Nadia Schürch

Screening of Swiss hot spring resorts for potentially pathogenic free-living amoebae.

Free-living amoebae (FLA) belonging to Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Sappinia pedata are known to cause infections in humans and animals leading to severe brain pathologies. Worldwide, warm aquatic environments have been found to be suitable habitats for pathogenic FLA. The present study reports on screening for potentially pathogenic FLA in four hot spring resorts in Switzerland. Water samples were taken from water filtration units and from the pools, respectively. Amoebae isolated from samples taken during, or before, the filtration process were demonstrated to be morphologically and phylogenetically related to Stenoamoeba sp., Hartmannella vermiformis, Echinamoeba exundans, and Acanthamoeba healyi. With regard to the swimming pools, FLA were isolated only in one resort, and the isolate was identified as non-pathogenic and as related to E. exundans. Further investigations showed that the isolates morphologically and phylogenetically related to A. healyi displayed a pronounced thermotolerance, and exhibited a marked in vitro cytotoxicity upon 5-day exposure to murine L929 fibroblasts. Experimental intranasal infection of Rag2-immunodeficient mice with these isolates led to severe brain pathologies, and viable trophozoites were isolated from the nasal mucosa, brain tissue, and lungs post mortem. In summary, isolates related to A. healyi were suggestive of being potentially pathogenic to immunocompromised persons. However, the presence of these isolates was limited to the filtration units, and an effective threat for health can therefore be excluded.

Anthrax Spore Detection by a Luminex Assay Based on Monoclonal Antibodies That Recognize Anthrose-Containing Oligosaccharides

ABSTRACT The similarity of endospore surface antigens between bacteria of the Bacillus cereus group complicates the development of selective antibody-based anthrax detection systems. The surface of B. anthracis endospores exposes a tetrasaccharide containing the monosaccharide anthrose. Anti-tetrasaccharide monoclonal antibodies (MAbs) and anti-anthrose-rhamnose disaccharide MAbs were produced and tested for their fine specificities in a direct spore enzyme-linked immunosorbent assay (ELISA) with inactivated spores of a broad spectrum of B. anthracis strains and related species of the Bacillus genus. Although the two sets of MAbs had different fine specificities, all of them recognized the tested B. anthracis strains and showed only a limited cross-reactivity with two B. cereus strains. The MAbs were further tested for their ability to be implemented in a highly sensitive and specific bead-based Luminex assay. This assay detected spores from different B. anthracis strains and two cross-reactive B. cereus strains, correlating with the results obtained in direct spore ELISA. The Luminex assay (detection limit 103 to 104 spores per ml) was much more sensitive than the corresponding sandwich ELISA. Although not strictly specific for B. anthracis spores, the developed Luminex assay represents a useful first-line screening tool for the detection of B. anthracis spores.

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 degrees C, subsequently displayed a high thermotolerance, being able to grow at 42 degrees C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.

Mycobacterium ulcerans Persistence at a Village Water Source of Buruli Ulcer Patients

Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.

Immuno‐detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains

Aims:  Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen‐like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium.

Comparison of rapid methods for detection of Giardia spp. and Cryptosporidium spp. (oo)cysts using transportable instrumentation in a field deployment.

Reliable, sensitive, quantitative, and mobile rapid screening methods for pathogenic organisms are not yet readily available, but would provide a great benefit to humanitarian intervention units in disaster situations. We compared three different methods (immunofluorescent microscopy, IFM; flow cytometry, FCM; polymerase chain reaction, PCR) for the rapid and quantitative detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in a field campaign. For this we deployed our mobile instrumentation and sampled canal water and vegetables during a 2 week field study in Thailand. For purification and concentrations of (oo)cysts, we used filtration and immunomagnetic separation. We were able to detect considerably high oo(cysts) concentrations (ranges: 15-855 and 0-240 oo(cysts)/liter for Giardia and Cryptosporidium, respectively) in 85 to 300 min, with FCM being fastest, followed by PCR, and IFM being slowest due to the long analysis time per sample. FCM and IFM performed consistently well, whereas PCR reactions often failed. The recovery, established by FCM, was around 30% for Giardia and 13% for Cryptosporidium (oo)cysts. It was possible to track (oo)cysts from the wastewater further downstream to irrigation waters and confirm contamination of salads and water vegetables. We believe that rapid detection, in particular FCM-based methods, can substantially help in disaster management and outbreak prevention.

Tapping the potential of intact cell mass spectrometry with a combined data analytical approach applied to Yersinia spp.: detection, differentiation and identification of Y. pestis.

In the everyday routine of an analytic lab, one is often confronted with the challenge to identify an unknown microbial sample lacking prior information to set the search limits. In the present work, we propose a workflow, which uses the spectral diversity of a commercial database (SARAMIS) to narrow down the search field at a certain taxonomic level, followed by a refined classification by supervised modelling. As supervised learning algorithm, we have chosen a shrinkage discriminant analysis approach, which takes collinearity of the data into account and provides a scoring system for biomarker ranking. This ranking can be used to tailor specific biomarker subsets, which optimize discrimination between subgroups, allowing a weighting of misclassification. The suitability of the approach was verified based on a dataset containing the mass spectra of three Yersinia species Yersinia enterocolitica, Y. pseudotuberculosis and Yersinia pestis. Thereby, we laid the emphasis on the discrimination between the highly related species Yersinia pseudotuberculosis and Y. pestis. All three species were correctly identified at the genus level by the commercial database. Whereas Y. enterocolitica was correctly identified at the species level, discrimination between the highly related Y. pseudotuberculosis and Y. pestis strains was ambiguous. With the use of the supervised modelling approach, we were able to accurately discriminate all the species even when grown under different culture conditions.

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species

BackgroundBrucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with ‘smooth’ lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay.MethodsTo complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed.ResultsMAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with ‘smooth’ LPS with detection limits of 2 × 102 to 8 × 104 cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample.ConclusionOverall, the robust Luminex assay should allow detection of Brucella spp. in both natural outbreak and bio-threat situations.

Molecular Epidemiology and Antibiotic Susceptibility of Livestock Brucella melitensis Isolates from Naryn Oblast, Kyrgyzstan

The incidence of human brucellosis in Kyrgyzstan has been increasing in the last years and was identified as a priority disease needing most urgent control measures in the livestock population. The latest species identification of Brucella isolates in Kyrgyzstan was carried out in the 1960s and investigated the circulation of Brucella abortus, B. melitensis, B. ovis, and B. suis. However, supporting data and documentation of that experience are lacking. Therefore, typing of Brucella spp. and identification of the most important host species are necessary for the understanding of the main transmission routes and to adopt an effective brucellosis control policy in Kyrgyzstan. Overall, 17 B. melitensis strains from aborted fetuses of sheep and cattle isolated in the province of Naryn were studied. All strains were susceptible to trimethoprim-sulfamethoxazole, gentamicin, rifampin, ofloxacin, streptomycin, doxycycline, and ciprofloxacin. Multilocus variable number tandem repeat analysis showed low genetic diversity. Kyrgyz strains seem to be genetically associated with the Eastern Mediterranean group of the Brucella global phylogeny. We identified and confirmed transmission of B. melitensis to cattle and a close genetic relationship between B. melitensis strains isolated from sheep sharing the same pasture.

Population Genomics of Francisella tularensis subsp. holarctica and its Implication on the Eco-Epidemiology of Tularemia in Switzerland

Whole genome sequencing (WGS) methods provide new possibilities in the field of molecular epidemiology. This is particularly true for monomorphic organisms where the discriminatory power of traditional methods (e.g., restriction enzyme length polymorphism typing, multi locus sequence typing etc.) is inadequate to elucidate complex disease transmission patterns, as well as resolving the phylogeny at high resolution on a micro-geographic scale. In this study, we present insights into the population structure of Francisella tularensis subsp. holarctica, the causative agent of tularemia in Switzerland. A total of 59 Fth isolates were obtained from castor bean ticks (Ixodes ricinus), animals and humans and a high resolution phylogeny was inferred using WGS methods. The majority of the Fth population in Switzerland belongs to the west European B.11 clade and shows an extraordinary genetic diversity underlining the old evolutionary history of the pathogen in the alpine region. Moreover, a new B.11 subclade was identified which was not described so far. The combined analysis of the epidemiological data of human tularemia cases with the whole genome sequences of the 59 isolates provide evidence that ticks play a pivotal role in transmitting Fth to humans and other vertebrates in Switzerland. This is further underlined by the correlation of disease risk estimates with climatic and ecological factors influencing the survival of ticks.