies is M302/10/6T (=LMG 29473T=CCUG 68650T).

Comparative Genomics of All Three Campylobacter sputorum Biovars and a Novel Cattle-Associated C. sputorum Clade.

Campylobacter sputorum is a nonthermotolerant campylobacter that is primarily isolated from food animals such as cattle and sheep. C. sputorum is also infrequently associated with human illness. Based on catalase and urease activity, three biovars are currently recognized within C. sputorum: bv. sputorum (catalase negative, urease negative), bv. fecalis (catalase positive, urease negative), and bv. paraureolyticus (catalase negative, urease positive). A multi-locus sequence typing (MLST) method was recently constructed for C. sputorum. MLST typing of several cattle-associated C. sputorum isolates suggested that they are members of a divergent C. sputorum clade. Although catalase positive, and thus technically bv. fecalis, the taxonomic position of these strains could not be determined solely by MLST. To further characterize C. sputorum, the genomes of four strains, representing all three biovars and the divergent clade, were sequenced to completion. Here we present a comparative genomic analysis of the four C. sputorum genomes. This analysis indicates that the three biovars and the cattle-associated strains are highly related at the genome level with similarities in gene content. Furthermore, the four genomes are strongly syntenic with one or two minor inversions. However, substantial differences in gene content were observed among the three biovars. Finally, although the strain representing the cattle-associated isolates was shown to be C. sputorum, it is possible that this strain is a member of a novel C. sputorum subspecies; thus, these cattle-associated strains may form a second taxon within C. sputorum.

Complete Genome Sequences of Campylobacter hyointestinalis subsp. hyointestinalis Strain LMG 9260 and C. hyointestinalis subsp. lawsonii Strain LMG 15993.

Campylobacter hyointestinalis is isolated primarily from ruminants and swine, but is also occasionally isolated from humans. C. hyointestinalis is currently divided into two subspecies, C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii This study describes the first closed whole-genome sequences of C. hyointestinalis subsp. hyointestinalis isolate LMG 9260 and C. hyointestinalis subsp. lawsonii isolate LMG 15993.

Complete Genome Sequence of Campylobacter iguaniorum Strain RM11343, Isolated from an Alpaca.

Campylobacter iguaniorum is a member of the C. fetus group of campylobacters and is one of two Campylobacter taxa isolated from reptiles. This study describes the whole-genome sequence of the C. iguaniorum strain RM11343, which was isolated from a California alpaca fecal sample.

Effect of sulfur dioxide fumigation on survival of foodborne pathogens on table grapes under standard storage temperature.

We examined the fate of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica Thompson inoculated on freshly-harvested table grapes under standard cold storage with initial and weekly sulfur dioxide (SO2) fumigation. L. monocytogenes and S. enterica Thompson were much more sensitive to cold temperature than E. coli O157:H7. Furthermore, L. monocytogenes was highly susceptible to SO2. Initial fumigation with 100 or 200 ppm-hr was sufficient to eliminate this pathogen on grapes with low (10(4) cells/grape) and high (10(6) cells/grape) inocula, respectively. Initial fumigation with 300 ppm-hr reduced S. enterica Thompson population about 300- and 10-fold on grapes with low and high inocula, respectively. Initial fumigation with 300 ppm-hr reduced E. coli O157:H7 population to less than 10-fold, regardless of inoculum density. When grapes were inoculated with the high inoculum and fumigated on days 0 and 7 with 200 or 300 ppm-hr SO2, S. enterica Thompson and E. coli O157:H7 were completely inactivated between days 8 and 14 of cold storage. Standard cold storage combined with SO2 fumigation was effective in reducing and eliminating all three pathogens on table grapes, however, depending on the dose, two or three fumigations were needed for elimination of S. enterica Thompson and E. coli O157:H7.

Comparative genomics of the Campylobacter lari group.

The Campylobacter lari group is a phylogenetic clade within the epsilon subdivision of the Proteobacteria and is part of the thermotolerant Campylobacter spp., a division within the genus that includes the human pathogen Campylobacter jejuni. The C. lari group is currently composed of five species (C. lari, Campylobacter insulaenigrae, Campylobacter volucris, Campylobacter subantarcticus, and Campylobacter peloridis), as well as a group of strains termed the urease-positive thermophilic Campylobacter (UPTC) and other C. lari-like strains. Here we present the complete genome sequences of 11 C. lari group strains, including the five C. lari group species, four UPTC strains, and a lari-like strain isolated in this study. The genome of C. lari subsp. lari strain RM2100 was described previously. Analysis of the C. lari group genomes indicates that this group is highly related at the genome level. Furthermore, these genomes are strongly syntenic with minor rearrangements occurring only in 4 of the 12 genomes studied. The C. lari group can be bifurcated, based on the flagella and flagellar modification genes. Genomic analysis of the UPTC strains indicated that these organisms are variable but highly similar, closely related to but distinct from C. lari. Additionally, the C. lari group contains multiple genes encoding hemagglutination domain proteins, which are either contingency genes or linked to conserved contingency genes. Many of the features identified in strain RM2100, such as major deficiencies in amino acid biosynthesis and energy metabolism, are conserved across all 12 genomes, suggesting that these common features may play a role in the association of the C. lari group with coastal environments and watersheds.

Multilocus sequence typing methods for the emerging Campylobacter Species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus.

Multilocus sequence typing (MLST) systems have been reported previously for multiple food- and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.

Effects of a hydrodynamic process on extraction of carotenoids from tomato.

We evaluated the results of using a proprietary hydrodynamic method, which was introduced with the hope of increasing accessibility of beneficial nutrition-enhancing fruit and vegetable products. Tomato, a major dietary source of carotenoids, notably lycopene, was tested because of its many health benefits to consumers. Samples before and after treatment were compared for lycopene, phytoene, and phytofluene contents. Extractable lycopene and other carotenoids increased significantly. In nature, lycopene exists almost exclusively as the all-trans stereoisomer. Cis-lycopene isomers form during cooking and digestion, resulting in higher percentages in plasma and tissues than ingested. Cis-lycopene isomers are more bioavailable than all-trans lycopene. Extraction using this proprietary method increased extracted cis-lycopene to as high as 43% of the total lycopene, indicating increased isomerisation. This method could therefore contribute significantly to the delivery of health benefits of biologically available lycopene from tomato products for metabolic functions.

Carotenoid extraction from plants using a novel, environmentally friendly solvent.

Few environmentally friendly solvents are available to extract carotenoids for use in foods. The most effective known solvents are products of the petroleum industry and toxic for human consumption. Yet carotenoid extracts are desirable for use in dietary supplements and as additives to enhance the health benefits of processed foods. Ethyl lactate is an excellent solvent to extract both trans- and cis-lycopene isomers from dried tomato powder, the extraction efficiency of which is enhanced by the addition of the antioxidants alpha-tocopherol and alpha-lipoic acid, both of which are known to benefit human health. It is also useful to extract lutein and beta-carotene from dried powders prepared from white corn and carrots. Because of its low flammability and its origin as a byproduct of the corn and soybean industries, it is more advantageous than ethyl acetate, which is a petroleum product.

Tangerine tomatoes increase total and tetra-cis-lycopene isomer concentrations more than red tomatoes in healthy adult humans.

Lycopene, or the foods that contain it, may prevent prostate cancer. Studies suggest that some cis-lycopene isomers are more bioavailable than the trans-lycopene isomer. We hypothesized that tangerine tomatoes, which predominantly contain the tetra-cis isomer, should be a good source of bioavailable lycopene. We fed lunches containing 300 g tangerine or red tomato sauce per day to 21 healthy adults in a double-blind crossover design. We collected blood at baseline and after each treatment and washout period. We measured tetra-cis, other cis, and trans lycopene, as well as other carotenoids, by reversed-phase high-performance liquid chromatography. Both tomato sauces increased lycopene concentrations in blood, but the tangerine tomato sauce caused a greater increase of total and tetra-cis-lycopene. The cis isomer(s) may also have facilitated absorption of the trans-lycopene isomer. Indices of oxidative damage decreased as serum lycopene concentrations increased. Our results suggest that total lycopene concentrations can be increased by substituting tetra-cis-lycopene-rich tangerine tomatoes for common red tomatoes in the diet.

Steam cooking significantly improves in vitro bile acid binding of collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage.

Bile acid binding capacity has been related to the cholesterol-lowering potential of foods and food fractions. Lowered recirculation of bile acids results in utilization of cholesterol to synthesize bile acid and reduced fat absorption. Secondary bile acids have been associated with increased risk of cancer. Bile acid binding potential has been related to lowering the risk of heart disease and that of cancer. Previously, we have reported bile acid binding by several uncooked vegetables. However, most vegetables are consumed after cooking. How cooking would influence in vitro bile acid binding of various vegetables was investigated using a mixture of bile acids secreted in human bile under physiological conditions. Eight replicate incubations were conducted for each treatment simulating gastric and intestinal digestion, which included a substrate only, a bile acid mixture only, and 6 with substrate and bile acid mixture. Cholestyramine (a cholesterol-lowering, bile acid binding drug) was the positive control treatment and cellulose was the negative control. Relative to cholestyramine, in vitro bile acid binding on dry matter basis was for the collard greens, kale, and mustard greens, 13%; broccoli, 10%; Brussels sprouts and spinach, 8%; green bell pepper, 7%; and cabbage, 5%. These results point to the significantly different (P < or = .05) health-promoting potential of collard greens = kale = mustard greens > broccoli > Brussels sprouts = spinach = green bell pepper > cabbage as indicated by their bile acid binding on dry matter basis. Steam cooking significantly improved the in vitro bile acid binding of collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage compared with previously observed bile acid binding values for these vegetables raw (uncooked). Inclusion of steam-cooked collard greens, kale, mustard greens, broccoli, green bell pepper, and cabbage in our daily diet as health-promoting vegetables should be emphasized. These green/leafy vegetables, when consumed regularly after steam cooking, would lower the risk of cardiovascular disease and cancer, advance human nutrition research, and improve public health.

A comparison of carotenoid content and total antioxidant activity in catsup from several commercial sources in the United States.

Samples of catsup from 13 commercial sources, representing at least 10 U.S. manufacturers, were analyzed for carotenoid content, antioxidant activity, and percentage solids. The solids content of these catsup brand samples varied from 26.31 to 38.06% solids. The lycopene content ranged from 59.42 to 183.36 microg, and total carotenoids were as high as 216.6 microg/g fresh weight, respectively. In addition, both hydrophilic and lipophilic antioxidant activities were measured using the Trolox equivalent antioxidant capacity (TEAC) assay. These measurements of samples of the various catsup brands ranged from 176.5 to 356.8 total TEAC units.

Fatty acid and carotenoid composition of gac (Momordica cochinchinensis Spreng) fruit.

In this study, we analyzed fatty acid and carotenoid composition of fruit tissues, including seed (which are surrounded by a bright red, oily aril) of Momordica cochinchinensis Spreng, known as gac in Vietnam. Carotenoid content was analyzed by reversed-phase HPLC, using a C(30) column and a method separating cis- and trans-isomers of the major carotenoids in this fruit. Mean values obtained in aril tissues were 1342 microg trans-, 204 microg cis-, and 2227 microg total lycopene; 597 microg trans-, 39 microg cis-, and 718 microg total beta-carotene; and 107 microg alpha-carotene/g FW. Mesocarp contained 11 microg trans-, 5 microg cis-beta-carotene/g FW, trace amounts of alpha-carotene, and no lycopene. Gac aril contained 22% fatty acids by weight, composed of 32% oleic, 29% palmitic, and 28% linoleic acids. Seeds contained primarily stearic acid (60.5%), smaller amounts of linoleic (20%), oleic (9%), and palmitic (5-6%) acids, and trace amounts of arachidic, cis-vaccenic, linolenic, and palmitoleic, eicosa-11-enoic acids, and eicosa-13-enoic (in one fruit only) acids.