Mechanically resolved imaging of bacteria using expansion microscopy.

Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (µExM) in which bacterial cells are physically expanded prior to imaging. We find that expansion patterns depend on the structural and mechanical properties of the cell wall, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of µExM to distinguish species through an in vitro defined community of human gut commensals and in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.

Chemosynthetic symbiont with a drastically reduced genome serves as primary energy storage in the marine flatworm Paracatenula.

Hosts of chemoautotrophic bacteria typically have much higher biomass than their symbionts and consume symbiont cells for nutrition. In contrast to this, chemoautotrophic Candidatus Riegeria symbionts in mouthless Paracatenula flatworms comprise up to half of the biomass of the consortium. Each species of Paracatenula harbors a specific Ca Riegeria, and the endosymbionts have been vertically transmitted for at least 500 million years. Such prolonged strict vertical transmission leads to streamlining of symbiont genomes, and the retained physiological capacities reveal the functions the symbionts provide to their hosts. Here, we studied a species of Paracatenula from Sant'Andrea, Elba, Italy, using genomics, gene expression, imaging analyses, as well as targeted and untargeted MS. We show that its symbiont, Ca R. santandreae has a drastically smaller genome (1.34 Mb) than the symbiont´s free-living relatives (4.29-4.97 Mb) but retains a versatile and energy-efficient metabolism. It encodes and expresses a complete intermediary carbon metabolism and enhanced carbon fixation through anaplerosis and accumulates massive intracellular inclusions such as sulfur, polyhydroxyalkanoates, and carbohydrates. Compared with symbiotic and free-living chemoautotrophs, Ca R. santandreae's versatility in energy storage is unparalleled in chemoautotrophs with such compact genomes. Transmission EM as well as host and symbiont expression data suggest that Ca R. santandreae largely provisions its host via outer-membrane vesicle secretion. With its high share of biomass in the symbiosis and large standing stocks of carbon and energy reserves, it has a unique role for bacterial symbionts-serving as the primary energy storage for its animal host.

Helmintosis y eosinofilia en España (1990-2015) / Helminthosis and eosinophilia in Spain (1990-2015)

La detección de eosinofilia periférica es un motivo relativamente frecuente para la remisión de un paciente a una Unidad/Servicio de Enfermedades Infecciosas. En general, se pretende descartar una enfermedad parasitaria, tanto en personas autóctonas como en viajeros o inmigrantes. Excepcionalmente la eosinofilia relacionada con parásitos corresponde a una protozoosis, siendo los helmintos los principales agentes causales de este hallazgo hematológico. La eosinofilia puede ser el único hallazgo anormal o formar parte del cuadro clínico-biológico del paciente. Por otro lado, no todas las helmintosis se asocian de forma sistemática a eosinofilia, y el grado de la misma difiere entre las fases de la infección y el tipo de helminto. El propósito de esta revisión es un estudio sistemático de la relación entre helmintosis y eosinofilia en la literatura española, distinguiendo los casos autóctonos e importados, así como la relación con situaciones de inmunodepresión (AU)

The finding of blood eosinophilia in a patient is a relatively frequent reason to refer him/her to a Clinical Department of Infectious Diseases. The doctor usually intends to rule out a parasitic disease in the autochthonous population, travelers or immigrants. It is uncommon for an eosinophilia to be produced by protozoa infection, whereas helminth parasites are more frequently associated with an increase of eosinophil counts in the infected patient. Eosinophilia can be the only abnormal finding, or it could be part of more complex clinical manifestations suffered by the patient. Furthermore, many, but not all, helminth infections are associated with eosinophilia, and the eosinophil level (low, high) differs according to parasite stages, helminth species, and worm co-infections. The purpose of the present article is to carry out a systematic review of cases and case series on helminth infections and eosinophilia reported in Spain from 1990 to 2015, making a distinction between autochthonous and imported (immigrants and travelers) cases, and studying their relationship with immunodepression situations (AU)

Biological and Chemical Diversity of Bacteria Associated with a Marine Flatworm.

The aim of this research is to explore the biological and chemical diversity of bacteria associated with a marine flatworm Paraplanocera sp., and to discover the bioactive metabolites from culturable strains. A total of 141 strains of bacteria including 45 strains of actinomycetes and 96 strains of other bacteria were isolated, identified and fermented on a small scale. Bioactive screening (antibacterial and cytotoxic activities) and chemical screening (ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)) yielded several target bacterial strains. Among these strains, the ethyl acetate (EA) crude extract of Streptomyces sp. XY-FW47 fermentation broth showed strong antibacterial activity against methicillin-resistant Staphylococcus aureus ATCC43300 (MRSA ATCC43300) and potent cytotoxic effects on HeLa cells. The UPLC-MS spectral analysis of the crude extract indicated that the strain XY-FW47 could produce a series of geldanamycins (GMs). One new geldanamycin (GM) analog, 4,5-dihydro-17-O-demethylgeldanamycin (1), and three known GMs (2-4) were obtained. All of these compounds were tested for antibacterial, cytotoxic, and antifungal activities, yet only GM (3) showed potent cytotoxic (HeLa cells, EC50 = 1.12 µg/mL) and antifungal (Setosphaeria turcica MIC = 2.40 µg/mL) activities. Their structure-activity relationship (SAR) was also preliminarily discussed in this study.

Deinococcus planocerae sp. nov., isolated from a marine flatworm.

A Gram-positive, non-motile and coccoid strain, designated XY-FW106T, was isolated from a marine flatworm identified to be Planocera sp. The 16S rRNA gene sequence of this pink organism was consistent with membership of the genus Deinococcus, with high sequence similarity to Deinococcus aetherius ST0316T (94.7%). The optimum growth temperature range of the strain XY-FW106T was found to be 25-30 °C and optimum growth occurs at pH 7.2-7.4 without NaCl. The strain XY-FW106T was found to contain unidentified glycolipids, unidentified phosphoglycolipids, unidentified phospholipids and unidentified lipids, which differed from those of closely related species. Menaquinone MK-8 was identified as the major respiratory quinone and the predominant cellular fatty acids were found to be Summed Feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0, iso-C15:0, and Summed Feature 8 (C18:1 ω7c/C18:1 ω6c). The DNA G+C content was determined to be 70.2 mol%. The biochemical and chemotaxonomic data together suggest that the strain represents a new species for which the name Deinococcus planocerae sp. nov. is proposed. The type strain is XY-FW106T (=MCCC 1K01499T=KCTC 33809T).

Real-time PCR detection and phylogenetic relationships of Neorickettsia spp. in digeneans from Egypt, Philippines, Thailand, Vietnam and the United States.

Neorickettsia (Rickettsiales, Anaplasmataceae) is a genus of obligate intracellular bacterial endosymbionts of digeneans (Platyhelminthes, Digenea). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of domestic animals, wildlife, and humans. In this study we report the results of screening digenean samples for Neorickettsia collected from bats in Egypt and Mindoro Island, Philippines, snails and fishes from Thailand, and fishes from Vietnam and the USA. Neorickettsia were detected using a real-time PCR protocol targeting a 152bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1853bp long region of the GroESL operon and a 1371bp long region of 16S rRNA. Eight unique genotypes of Neorickettsia were obtained from digenean samples. Neorickettsia sp. 8 obtained from Lecithodendrium sp. from Egypt; Neorickettsia sp. 9 and 10 obtained from two species of Paralecithodendrium from Mindoro, Philippines; Neorickettsia sp. 11 from Lecithodendrium sp. and Neorickettsia sp. 4 (previously identified from Saccocoelioides lizae, from China) from Thailand; Neorickettsia sp. 12 from Dicrogaster sp. Florida, USA; Neorickettsia sp. 13 and SF agent from Vietnam. Sequence comparison and phylogenetic analysis demonstrated that the forms, provisionally named Neorickettsia sp. 8-13, represent new genotypes. We have for the first time detected Neorickettsia in a digenean from Egypt (and the African continent as a whole), the Philippines, Thailand and Vietnam based on PCR and sequencing evidence. Our findings suggest that further surveys from the African continent, SE Asia, and island countries are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

Pathogenic shifts in endogenous microbiota impede tissue regeneration via distinct activation of TAK1/MKK/p38.

The interrelationship between endogenous microbiota, the immune system, and tissue regeneration is an area of intense research due to its potential therapeutic applications. We investigated this relationship in Schmidtea mediterranea, a model organism capable of regenerating any and all of its adult tissues. Microbiome characterization revealed a high Bacteroidetes to Proteobacteria ratio in healthy animals. Perturbations eliciting an expansion of Proteobacteria coincided with ectopic lesions and tissue degeneration. The culture of these bacteria yielded a strain of Pseudomonas capable of inducing progressive tissue degeneration. RNAi screening uncovered a TAK1 innate immune signaling module underlying compromised tissue homeostasis and regeneration during infection. TAK1/MKK/p38 signaling mediated opposing regulation of apoptosis during infection versus normal tissue regeneration. Given the complex role of inflammation in either hindering or supporting reparative wound healing and regeneration, this invertebrate model provides a basis for dissecting the duality of evolutionarily conserved inflammatory signaling in complex, multi-organ adult tissue regeneration.

Pseudovibrio stylochi sp. nov., isolated from a marine flatworm.

A Gram-stain-negative, motile, rod-shaped bacterial strain, designated UST20140214-052T, was isolated from a marine flatworm (polyclad). The bacterium was found to be catalase-positive and weakly oxidase-positive, and motile by means of several peritrichous or subpolar flagella. Strain UST20140214-052T grew optimally at 28 °C, at pH 7.0 and in the presence of 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain UST20140214-052T belongs to the genus Pseudovibrio, with highest sequence similarity to Pseudovibrio hongkongensis UST20140214-015BT (98.8 %), followed by Pseudovibrio japonicus WSF2T (96.2 %), Pseudovibrio ascidiaceicola F423T (96.2 %), Pseudovibrio denitrificans DN34T (95.9 %), Pseudovibrio axinellae Ad2T (95.9 %). All the other species shared < 95.5 % sequence similarity. The DNA-DNA hybridization estimate value between strain UST20140214-052T and P. hongkongensis UST20140214-015BT was 24.7 ± 2.4 %. The major fatty acid was summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c, as defined by the MIDI system; 62.6 %). The DNA G+C content was 47.0 mol%. The combined genotypic and phenotypic data show that strain UST20140214-052T represents a novel species within the genus Pseudovibrio, for which the name Pseudovibrio stylochi sp. nov. is proposed, with the type strain UST20140214-052T ( = KCTC 42384T = MCCC 1K00452T).

Pseudovibrio hongkongensis sp. nov., isolated from a marine flatworm.

A Gram-negative, motile, rod-shaped bacterial strain, designated UST20140214-015B(T), was isolated from a marine flatworm (Polyclad). The bacterium was found to be Gram-negative, oxidase and catalase positive, long-rod shaped, and motile by means of several peritrichous or subpolar flagella. Strain UST20140214-015B(T) grew optimally at 28 °C, at pH 7.0 and in the presence of 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain UST20140214-015B(T) belongs to the genus Pseudovibrio, with highest sequence similarity to Pseudovibrio japonicus WSF2(T) (96.9 %), followed by P. ascidiaceicola F423(T) (96.7 %), P. denitrificans DN34(T) (96.6 %), P. axinellae Ad2(T) (96.3 %). All the other species shared <95.5 % sequence similarity. The principal fatty acids were identified as summed feature 8 (C18:1 ω7c/ω6c, as defined by the MIDI system; 70.8 %), C16:0 (9.1 %), summed feature 3 (C16:1 ω6c/C16:1 ω7c; 5.7 %). The G+C content of the chromosomal DNA was determined to be 53.3 mol%. The combined genotypic and phenotypic data show that strain UST20140214-015B(T) represents a novel species within the genus Pseudovibrio, for which the name Pseudovibrio hongkongensis sp. nov. is proposed, with the type strain UST20140214-015B(T) (=KCTC 42383(T) = MCCC 1K00451(T)).

No evidence for a culturable bacterial tetrodotoxin producer in Pleurobranchaea maculata (Gastropoda: Pleurobranchidae) and Stylochoplana sp. (Platyhelminthes: Polycladida).

Tetrodotoxin (TTX) is a potent neurotoxin found in the tissues of many taxonomically diverse organisms. Its origin has been the topic of much debate, with suggestions including endogenous production, acquisition through diet, and symbiotic bacterial synthesis. Bacterial production of TTX has been reported in isolates from marine biota, but at lower than expected concentrations. In this study, 102 strains were isolated from Pleurobranchaea maculata (Opisthobranchia) and Stylochoplana sp. (Platyhelminthes). Tetrodotoxin production was tested utilizing a recently developed sensitive method to detect the C9 base of TTX via liquid chromatography-mass spectrometry. Bacterial strains were characterized by sequencing a region of the 16S ribosomal RNA gene. To account for the possibility that TTX is produced by a consortium of bacteria, a series of experiments using marine broth spiked with various P. maculata tissues were undertaken. Sixteen unique strains from P. maculata and one from Stylochoplana sp. were isolated, representing eight different genera; Pseudomonadales, Actinomycetales, Oceanospirillales, Thiotrichales, Rhodobacterales, Sphingomonadales, Bacillales, and Vibrionales. Molecular fingerprinting of bacterial communities from broth experiments showed little change over the first four days. No C9 base or TTX was detected in isolates or broth experiments (past day 0), suggesting a culturable microbial source of TTX in P. maculata and Stylochoplana sp. is unlikely.

Neorickettsial endosymbionts of the digenea: diversity, transmission and distribution.

Digeneans are endoparasitic flatworms with complex life cycles and distinct life stages that parasitize different host species. Some digenean species harbour bacterial endosymbionts known as Neorickettsia (Order Rickettsiales, Family Anaplasmataceae). Neorickettsia occur in all life stages and are maintained by vertical transmission. Far from benign however, Neorickettsia may also be transmitted horizontally by digenean parasites to their vertebrate definitive hosts. Once inside, Neorickettsia can infect macrophages and other cell types. In some vertebrate species (e.g. dogs, horses and humans), neorickettsial infections cause severe disease. Taken from a mostly parasitological perspective, this article summarizes our current knowledge on the transmission ecology of neorickettsiae, both for pathogenic species and for neorickettsiae of unknown pathogenicity. In addition, we discuss the diversity, phylogeny and geographical distribution of neorickettsiae, as well as their possible evolutionary associations with various groups of digeneans. Our understanding of neorickettsiae is at an early stage and there are undoubtedly many more neorickettsial endosymbioses with digeneans waiting to be discovered. Because neorickettsiae can infect vertebrates, it is particularly important to examine digenean species that regularly infect humans. Rapid advances in molecular tools and their application towards bacterial identification bode well for our future progress in understanding the biology of Neorickettsia.

Bacterial symbiosis maintenance in the asexually reproducing and regenerating flatworm Paracatenula galateia.

Bacteriocytes set the stage for some of the most intimate interactions between animal and bacterial cells. In all bacteriocyte possessing systems studied so far, de novo formation of bacteriocytes occurs only once in the host development, at the time of symbiosis establishment. Here, we present the free-living symbiotic flatworm Paracatenula galateia and its intracellular, sulfur-oxidizing bacteria as a system with previously undescribed strategies of bacteriocyte formation and bacterial symbiont transmission. Using thymidine analogue S-phase labeling and immunohistochemistry, we show that all somatic cells in adult worms - including bacteriocytes - originate exclusively from aposymbiotic stem cells (neoblasts). The continued bacteriocyte formation from aposymbiotic stem cells in adult animals represents a previously undescribed strategy of symbiosis maintenance and makes P. galateia a unique system to study bacteriocyte differentiation and development. We also provide morphological and immunohistochemical evidence that P. galateia reproduces by asexual fragmentation and regeneration (paratomy) and, thereby, vertically transmits numerous symbiont-containing bacteriocytes to its asexual progeny. Our data support the earlier reported hypothesis that the symbiont population is subjected to reduced bottleneck effects. This would justify both the codiversification between Paracatenula hosts and their Candidatus Riegeria symbionts, and the slow evolutionary rates observed for several symbiont genes.

Virus-like particles in a monogenean (Platyhelminthes) parasitic in a marine fish.

In Microcotyle sp., a gill parasite in the marine fish Abudefduf analogus near Dakar, Senegal, various organs were studied by transmission electron microscopy. One of the six worms studied contained virus-like particles located only in the outer layer and in the cytons (deep cell bodies) of the tegument. The tegument ultrastructure is described for both healthy and infected monogeneans. The outer layer of the tegument, 2-5 microns thick, does not have microvilli. The virus-like particles are cytoplasmic, about 70 nm in diameter with a single-layered capsid-like structure 10-12 nm thick, and have an icosahedral symmetry. They originate from viroplasms and accumulate in paracrystalline arrays up to 1 micron in size. By their ultrastructural characteristics, these virus-like particles are related to the Reoviridae or, more probably, the Birnaviridae. This is the third report of viruses in monogeneans, and the first in a polyopisthocotylean monogenean. It is hypothesized that monogeneans could act as vectors of viral diseases among their host fishes.

Rickettsiae and giant lysosomes in the testes of Temnocephala novaezealandiae (Platyhelminthes: Temnocephaloidea).

Rickettsiae are concentrated in the testes of Temnocephala novaezealandiae, where they occupy the cytoplasm of spermatogenetic stages and the testis epithelia. They have gram negative ultrastructural characteristics and are surrounded by clear zones which are not membrane-limited; indented mitochondria are associated with the clear zones. Propagation occurs by simple division. Extensions of the testis epithelium envelop cloned groups of spermatocytes, spermatids and spermatozoa. Heterophagosomes isolating unmodified rickettsiae, and giant lysosomes enclosing bacteria in advanced stages of degradation, are contained within the epithelial processes. Rickettsiae and large lysosomes were found also in testes of Troglocaridicola mrazeki, a scutariellid. Peririckettsial lucid zones are interpreted as areas of histopathy where host cytoplasm has been catabolized by bacterial enzymes. The rickettsiae evidently are true parasites, although they have no apparent harmful effect on the general condition of their hosts. Digestion of bacteria in lysosomes may provide nutrients for spermatogenetic stages. Transmission of rickettsiae to juvenile temnocephalids does not occur via the spermatozoa.