Accurate flow cytometric monitoring of Escherichia coli subpopulations on solid food treated with high pressure carbon dioxide.

AIMS: Evaluation of flow cytometry coupled with viability markers to monitor the inactivation of Escherichia coli cells spiked on solid food following High Pressure Carbon Dioxide (HPCD), a mild processing technology. METHODS AND RESULTS: Flow cytometry (FCM) coupled with SYBR-Green I and Propidium Iodide was applied to monitor the effect of HPCD treatment on E. coli cells spiked on fresh cut carrots, therefore mimicking contamination of food products by faecal coliforms. FCM allowed to distinguish E. coli cells from carrot debris and natural flora, to evaluate the reduction of total cells, and to quantify viable and dead cells based on their membrane integrity after HPCD treatment. The comparison of FCM results with conventional cultivation methods revealed that HPCD treatments performed at 120 bar, 22 or 35°C for 5 min disrupted 43 and 53% of bacterial cells, respectively, and produced a large percentage of partially permeabilized (96·5 and 98%) cells. CONCLUSIONS: Although treatments at 22°C for 10 min and at 35°C for 7 min were sufficient to inhibit the ability of all E. coli cells to replicate with an inactivation of 8 Log, FCM analysis showed that the inactivation of intact cells was only 2-2·5 Log. A fraction of HPCD-treated cells maintained their metabolic activity and re-growth capacity, indicating that the treatment induces a transitory Viable But Not Cultivable (VNBC) state. SIGNIFICANCE AND IMPACT OF THE STUDY: Under stress conditions many pathogens enter in a VBNC state, thus escaping detection by traditional cultivation methods. We provide the first evaluation of HPCD-mediated bacterial inactivation on fresh food using FCM coupled with viability markers, which should assist in the prevention of food-associated health risks.

Antimicrobial susceptibility and mechanism of resistance to fluoroquinolones in Staphylococcus intermedius and Staphylococcus schleiferi.

The objective of the present study was to determine the antimicrobial susceptibility of 136 canine isolates of Staphylococcus intermedius and 10 canine isolates of S. schleiferi subspecies coagulans to 16 fluoroquinolones (FQs), and to investigate the mechanisms of resistance in the nonsusceptible isolates. Of the 136 of S. intermedius tested 98.5% were susceptible to all 16 FQs whereas only 40% of the 10 isolates of S. schleiferi subspecies coagulans were susceptible. Two isolates of S. intermedius and six isolates of S. schleiferi, were found to be resistant to 13 out of 16 FQs, while they retained their susceptibility to fourth generation FQs such as gatifloxacin, moxifloxacin and trovafloxacin. Sequencing of the quinolone-resistance determining regions of gyrA and grlA genes showed that in S. intermedius, dichotomous resistance to FQs was associated with the occurrence of one alteration in GyrA-84 and one in GrlA-80, while in S. schleiferi the same pattern of resistance was observed in isolates showing these changes only in gyrA. This study is the first to screen FQs of the second, third and fourth generation for antimicrobial resistance in clinical isolates of S. intermedius and S. schleiferi of canine origin, and to describe mutations in gyrA and grlA associated with FQ resistance in these bacterial species.

Species diversity among the genus Monorchis (Digenea: Monorchiidae) parasitic in marine teleosts: molecular, morphological and morphometrical studies with a description of Monorchis blennii n. sp.

Molecular, morphological and morphometrical studies were conducted on two species of the genus Monorchis (Monorchiidae), Monorchis parvus and Monorchis monorchis, collected in different fish hosts from the Mediterranean Sea. The analysis of internal transcribed spacer 1 sequences of ribosomal DNA showed that M. monorchis specimens from Parablennius gattorugine were strongly divergent (12.9%) from specimens of this species collected in Spondyliosoma cantharus and Diplodus puntazzo. This high genetic variation was confirmed by the analysis of morphological structures and morphometrics, which showed that M. monorchis specimens from P. gattorugine can be distinguished from those of S. cantharus and D. puntazzo by several morphological characteristics, including body size, number and distribution of vitelline follicles, testis shape, structure of the cirrus pouch, and number of eggs. Our results show that M. monorchis specimens isolated from P. gattorugine represent a clearly distinct entity from M. monorchis found in the other hosts, which has enabled us to describe a new species, Monorchis blennii n. sp.

Genetic variability among cercariae of the Schistosomatidae (Trematoda: Digenea) causing swimmer's itch in Europe.

Ribosomal DNA sequences of the internal transcribed spacer 1 (ITS1) were obtained from schistosome cercariae responsible for swimmer's itch in Europe. Two types of ITS1 (1100 and 1400), which differ by the number of repeated patterns were found among cercariae shedded by Lymnaea ovata and L. auricularia (Lymnaeidae). A phylogenetic analysis of the ITS1 region showed that sequences of each type form two well-defined clades. An adult of Trichobilharzia regenti isolated from the nasal vessels of Anas platyrhynchos (Anatidae) was found to correspond to the cercaria type 1400. The sequencing of several ITS1 clones from a single cercaria of each type, as well as a specific PCR-based test suggested that both ITS1 types do not co-occur within a single individual.

Molecules, morphology and morphometrics of Cainocreadium labracis and Cainocreadium dentecis n. sp. (Digenea: Opecoelidae) parasitic in marine fishes.

Molecular, morphological and morphometric analyses were conducted on several samples of Cainocreadium labracis (Opecoelidae), a trematode parasitic in marine teleosts. The samples were isolated from several specimens of Dicentrarchus labrax, the type host, and Dentex dentex. The molecular analysis of complete Internal Transcribed Spacer sequences of ribosomal DNA revealed that specimens isolated from each host species form two well-defined groups, whose sequence divergence reaches 7.5%. The morphological study showed that the two groups can be distinguished by several characters, including the level of maximum body breadth, the relative position of the testes, the shape of the cirrus pouch, and the extent of the uterus. Multivariate analyses of morphometrics demonstrated consistency of most of the characters for discriminating the two groups. Our results show that C. labracis specimens isolated from D. labrax and D. dentex represent clearly distinct entities from molecular, morphological and statistical points of view, which has enabled us to describe a new species, Cainocreadium dentecis n. sp.

The murine orthologue of the Golgi-localized TPTE protein provides clues to the evolutionary history of the human TPTE gene family.

The human TPTE gene encodes a testis-specific protein that contains four potential transmembrane domains and a protein tyrosine phosphatase motif, and shows homology to the tumor suppressor PTEN/MMAC1. Chromosomal mapping revealed multiple copies of the TPTE gene present on the acrocentric chromosomes 13, 15, 21 and 22, and the Y chromosome. Zooblot analysis suggests that mice may possess only one copy of TPTE. In the present study, we report the isolation and initial characterization of the full-length cDNA of the mouse homologue Tpte. At least three different mRNA transcripts ( Tpte.a, b, c) are produced via alternative splicing, encoding predicted proteins that would contain four potential transmembrane domains and a protein tyrosine phosphatase motif. Transfection of a 5'EGFP-TPTE fusion protein in Hela cells revealed an intracellular localization within the Golgi apparatus. Tpte was mapped by radiation hybrid to a region of mouse chromosome 8 that shows conserved synteny with human 13q14.2-q21 between NEK3 and SGT1. This region of the human genome was found to contain a partial, highly diverged copy of TPTE that is likely to represent the ancestral copy from which the other copies of TPTE arose through duplication events. The Y chromosome copy of TPTE is a pseudogene and is not therefore involved in the testis expression of this gene family.

Comparison of ribosomal DNA sequences of Lamellodiscus spp. (monogenea, diplectanidae) parasitising Pagellus (sparidae, teleostei) in the North Mediterranean Sea: species divergence and coevolutionary interactions.

We sequenced DNA fragments from four monogenean species of the genus Lamellodiscus and their three fish host species from the genus Pagellus in the North Mediterranean Sea, in order to estimate the molecular divergence and the coevolutionary interactions in this association. By comparing the ITS1 sequences of the parasites, we assessed their level of interspecific differences and tested the phylogenetic status of Lamellodiscus virgula and Lamellodiscus obeliae, formerly described as two different species. Moreover, we wanted to know if closely related parasites used closely related hosts, to investigate the coevolutionary interactions in this complex. Phylogenetic relationships among Lamellodiscus species were estimated with partial 18S ribosomal DNA sequences while mitochondrial cytochrome-b DNA sequences were used for their fish hosts. The ITS1 sequences appear to be highly variable among Lamellodiscus species, except L.virgula and L.obeliae, suggesting an old divergence time or a rapid molecular evolution within this genus. This fish-parasite association seems to exhibit coevolutionary interactions. L.virgula and L.obeliae are proposed to be a single species on the basis of their almost identical ITS1 sequences.

The life cycle of Opecoeloides columbellae (Pagenstecher, 1863) n. comb. (Digenea, opecoelidae): evidence from molecules and morphology.

The cosmopolitan digenean family Opecoelidae comprises several hundred species, whose adults live in the digestive tract of marine and freshwater fishes. The genus Opecoeloides Odhner, 1928 is represented in the Mediterranean by a single species, Opecoeloides furcatus (Bremser in Rudolphi, 1819), that has been recorded from six definitive hosts species. To see if this broad host range could be the result of an underestimation of species diversity, we obtained ITS1 ribosomal DNA sequences as well as morphological data from adult specimens of O. furcatus isolated from two definitive hosts species: Mullus surmuletus and Gaidropsarus mediterraneus. Sequence and morphological data were also obtained from several opecoelid cercariae and metacercariae occurring in different invertebrate hosts. The data presented here provide striking evidence that O. furcatus specimens isolated from the two host fishes represent distinct species. This argument is reinforced by the fact that cercariae corresponding to each of these adult species were found in two molluscan host-species, Columbella rustica and Mitrella scripta. These parasite species differ by several nucleotide substitutions and a 60 bp-long insertion in the ITS1. They also show clear morphological differences in testis and ovary shape, as well as in their mean dimensions. Here, we attribute the adult specimens found in G. mediterraneus to Opecoeloides columbellae (Pagenstecher, 1863) n. comb. This species was described and compared with O. furcatus from M. surmuletus. ITS1 sequence comparison allowed identification of the cercaria (occurring in C. rustica) and metacercaria (occurring in Hippolyte inermis) of O. columbellae n. comb.

The life cycle of Monorchis parvus (Digenea: Monorchiidae) demonstrated by developmental and molecular data.

Cercaria cerastodermae I, a digenean parasite of Cerastoderma edule, was recorded for the first time in the Atlantic Ocean off the Iberian peninsula. Sporocysts were present in the hemolymph of the digestive gland, gonad, gills, and foot of the mollusc. Most of the cercariae present within sporocysts were encysted as metacercariae. The corresponding adult stages were obtained after experimental infection of several Diplodus sargus artificially reared in fish farms and that had previously been protected against natural infections. Numerous adult specimens of Monorchis parvus were collected in Diplodus annularis along the French Mediterranean coast. Comparison of wild and experimental adults allowed the adult stage of Cercaria cerastodermae I to be identified as M. parvus. Another monorchid, Monorchis monorchis, a parasite of Spondyliosoma cantharus, was found in the same Mediterranean area and compared with M. parvus. Additionally, ITS1 nuclear ribosomal DNA sequences of C. cerastodermae I and of the adults collected in naturally infected D. annularis and S. cantharus were obtained. Sequence data indicate that C. cerastodermae I corresponds to the adult of M. parvus found in D. annularis and is clearly distinct from M. monorchis found in S. cantharus.

The life-cycle of three species of the Mesometridae (Digenea) with comments on the taxonomic status of this family.

The intra-molluscan stages of three species of the Mesometridae Poche, 1926 are described. The corresponding adult stages are intestinal parasites of herbivorous sparid teleosts. The cercariae develop in prosobranch gastropods. The larvae of Elstia stossichianum occur in Vermetus triqueter, those of Wardula capitellata are parasites of Barleeia rubra and those of Centroderma spinosissima are found in three related rissoid hosts species: Rissoa ventricosa, R. auriscalpium and R. similis. The phylogenetic status of the Mesometridae in the Digenea is discussed in relation to larval and life-cycle characters.

Molecular identification of developmental stages in Opecoelidae (Digenea).

Nuclear ribosomal DNA sequences represent a useful tool for distinction of poorly differentiated developmental stages, such as trematode cercariae or metacercariae. Here, the complete internal transcribed spacer region of the ribosomal DNA (ITS 1 + 5.8S + ITS 2) was sequenced for 29 specimens of the digenean family Opecoelidae, including 16 adult specimens and 13 undescribed larval stages (nine cercariae and four metacercariae) occurring in various marine host organisms. Six cercariae and three metacercariae were found to match their corresponding adult form. This work also revealed that cercariae of the same species are able to infect more than one gastropod host species, suggesting that the specificity for the first intermediate host within the Digenea may be lower than previously thought.

Use of the ITS rDNA for elucidation of some life-cycles of Mesometridae (Trematoda, Digenea).

Identification of larval stages is crucial for elucidating the life-cycles of various Digenea. However, in many digenean species, the larvae lack distinctive morphological features and it is impossible to establish the affiliation between the larval and adult stages by using morphological criteria. Molecular methods, based on DNA sequencing or PCR-restriction fragment length polymorphism analysis, can offer a new tool for larval-stage identification. In this study, the sequences of internal transcribed spacer of the ribosomal DNA were used to identify the cercariae of three out of five species of the family Mesometridae (Centroderma spinosissima, Elstia stossichianum and Wardula capitellata). The three species differ from one another by number of repeats in the region of internal transcribed spacer 1. The phylogeny of Mesometridae was inferred from their internal transcribed spacer ribosomal DNA sequences. The PCR-linked restriction fragment length polymorphism approach was developed for future life-cycle and ecological studies of this family.

Molecular phylogeny of Mesometridae (Trematoda, Digenea) with its relation to morphological changes in parasites.

Complete ITS (Internal Transcribed Spacer) ribosomal DNA sequences were obtained for the six species know at present time within the Mesometridae Poche, 1926. The adult stages are intestinal parasites of herbivorous sparid teleosts. Aligned sequences were analysed with Maximum Parsimony, Maximum Likelihood and Neighbor-Joining phylogenetic methods to infer evolutionary relationships among mesometrid species. The ITS-based phylogeny obtained showed the two Wardula species as a sister group to other Mesometridae, and as compared to morphological data, suggest some general tendencies in the morphological evolution of this group. It consists mainly in changes from elongated to subcircular forms, regression of the pharynx, and the development of a strong accessory attachment organ.