Bacteriolytic activity in saliva of the hematophagous Triatoma infestans (Reduviidae) and novel characterization and expression site of a third lysozyme.

Saliva of hematophagous insects contains many different compounds, mainly acting as anticoagulants. Investigating the bacteriolytic compounds of the saliva of the bloodsucking Triatoma infestans photometrically between pH 3 and pH 10 using unfed fifth instars and nymphs up to 15 days after feeding, we found bacteriolytic activity against lyophilized Micrococcus luteus was stronger at pH 4 and pH 6. After feeding, the activity level at pH 4 was unchanged, but at pH 6 more than doubled between 3 and 7 days after feeding. In zymographs of the saliva and after incubation at pH 4, bacteriolytic activity against Micrococcus luteus was present at eight lysis zones between 14.1 and 38.5 kDa, showing the strongest activity at 24.5 kDa. After incubation at pH 6, lysis zones only appeared at 15.3, 17, and 31.4 kDa. Comparing zymographs of the saliva of unfed and fed nymphs, bacteriolytic activity at 17 kDa increased after feeding. In total nine lysis bands appeared, also at >30 kDa, so far unreported in the saliva of triatomines. Reverse transcription polymerase chain reaction using oligonucleotides based on the previously described lysozyme gene of T. infestans, TiLys1, verified expression of genes encoding TiLys1 and TiLys2 in the salivary glands, but also of an undescribed third lysozyme, TiLys3, of which the cloned cDNA shares characteristics with other c-type lysozymes of insects. While TiLys1 was expressed in the tissue of all three salivary glands, transcripts of TiLys2 and of TiLys3 seem to be present only in the gland G1 and G3, respectively.

Vermamoeba vermiformis as etiological agent of a painful ulcer close to the eye.

In the present article, we report on the identification of Vermamoeba (Hartmannella) vermiformis as the etiological agent of a tissue infection close to the eye of a female patient. Laboratory examination revealed no involvement of any pathogenic bacteria or fungi in the tissue infection. V. vermiformis was identified by cultivation and morphology of trophozoites and cysts as well as phylogenetic analysis of nuclear 18S rDNA. The lesion improved in the course of 4 weeks by application of zinc paste.

Lyophilisation as a simple and safe method for long-term storage of free-living amoebae at ambient temperature.

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. As some species or strains of these FLA are pathogenic for humans and animals, they represent objects of medical and parasitological research worldwide. Storage of valuable FLA strains in laboratories is often time- and energy-consuming and expensive. The shipment of such strains as frozen stocks is cumbersome and challenging in terms of cooling requirements as well as of transport regulations. To overcome these difficulties and challenges in maintenance and transport, we present a new method to generate lyophilised samples of non-cyst-forming FLA (Ripella (Vannella) spp.) and cyst-forming FLA (Acanthamoeba spp.) strains which guarantees a simple mechanism for long-term storage at ambient temperature, as well as easy handling and/or shipment. The survival rate of all FLA lyophilisates after short-term storage (2 months) was comparable to the survival rate of freeze cultures of the respective strains. Furthermore, the viability of Acanthamoeba spp. cysts after storage for 29 months was 20 to 40% following lyophilisation and rehydration, with strain variation.

Failure of molecular diagnostics of a keratitis-inducing Acanthamoeba strain.

An otherwise healthy 49-year-old female patient presented at the local hospital with severe keratitis in both inflamed eyes. She was a contact lens wearer and had no history of a corneal trauma. In our laboratory for medical parasitology Acanthamoebae were detected microscopically from the cornea scraping and from the fluid of the contact lens storage case after xenical culture and showed the typical cyst morphology of Acanthamoebae group II. The diagnosis of "Acanthamoeba keratitis" was established and successful therapy was provided. While the morphological microscopic method led to the correct diagnosis in this case, an in-house multiplex qPCR and a commercial qPCR showed false negative results regarding Acanthamoeba sp. The subsequent sequencing revealed the Acanthamoeba genotype T4. In the present case report, the inability to detect Acanthamoebae using qPCR only is presented. Therefore, we recommend the utilization of combined different assays for optimal diagnostic purposes.

Detection of Balamuthia mandrillaris DNA in the storage case of contact lenses in Germany.

Acanthamoeba spp. are frequently the etiological agents of a severe form of sight-threatening keratitis, called Acanthamoeba keratitis. The contact lens storage solution of a patient with keratitis of unknown genesis was screened using our diagnostic tools to detect potentially pathogenic free-living amoebae (FLA). Culture methods and a triplex quantitative real-time polymerase chain reaction (qPCR) targeting Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris were used in context of this routine screening. While no amoebae were detected by culture, qPCR specifically detected DNA of B. mandrillaris. This FLA is known as the etiological agent of a fatal form of encephalitis in humans and other mammals, Balamuthia amoebic encephalitis (BAE). A fragment of the 18S rDNA gene was amplified from the sample and showed 99 % sequence identity to B. mandrillaris sequences from GenBank. To the best of our knowledge, this is the first report of B. mandrillaris found in association with contact lenses. Although no viable amoeba was obtained by culturing efforts, the verification of B. mandrillaris DNA in the contact lens storage solution demonstrates how easily this pathogen might come into close contact with humans.

Rhinosporidiosis in African reed frogs Hyperolius spp. caused by a new species of Rhinosporidium.

We report the identification of a new Rhinosporidium species (Dermocystida, Mesomycetozoea) infecting amphibian hosts, while showing a species specificity for African reed frogs of the genus Hyperolius. Large dermal cysts (sporangia) of R. rwandae sp. nov. were observed in 18% of H. lateralis and similar cysts in 0.7% of H. viridiflavus surveyed. Fully developed R. rwandae cysts are about 500 to 600 µm in diameter and sealed from the frog tissue by a thick chitinous wall. Some cysts were filled with numerous round-oval basophilic microspores of 8 to 12 µm diameter. With the exception of legs, nodules were visible over the complete torso surface including the vocal sac of males, but the most affected skin region was the area around the cloaca. Behavior, condition, and lifespan of infected frogs do not seem to be distinct from that of healthy individuals. The mode of infection remains unknown, but we hypothesize that the infectious life stage reaches the dermis via the intraepidermal ducts of the skin glands. Molecular evidence places the new frog pathogen as a sister species of the human pathogen R. seeberi.

Some secrets are revealed: parasitic keratitis amoebae as vectors of the scarcely described pandoraviruses to humans.

In this article, the results of a long effort to derive valuable phylogenetic data about an extraordinary spore-like infectious particle (endocytobiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens and the inflamed eye of a patient with keratitis are presented. The development of these endocytobionts has already been demonstrated with electron microscopic photo sequences, leading to a relevant model of its development presented here. The molecular biological investigation following the discovery of two other Pandoravirus species within aquatic sediments in 2013 led to the taxonomic affiliation of our endocytobiont with the genus Pandoravirus. A range of endocytobionts (intracellular biofilms) have been found in recent years, among which are several viruses which obligatorily proliferate within free-living amoebae. In human medicine, foreign objects which are placed in or on humans cause problems with microorganisms in biofilms. Contact lenses are especially important, because they are known as a source of a rapid formation of biofilm. These were the first Pandoraviruses described, and because this is additionally the first documented association with humans, we have clearly demonstrated how easily such (viral) endocytobionts can be transferred to humans. This case counts as an example of parasites acting as vectors of phylogenetically different microorganisms especially when living sympatric within their biocoenosis of biofilms. As the third part of the "Pandoravirus trilogy", it finally reveals the phylogenetic nature of these "extraordinary endocytobionts" within Acanthamoebae.

A Case of Pseudomonas straminea Blood Stream Infection in an Elderly Woman with Cellulitis.

Here, we report the simultaneous isolation of Pseudomonas straminea from blood cultures and from a skin ulcer in an elderly woman who suffered from atopic dermatitis and psoriasis and developed acute cellulitis of both arms requiring hospital treatment. To the best of our knowledge, P. straminea has not been previously reported to cause invasive infections in humans. This case highlights how chronic diseases and older age increase the susceptibility to bacterial infections with environmental bacteria of low virulence. Our study describes the microbiological identification of the blood culture isolate, including morpho-molecular characterization and virulence demonstration in a Galleria mellonella model.

Repeated introduction of Aedes albopictus into Germany, July to October 2012.

During a small-scale surveillance project to identify possible routes of entry for invasive mosquitoes into Germany, 14 adult Aedes (Stegomyia) albopictus (Skuse) were discovered between July and October 2012. They were trapped at three different service stations in Bavaria and Baden-Wuerttemberg located along two motorways that connect Germany with southern Europe. This indicates regular introduction of A. albopictus into Germany and highlights the need for a continuous surveillance and control programme.

Molecular detection of Leishmania (Sauroleishmania) adleri (Trypanosomatida: Trypanosomatidae) in Sergentomyia sp. sand flies (Diptera: Psychodidae) in Mali and Niger.

Phlebotomine sand flies of the genus Sergentomyia are considered to be of minor importance as vectors of Leishmania parasites pathogenic to humans, but are known to transmit lizard parasites of the subgenus Sauroleishmania, including L. (S.) adleri. However, knowledge on the geographic distribution of Sauroleishmania spp. and the infection rates in the vectors is very limited. Therefore, our study aimed (1) to further elucidate the distribution and prevalence of Sauroleishmania spp. in their respective vectors and (2) to assess the potential risk for occasional transmission of Leishmania parasites to international military personnel deployed in camps in Mali and Niger. A total of 1,482 wild-caught sand flies (Sergentomyia spp. and closely related Grassomyia spp.) were screened by real-time PCR for the presence of Leishmania DNA. Thirty-two sand fly pools were tested positive, with six from Mali and 26 from Niger. The DNA of four representative isolates was sequenced. The resulting sequences revealed a homology to L. adleri, which leads to the first report of this species from Mali and Niger to the best of our knowledge. The results suggest that Sergentomyia (Sintonius) clydei might be the natural sand fly vector, while Grassomyia spp. appear to be refractory. No Leishmania sp. pathogenic to humans was detected in these sand flies.

Molecular Epidemiology of Escherichia coli with Resistance against Third-Generation Cephalosporines Isolated from Deployed German Soldiers-A Retrospective Assessment after Deployments to the African Sahel Region and Other Sites between 2007 and 2016.

Colonization and infection with bacteria with acquired antibiotic resistance are among the risks for soldiers on international deployments. Enterobacterales with resistance against third-generation cephalosporines are amongst the most frequently imported microorganisms. To contribute to the scarcely available epidemiological knowledge on deployment-associated resistance migration, we assessed the molecular epidemiology of third-generation cephalosporine-resistant Escherichia coli isolated between 2007 and 2016 from German soldiers after deployments, with a particular focus on the African Sahel region. A total of 51 third-generation cephalosporine-resistant E. coli isolated from 51 military returnees from deployment collected during the assessment period between 2007 and 2016 were subjected to short-read next-generation sequencing analysis. Returnees from the Sahel region (Djibouti, Mali, South Sudan, Sudan, Sudan, and Uganda) comprised a proportion of 52.9% (27/51). Repeatedly isolated sequence types according to the Warwick University scheme from returnees from the Sahel region were ST38, ST131, and ST648, confirming previous epidemiological assessments from various sub-Saharan African regions. Locally prevalent resistance genes in isolates from returnees from the Sahel region associated with third-generation resistance were blaCTX-M-15, blaCTX-M-27, blaCTX-M-1, blaTEM-169, blaCTX-M-14, blaCTX-M-99-like, blaCTX-M-125, blaSHV-12, and blaDHA-1, while virulence genes were east1, sat, and tsh in declining order of frequency of occurrence each. In line with phenotypically observed high resistance rates for aminoglycosides and trimethoprim/sulfamethoxazole, multiple associated resistance genes were observed. A similar, slightly more diverse situation was recorded for the other deployment sites. In summary, this assessment provides first next-generation sequencing-based epidemiological data on third-generation cephalosporine-resistant E. coli imported by deployed German soldiers with a particular focus on deployments to the Sahel region, thus serving as a small sentinel. The detected sequence types are well in line with the results from previous epidemiological assessments in sub-Saharan Africa.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Strains Isolated at the German Military Field Laboratory in Mazar-e Sharif, Afghanistan.

The study was performed to provide an overview of the molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in Afghanistan isolated by the German military medical service during the Afghanistan conflict. A total of 18 isolates were collected between 2012 and 2018 at the microbiological laboratory of the field hospital in Camp Marmal near Mazar-e Sharif, Afghanistan, from Afghan patients. The isolates were subjected to phenotypic and genotypic differentiation and antimicrobial susceptibility testing as well as to a core genome multi-locus sequence typing (cgMLST) approach based on whole-genome next-generation sequence (wgNGS) data. Next to several sporadic isolates, four transmission clusters comprising strains from the international clonal lineages IC1, IC2, and IC9 were identified. Acquired carbapenem resistance was due to blaOXA-23 in 17/18 isolates, while genes mediating resistance against sulfonamides, macrolides, tetracyclines, and aminoglycosides were frequently identified as well. In conclusion, the assessment confirmed both the frequent occurrence of A. baumannii associated with outbreak events and a variety of different clones in Afghanistan. The fact that acquired carbapenem resistance was almost exclusively associated with blaOXA-23 may facilitate molecular resistance screening based on rapid molecular assays targeting this resistance determinant.

Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection of Leishmania spp. in Human Serum.

To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results.

Molecular phylogeny of Megalobatrachonema (Nematoda: Ascaridida), with description of a new species based on morphological and molecular evidence.

Species of MegalobatrachonemaYamaguti, 1941 (Ascaridida: Cosmocercoidea) are important nematode parasites in amphibians and reptiles. However, the phylogenetic relationship of its included two subgenera Megalobatrachonema and Chabaudgolvania remains unclear. In the present study, a new species of Megalobatrachonema, M. (Chabaudgolvania) wangi sp. nov., was described based on the specimens collected from the lesser spiny frog Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura) in China. The ribosomal [large ribosomal DNA (28S) and internal transcribed spacer (ITS1-5.8S-ITS2)] and mitochondrial [12S small subunit ribosomal DNA and cytochrome c oxidase subunit 1 (cox1)] target regions of the new species and M. (Chabaudgolvania) terdentatum, together with the 12S region of M. (Megalobatrachonema) hainanensis, were amplified and sequenced for molecular identification and phylogeny. Moreover, in order to clarify the systematic position of the new species and the phylogenetic relationship of the two subgenera Megalobatrachonema and Chabaudgolvania, phylogenetic analyses based on 28S + ITS1-5.8S-ITS2 + 12S sequence data were performed using maximum likelihood (ML) inference and Bayesian inference (BI). The molecular phylogenetic results conflicted with the current classification and challenged the validity of the subgenus Chabaudgolvania, that should be a synonym of the subgenus Megalobatrachonema. The presence or absence of valves in the oesophageal bulb as a key criterion for delimitation of the two subgenera Megalobatrachonema and Chabaudgolvania seems to be unreliable.

Changes of the abundance of Culicoides obsoletus s.s. and Culicoides scoticus in Southwest Germany identified by a PCR-based differentiation.

The outbreak of bluetongue disease in Central Europe necessitates new approaches in the identification of vectors to follow-up changes of populations of species and not of complexes. Since females of species of the complex of Culicoides obsoletus are difficult to be identified according to morphological criteria, we applied a polymerase chain reaction (PCR)-based strategy targeting the mitochondrial cytochrome oxidase subunit I to differentiate between the species Culicoides obsoletus s.s. and Culicoides scoticus. Catches of culicoids obtained from May to November 2007 in an ultraviolet lamp trap at a cattle farm in Rhineland-Palatinate, Southern Germany were surveyed for changes of the abundance of both species. Only in May 2007, the samples contained similar proportions of both species. Afterwards, C. scoticus dominated with up to 88%. Calculating the number of specimens of both species within the total catches of culicoids, the numbers of C. obsoletus s.s. slightly decreased from May to July and increased to a little maximum in August. C. scoticus seemed to have three maxima in this period of time, the strongest one in August, presumably due to different generations and not to climatic conditions. These results indicate that the applied PCR strategy can be used for a detailed analysis of culicoids as basis for the estimation of the transmission risk of the bluetongue virus by different species of the Obsoletus complex.

Monitoring of ceratopogonidae in southwest Germany.

Within the entomological monitoring program of the German federal ministry of food, agriculture, and user protection (BMELV), at 12 cattle farms in Rhineland-Palatinate and two in Saarland, ultraviolet lamp traps were used to monitor the distribution and seasonal appearance of potential vectors of the bluetongue virus, with special consideration of species of Culicoides. Using the traps during the first seven nights of each month from April 2007 to May 2008, 5,000-120,000 ceratopogonids were caught at different locations, in total about 500,000 and mainly females. Ninety-four percent belonged to the genus Culicoides, and of these, 90% were Culicoides obsoletus s.l., 6% were Culicoides pulicaris s.l., and 4% were other species of this genus. In all traps, the first ceratopogonids were caught in April 2007, the total number peaking in August 2007. After a reduction in September, a lower peak occurred in October. During the whole winter, some ceratopogonids were active. At nearly all locations, the total numbers of C. obsoletus s.l., C. pulicaris s.l., and of other ceratopogonids were significantly correlated with the temperatures, and higher population densities of C. obsoletus s.l. seemed to occur at altitudes of about 300 m above sea level.

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii.

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont.

Free-Living Amoebae as Hosts for and Vectors of Intracellular Microorganisms with Public Health Significance.

Free-living amoebae (FLA) are parasites within both humans and animals causing a wide range of symptoms and act as hosts of, and vehicles for phylogenetically diverse microorganisms, called endocytobionts. The interaction of the FLA with sympatric microorganisms leads to an exceptional diversity within FLA. Some of these bacteria, viruses, and even eukaryotes, can live and replicate intracellularly within the FLA. This relationship provides protection to the microorganisms from external interventions and a dispersal mechanism across various habitats. Among those intracellularly-replicating or -residing organisms there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals and are therefore of interest to Public Health Authorities. Mimiviruses, Pandoraviruses, and Pithoviruses are examples for interesting viral endocytobionts within FLA. Future research is expected to reveal further endocytobionts within free-living amoebae and other protozoa through co-cultivation studies, genomic, transcriptomic, and proteomic analyses.

DNA macroarray and real-time PCR analysis of two nuclear photosystem I mutants from Chlamydomonas reinhardtii reveal downregulation of Lhcb genes but different regulation of Lhca genes.

In photoautotrophic organisms, the expression of nuclear genes encoding plastid proteins is known to be regulated at various levels. In this study, we present the analysis of two non-photosynthetic mutants (CC1051 and TR72) from the unicellular green alga Chlamydomonas reinhardtii. Both mutant strains show a defect in the processing of chloroplast psaA mRNA, and therefore they are assumed to be defective in photosystem I (PSI) assembly. We have performed macroarray experiments with trans-splicing mutants CC1051 and TR72 in order to analyse putative pleiotropic effects of nuclear-located mutations leading to a non-functional PSI. To the best of our knowledge, this is the first example of Chlamydomonas cDNA macroarray analysis comparing the transcriptional regulation of nuclear genes in wild-type and photosystem I mutants. The macroarray results demonstrated a transcriptional downregulation of members of the Lhcb gene family more than 2-fold in both mutant strains. In addition, real-time RT-PCR experiments found a 4- to 16-fold reduction in transcript levels of several Lhca genes in TR72; whereas in CC1051, no significant change in transcript levels was observed. Taken together, our data suggest that a signal is transmitted from the chloroplast to the nucleus that serves to regulate the level of light harvesting polypeptides in the organelle.