Untersuchung von Wasserproben aus der Industrie auf freilebende Amöben

Das Ziel vorliegender Studie bestand in der qualitativen Erfassung von Akanthamöben, Hartmannellen und Näglerien in Wasserproben aus der Papierindustrie. Diese aeroben Amöben sind primär freilebend (FLA) und ausgesprochene Kosmopoliten. Naegleria fowleri und Acanthamoeba spp. sind potentielle Humanpathogene, die schwere, meist tödlich verlaufende, Entzündungen des Gehirns, verursachen können. Darüber hinaus können insbesondere Akanthamöben und Hartmannellen als Vektoren und Reservoir für den Erreger der Legionärskrankheit, Legionella pneumophila, fungieren. Aufgrund von Legionellose-Ausbrüchen in europäischen Papierfabriken wurde ein fächerübergreifendes Forschungsprojekt zur Untersuchung von Prozesswasser-Proben auf Legionellen initiiert. Um einen zuverlässigen Nachweis der Amöben zu ermöglichen, sollten alle Proben mittels Kultur und molekularbiologischer Methoden im Parallelansatz untersucht werden. Hierfür wurden neue genus-spezifische PCR-Systeme etabliert. Die Acanthamoeba-Spezies sollten zusätzlich mittels Sequenz-Analyse genotypisiert werden. Insgesamt wurden 28 Wasserproben aus 16 verschiedenen Betrieben untersucht. Dabei konnten in 27 von 28 Wasserproben FLA nachgewiesen werden. Acanthamoeba war mit 89% die mit Abstand häufigste Gattung, gefolgt von diversen FLA, wie Echinamöben und Vahlkampfien in 32% der Proben. Vertreter der Gattung Hartmannella wurden in 21% der Wasserproben gefunden und Naegleria war nur in einer Probe nachweisbar. In 39% der Proben wurden mehrere Amöbengenera detektiert. Insgesamt waren fünf von 28 Wasserproben (18%) positiv auf Legionella spp. In allen fünf Proben waren auch Akanthamöben nachweisbar. In einer dieser fünf Legionella-positiven Proben wurden außerdem Hartmannellen detektiert und in einer Probe war Naegleria sp. nachweisbar. Insgesamt konnte gezeigt werden, dass FLA, insbesondere Akanthamöben, in Industriewässern häufig sind und auch syntop mit Legionellen auftreten. Diese Ergebnisse bestätigen die Rolle der FLA für die Verbreitung von Legionellen.Due to recent cases of legionellosis in European paper factories, an interdisciplinary project for an investigation of process water has been initiated. The aim of the study was to investigate the occurrence of Acanthamoeba, Hartmannella and Naegleria in water samples from Austrian paper factories. Naegleria fowleri is known as the causative agent of primary amoebic meningoencephalitis. Acanthamoeba species can cause infections in immunocompromised hosts (granulomatous amoebic encephalitis) as well as in immunocompetent humans (Acanthamoeba-keratitis). Furthermore, free-living amoebae (FLA), particularly acanthamoebae and hartmannellae, can serve as vectors and reservoir for Legionella pneumophila, the causative agent of Legionnaires´ disease. To ensure a reliable detection, all samples were investigated by culture and molecular biological techniques in parallel. For this reason genus-specific PCR protocols were established. Additionally, employing sequence analysis Acanthamoeba genotypes were determined. A total of 28 water samples from 16 factories were examined, with 27 samples being positive for FLA. Acanthamoeba species were most prevalent with 89%, followed by 32% of diverse FLA, such as echinamoebae and vahlkampfiae. Hartmannella was observed in 21% of the samples and Naegleria sp. was only detected once. 39% of the samples were positive for several genera of amoebae. In 18% (five of 28) of the samples Legionella spp. were present and co-occurred with Acanthamoeba. Hartmannella and Naegleria were present in one of five Legionella-positive samples, respectively. These results show that FLA, particularly Acanthamoeba, are common in process waters and occur syntopically with Legionella spp. These findings confirm the role of FLA for the distribution of legionellae

Free-living amoebae as bacterial reservoirs and human pathogens

Freilebende Amöben (FLA) sind in der Umwelt weit verbreitet und sind die Erreger von sehr seltenen, aber schweren Infektionen. Akanthamöben können insbesondere bei Kontaktlinsenträgern eine rasch fortschreitende Keratitis (AK) auslösen. Darüber hinaus können Akanthamöben opportunistische Infektionen, wie etwa die Granulomatöse Amöbenenzephalitis (GAE) verursachen. Die durch Balamuthia mandrillaris verursachte GAE und auch die Primäre Amöbenmeningoenzephalitis (PAME) durch Naegleria fowleri können hingegen auch beim Immungesunden auftreten. Neben ihrer Rolle als Pathogene weiß man, dass FLA auch als Reservoir für verschiedene Bakterien, wie etwa für Legionella pneumophila dienen, die durch die intrazelluläre Replikation sogar ihre Infektivität steigern. Industriewässer und insbesonder Kühltürme waren in der Vergangenheit immer wieder die Quelle von Legionellen-Ausbrüchen und werden daher regelmäßigen Untersuchungen unterzogen, allerdings ohne dabei die FLA und ihre Funktion als Vehikel und Reservoir zu berücksichtigen. Das Ziel der ersten Studie war daher ein FLA-Screening von Industriewässern durch Einsatz von Kultur, PCR und Sequenzieren parallel zu einer Standard-Untersuchung auf Legionellen (ÖNORM ISO 11731:1998). Insgesamt wurden 201 Wasserproben untersucht, wobei 129 Wasserproben von Kühltürmen, 72 Prozeßwasserproben von österreichischen Papiermaschinen und 30 Proben von Kühlschmierstoffen untersucht wurden. In 72.6% der Wasserproben konnten FLA nachgewiesen werden, wobei Acanthamoeba spp. mit 23.9% am Häufigsten waren. 34.8% der Wasserproben waren positiv auf Legionella spp., wovon 81.4% positiv auf FLA waren. Die Kühlschmierstoffe waren dagegen negativ auf Legionellen und nur in einer Probe konnten Akanthamöben nachgewiesen werden. Basierend auf diesen Ergebnissen, sollte in der zweiten Studie ein System zur Detektion der Wirtsamöben etabliert werden, dass man in ein Legionella Routine Screening implementieren kann. Dafür wurde ein für verschiedene FLA-Taxa spezifisches real-time PCR-System entwickelt. Insgesamt wurden drei Kühltürme über den Zeitraum von einem Jahr regelmäßig untersucht, jeweils mit Kulturmethoden und parallel mit molekularbiologischen Methoden. Zum Vergleich wurden Trinkwasserproben in die Studie mit einbezogen. Das Legionellen-Screening wurde nach einem Standard Protokoll durchgeführt (ISO 11731-2:2004). Insgesamt waren 83.3% der Kühlturmproben und 50% der Trinkwasserproben positiv auf FLA wobei wiederum Acanthamoeba die häufigste Gattung war. Bemerkenswert ist, dass aufgrund der hohen organischen Belastung 69.7% der Kühlturmproben für die Standard Legionellen-Untersuchung ungeeignet waren. Von den übrigen Proben waren in Kultur 25% positiv auf Legionella spp. und durch molekularbiologische Methoden waren insgesamt 50% der Proben positiv. Diese Ergebnisse zeigen, dass ein Verbesserungsbedarf bei den derzeit verwendeten Untersuchungsmethoden besteht. Im Zuge der letzten Studie erstellten wir einen Überblick über die letzten 20 Jahre der Acanthamoeba-Diagnostik in Österreich. Alle Patientenproben mit Verdacht auf AK oder GAE wurden durch Kultivierungsmethoden und/oder PCR auf Acanthamoeba untersucht und alle Amöbenisolate wurden genotypisiert. Insgesamt wurden 154 Fälle von AK und 3 Fälle von GAE diagnostiziert, wobei der häufigste Genotyp T4 war.Free-living amoebae (FLA) are widely distributed in the environment and are known causative agents of rare but often serious diseases. Acanthamoebae can cause a rapidly progressing keratitis (AK) especially in contact lens wearers. Moreover, acanthamoebae can cause opportunistic infections, such as granulomatous amoebic encephalitis (GAE). The GAE caused by Balamuthia mandrillaris and the fatal primary amoebic meningoencephalitis (PAME) caused by Naegleria fowleri can both affect also healthy humans. In addition to their role as pathogens, FLA also serve as a reservoir for various bacteria, such as Legionella pneumophila, which increase their infectivity through intracellular replication. Industrial waters and, in particular, cooling towers have been the source of Legionella outbreaks in the past and are therefore subject to periodic screenings, not considering, however, FLA and their reservoir function. Therefore, the aim of the first study was to screen industrial waters for FLA by culture, PCR and sequencing in parallel to a standard investigation on legionellae (ÖNORM ISO 11731:1998). A total of 201 samples were investigated, including 129 cooling waters, 72 process waters and, in addition, 30 cooling lubricants from Austrian paper plants. Altogether, FLA were detected in 72.6% of the water samples, with acanthamoebae being most prevalent (in 23.9% of all samples). Moreover, 34.8% of the water samples were positive for Legionella spp., of which 81.4% were positive for FLA. The cooling lubricants were negative for legionellae and only one sample was positive for Acanthamoeba. Based on these results, the aim of the second study was to incorporate a screening system for host amoebae into a Legionella routine screening. A new real-time PCR screening system specific for different groups of FLA was established for this purpose. Three cooling towers were regularly screened over the period of one year and investigated by culture and molecular biological methods in parallel. Additionally, 12 tap water samples were included in the study. The Legionella screening was performed according to the standard protocol ISO 11731-2:2004. Altogether, 83.3% of the cooling tower samples and 50% of the tap water samples were positive for FLA, and again Acanthamoeba was the most common genus. It is noteworthy that, due to the high organic burden, 69.7% of the cooling tower samples were unsuitable for the standard Legionella screening. In the remaining samples, positivity for Legionella spp. was 25% by culture, but overall positivity was 50% by molecular biological methods, demonstrating a need for improvement of the currently established screening methods. In the last study, we aimed to give an overview of the cases of Acanthamoeba infections diagnosed in Austria during the past 20 years. All samples of patients with suspected AK or GAE were screened for Acanthamoeba spp. by culture, PCR or both, and the detected amoebae were genotyped. A total of 154 cases of AK and three cases of GAE were diagnosed, involving various Acanthamoeba genotypes, but with the most common genotype being T4.submitted by Mag. rer. nat. Ute ScheiklZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2016Wien, Univ., Diss., 2016OeBB(VLID)189592

Twenty Years of Acanthamoeba Diagnostics in Austria

Acanthamoebae are the causative agents of an often seriously progressing keratitis (AK) occurring predominantly in contact lens wearers and can cause several disseminating infections potentially resulting in granulomatous amoebic encephalitis (GAE) in the immunocompromised host. Our institution is the Austrian reference laboratory for Acanthamoeba diagnostics and the aim of this study was to give an overview of proven cases of Acanthamoeba infections in Austria during the past 20 yr. All samples of patients with suspected AK or GAE were screened for Acanthamoeba spp. by culture and/or PCR and the detected amoebae were genotyped. Altogether, 154 cases of AK and three cases of GAE were diagnosed. Age of the AK patients ranged from 8 to 82 yr (mean 37.8) and 58% of the patients were female. Approximately 89% of the AK patients were contact lens wearers, almost all cases were unilateral and 19% of the patients required a keratoplasty. Age of the GAE patients ranged from 2 to 25 yr (mean 14.7), all were HIVnegative, but two were severely immunosuppressed at the time of diagnosis. The predominant genotype in the AK cases was T4, other genotypes found were T3, T5, T6, T10 and T11. The three GAE cases involved genotypes T2, T4 and T5.(VLID)484741

Free-living amoebae and their associated bacteria in Austrian cooling towers: a 1-year routine screening

Free-living amoebae (FLA) are widely spread in the environment and known to cause rare but often serious infections. Besides this, FLA may serve as vehicles for bacterial pathogens. In particular, Legionella pneumophila is known to replicate within FLA thereby also gaining enhanced infectivity. Cooling towers have been the source of outbreaks of Legionnaires' disease in the past and are thus usually screened for legionellae on a routine basis, not considering, however, FLA and their vehicle function. The aim of this study was to incorporate a screening system for host amoebae into a Legionella routine screening. A new real-time PCR-based screening system for various groups of FLA was established. Three cooling towers were screened every 2 weeks over the period of 1 year for FLA and Legionella spp., by culture and molecular methods in parallel. Altogether, 83.3 % of the cooling tower samples were positive for FLA, Acanthamoeba being the dominating genus. Interestingly, 69.7 % of the cooling tower samples were not suitable for the standard Legionella screening due to their high organic burden. In the remaining samples, positivity for Legionella spp. was 25 % by culture, but overall positivity was 50 % by molecular methods. Several amoebal isolates revealed intracellular bacteria.TRP 209-B20(VLID)308958

Free-living amoebae (FLA) co-occurring with legionellae in industrial waters

AbstractLegionella pneumophila is known as the causative agent of Legionnaires’ disease and free-living amoebae (FLA) can serve as vehicles for legionellae. The aim of this study was to screen industrial waters for the occurrence of FLA and their co-occurrence with legionellae. A total of 201 water samples, including 129 cooling waters and 72 process waters, and 30 cooling lubricants were included in the study. Treated waters were screened periodically, pre and post treatment. Altogether, 72.6% of the water samples were positive for FLA, acanthamoebae being most prevalent (in 23.9% of the samples) followed by Vermamoeba vermiformis (19.4%). Only one cooling lubricant was positive (Acanthamoeba genotype T4). Legionella spp. were detected in 34.8% of the water samples and in 15% in high concentrations (>1000CFU/100ml). Altogether, 81.4% of the Legionella-positive samples were positive for FLA by standard methods. By applying a highly sensitive nested PCR to a representative set of random samples it was revealed that Legionella spp. always co-occurred with Acanthamoeba spp. Although the addition of disinfectants did influence amoebal density and diversity, treated waters showed no difference concerning FLA in the interphases of disinfection. It appears that FLA can re-colonize treated waters within a short period of time

‘Candidatus Cochliophilus cryoturris’ (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus

Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as ‘Candidatus Cochliophilus cryoturris’. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.© The Author(s) 201