Influence of tides on the dissemination and related health risks of intestinal helminths along the Kribi beaches (Atlantic Coast, Southern Cameroon).

Kribi is a seaside town that welcomes thousands of tourists each year. However, the poor sanitation condition of its beaches along the Atlantic coast is not without risk for visitors. In this study, we used the formol-ether concentration technique to identify and quantify larvae or eggs of intestinal helminths in waters of three regularly visited Kribi beaches (Mpalla, Ngoyè, and Mboamanga). Results revealed that all identified larvae and eggs were cestodes (Hymenolepis nana) and nematodes (Strongyloides sp., Ascaris sp., Ancylostoma duodenale and Trichuris trichiura). All the helminth eggs and larvae showed high abundance at low tide during rainy seasons. Ancylostoma duodenale eggs, totally absent at Mpalla, were densely present at low tide at Ngoyè (301 ± 15 eggs/L). Trichuris trichiura eggs showed the lowest abundance (0 to 62 eggs/L) at all sites. Abiotic variables indicated that waters at the various beaches were basic (pH: 8.75-9.77), generally warmer (32.44°C at Mpalla in the Short Rainy Season), more oxygenated at low tide, and moderately mineralized at high tide. Positive and significant correlations were observed at Ngoyè at low tide between Strongyloides sp. larvae and dissolved oxygen (P ˂ 0.05); and between Ancylostoma duodenale eggs and temperature (P ˂ 0.05). The overall results indicated that the beaches studied are subjected to fecal pollution. This pollution is more accentuated during low tides than during high tides. Depending on tidal movements, swimmers risk exposure to helminth eggs and larvae known to be responsible for gastroenteritis.

Biofilms in hospital effluents as a potential crossroads for carbapenemase-encoding strains.

Bacterial resistance to carbapenem, which is mainly due to the successful dissemination of carbapenemase-encoding genes, has become a major health problem. Few studies have aimed to characterize the level of resistance in the environment, notably in hospital wastewater, which is a likely hotspot for exchange of antibiotic resistance genes. In this work, we looked for the presence of imipenem-resistant bacteria and imipenem in the effluent of the teaching hospital of Clermont-Ferrand, France. Selective growth of bacteria from 14-day old biofilms formed in the pipe sewer showed that 22.1% of the isolates were imipenem-resistant and identified as Aeromonas (n = 23), Pseudomonas (n = 10), Stenotrophomonas (n = 4) and Acinetobacter (n = 1). Fifteen of these strains harbored acquired carbapenemase-encoding genes blaVIM (n = 11), blaOXA-48 (n = 2), blaGES (n = 1), blaNDM (n = 1). All isolates also harbored associated resistances to aminoglycosides, fluoroquinolones and/or tetracyclin. S1-nuclease pulsed-field gel electrophoresis analysis of eight selected isolates showed that four of them harbored one to two plasmids of molecular weight between 48.5 Kb and 194 Kb. In vitro transformation assays evidenced the presence of blaVIM and blaNDM on plasmids with the blaVIM harboring 80 Kb plasmid having conjugative capacity. The predicted environmental concentration of imipenem in the hospital effluent was 3.16 µg/L, suggesting that biofilm bacteria are subjected to sub-MICs of imipenem within the effluent. However, no imipenem molecule was detected in the hospital effluent, probably owing to its instability: in vitro assays indicated that imipenem's biological activity was no longer detectable after 45 h of storage. However, the predictive value of the hazard quotient relative to the development of resistance was >1.0 (HQr = 28.9 ±â€¯1.9), which indicates a possible risk. The presence of carbapenemase-encoding genes in hospital effluent biofilm strains and their ability to transfer are therefore a potential hazard that should not be neglected and points to the need for monitoring antibiotic resistance in hospital wastewater.

[Revision of of the subfamily of Metaracoelophryinae de Puytorac 1972 (Oligohymenophora: Hoplytophryida: Hoplytophryidae), astome ciliates of the digestive tract of Oligochaeta worms of Africa: description of five new species]. / Révision de la sous-famille des Metaracoelophryinae de Puytorac 1972 (Oligohymenophora : Hoplytophryida : Hoplytophryidae), ciliés astomes du tube digestif d'oligochètes terricoles d'Afrique : description de cinq espèces nouvelles.

Five new species belonging to the astome ciliates, living in the digestive tract of Oligochaeta worms belonging to the genus Alma from Cameroon, have been described. The techniques used are: vital staining, staining of the nucleus with Diamidino Phenyl Indol (DAPI), scanning electron microscopy and silver staining method (Fernandez Galiano, 1976, 1994). This work confirms the presence of the genus Paracoelophrya and Dicoelophrya in the digestive track of the oligochaete Alma from Gabon and Cameroon; it helps to understand the general taxonomy of this Metaracoelophryinae subfamily. Moreover, the homogeneity of this group is confirmed and the phylogenetic relationship inside the Hoplitophryida order need more studies to be solved.

[Morphological variations of the nuclear apparatus of astome ciliates Almophrya bivacuolata and A. maediovacuolata (protozoa: ciliophora) endocommensal of terricolous oligochaetes in Cameroon]. / Variations morphologiques de l'appareil nucléaire chez les ciliés Astomes Almophrya bivacuolata et A. mediovacuolata (protozoa: ciliophora) endocommensaux d'oligochites terricoles du Cameroun.

The silver impregnation supplemented by DAPI and Feulgen nuclear coloration enabled us to study the morphological variations of the nuclear apparatus of two species of endocommensal Astome ciliates, Almophrya bivacuoloata (de Puytorac & Dragesco, 1968) and A. mediovocuolata (Ngassam, 1983). We highlighted important digitations and the presence of dark bands in the structure of the "H" macronucleus of the small cellular types as well as the presence of intermediate forms between "H" and "X" in these two species.

Characterization, cloning and immunolocalization of a coronin homologue in Trichomonas vaginalis.

On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments. We have undertaken the identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.

Centrin protein and genes in Trichomonas vaginalis and close relatives.

Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Clamydomononas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina. Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina. Centrin was not demonstrated in the dividing spindle and paradesmosis. Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma-membrane. Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta-axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules. There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T. vaginalis. Two proteins of 22-20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined. By screening a T. vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found. The sequence comprises the 4 typical EF-hand Ca++-binding domains present in every known centrin. Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity).

Immunolocalization of two hydrogenosomal enzymes of Trichomonas vaginalis.

Three monoclonal antibodies specific for malic enzyme and for the alpha- and beta-subunits, respectively, of the succinyl-coenzyme A (CoA) synthetase of Trichomonas vaginalis were used to immunolocalize these proteins in the cell. All antibodies labeled the hydrogenosome matrix as determined both by immunofluorescence and by immunogold staining. There was no labeling on the cell surface or in any other cell compartment. These results support the idea that these proteins are restricted to a hydrogenosomal function and do not play a role as adhesins at the plasma membrane surface.

Evidence for an uncommon alpha-actinin protein in Trichomonas vaginalis.

As part of our ongoing project of identification of actin-binding proteins implicated in the cell transition (flagellate to amoeboid/adherent) of Trichomonas vaginalis, we have characterized an alpha-actinin-related protein in this parasite. The protein (P100) has a molecular mass of 100 kDa and an isoelectric point of 5.5. A monoclonal antibody raised against this protein co-localizes with the actin network. P100 gene transcripts are co-expressed with actin throughout the cell cycle. Analysis of the deduced protein sequence reveals three domains: an N-terminal actin-binding region; a central region rich in alpha-helix; and a C-terminal domain with Ca(2+)-binding capacity. Whereas the N- and C-terminal regions are well-conserved as compared to other alpha-actinins, we observe in the central region an atypical distribution of residues in five repeats. The sequence of the repeats does not show any homology with the rod domain of the other alpha-actinins, except for the first repeat which shows some similarity. The four other repeats of T. vaginalis P100 appear to result from a duplication event which is not detectable in the other sequences.

Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences.

The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.

Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads.

The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads.

Evidence for common epitopes among proteins of the membrane skeleton of a ciliate, an euglenoid and a dinoflagellate.

The cytoskeleton of many protists comprises an extensive submembranous epiplasm which contributes to cell shape and integration of cell membranes with underlying structures according to the species-specific cortical architecture. Using various extraction procedures, epiplasm-enriched fractions have been isolated from the ciliate Pseudomicrothorax dubius, the euglenoid Euglena acus and the dinoflagellate Noctiluca scintillans. Comparative gel electrophoretic analysis of such preparations reveals heterogeneity of protein composition, the major polypeptides differing in size. Antibodies raised against epiplasmic proteins from these three organisms have permitted the confirmation of submembranous localization of the antigens by immunoelectron microscopy. Heterologous reactions performed by means of combined immunocytochemical and immunoblotting procedures indicate the existence of common epitopes among major proteins making up the bulk of the epiplasm of the three species examined. These findings suggest that proteins of the epiplasm have significantly diverged during evolution while conserving structural domains essential for their cytoskeletal function. It is postulated that these common domains may underly the ability of epiplasmic proteins to assemble into an ordered spatial organization, typical of the highly differentiated cortex of unicellular micro-organisms.