Discrimination of Euglena gracilis strains Z and bacillaris by MALDI-TOF MS.

AIMS: Euglena gracilis is used as model organism for various microbiological, molecular biological and biotechnological studies. Its most studied wild-type strains are Z and bacillaris, but their discrimination by standard molecular methods is difficult. Therefore, we decided to test the suitability of MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) for identification of E. gracilis and for discrimination of these two strains possessing functional chloroplasts. MALDI-TOF MS profiling was also tested for two white (non-photosynthetic) stable E. gracilis mutant strains Wgm ZOflL and W10 BSmL. METHODS AND RESULTS: We have successfully obtained main spectrum profiles (MSPs) of E. gracilis strains Z, SAG 1224-5/25 and bacillaris, SAG 1224-5/15 using protein extraction procedure. Subsequent MALDI-TOF MS profiling of a number of tested samples and the comparison of the obtained protein profiles with our in-house database including MSPs of both strains have revealed that these two strains can be easily distinguished by MALDI-TOF MS based on score values over two in most cases. This method has also confirmed the ancestry of white mutant strains Wgm ZOflL and W10 BSmL, originally derived from strains Z and bacillaris, respectively. CONCLUSIONS: MALDI-TOF MS is suitable, accurate and rapid method for discrimination of E. gracilis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results can have broad practical implications for laboratories cultivating various strains of euglenids, and they can be applied for their discrimination by MALDI-TOF MS.

Legionnaires' Disease in Pediatric Patients, Control Measures and 5-Year Follow-up.

BACKGROUND: Legionnaires' disease (LD) occurs predominantly in adults and elderly people. Its incidence in Europe has been increasing in recent years. It is rare in younger age groups and prone to be reported as healthcare-associated infection with a higher risk of fatal outcome. Hospital-acquired LD is mostly associated with a colonized hospital water system. We describe 5 LD cases in a children's hospital in Slovakia, subsequent environmental investigation, control measures, and 5-year monitoring of Legionella colonization in hospital's water system. METHODS: In 2014-2019, we tested clinical specimens from 75 hospitalized patients. Respiratory samples were cultured for Legionella, patient's urine was tested for Legionella urinary antigens, and the microagglutination test was used for serologic testing. Samples of water were collected in 2015-2019 and processed according ISO11731. RESULTS: We identified 5 Legionella infections in 2014-2015. Median age of patients was 15 years. All were high-risk patients hospitalized for their underlying diseases. All patients required admission to intensive care unit, and artificial ventilation due to general deterioration and respiratory failure. Legionella pneumophila was isolated from 72% of water samples. Chlorine dioxide dosing into water system above 0.3 ppm caused significant decrease of Legionella concentration in water samples. Samples taken from outlets with antimicrobial filter installed were legionellae-negative. CONCLUSIONS: Control measures led to decreased risk of infection, but not to eradication of Legionellae. It is necessary to extend the diagnostics for Legionella infection in hospitalized children with pneumonia, especially in hospitals with colonized water system.

A preliminary checklist of Chironomidae (Diptera) from Albania with first records for the Balkan Peninsula.

The Chironomidae of Albania have so far received limited attention and only 39 species have been recorded prior to the present study. Here we bring the results of random and non-intensive samplings of chironomid pupal exuviae and adults, at five localities in 2012, that provided 55 species and 5 additional taxa, with 51 being new for the Albanian fauna, out of which 7 were new for the Balkan Peninsula. In addition to that, we present a preliminary checklist of Chironomidae based on the data from Fauna Europaea complemented by the results of the recent investigation. The catalogued fauna now contains 85 species in 44 genera and 6 sub-families.

MALDI-TOF MS analysis as a useful tool for an identification of Legionella pneumophila, a facultatively pathogenic bacterium interacting with free-living amoebae: A case study from water supply system of hospitals in Bratislava (Slovakia).

Legionellae, i.e. Legionella pneumophila, are human bacterial hydrophilic facultative pathogens causing pneumonia (Legionnaires' disease). Free-living amoebae (FLA) can serve as natural hosts and thus as reservoirs of many amoebae-resistant bacteria. An encysted amoeba can contribute to the resistance of intracellular L. pneumophila to various chemical and physical treatments. Humans can be infected by droplets containing bacteria from an environmental source or human-made devices such as shower heads, bathtubs, air-conditioning units or whirlpools. In this study, we were investigating the presence of FLA and L. pneumophila in plumbing systems of healthcare facilities in Bratislava (Slovakia) by standard diagnostic methods, while the presence of L. pneumophila was verified also by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis. The results showed the occurrence of L. pneumophila and FLA in 62.26% and 66.4% of samples taken from four paediatric clinics, respectively. Both standard methods and MALDI-TOF MS showed comparable results and they can be successfully applied for the identification of L. pneumophila strains in environmental samples. Our approach could be useful for further monitoring, prevention and decreasing risk of Legionella infection also in other hospitals.

A real-time PCR diagnostic method for detection of Naegleria fowleri.

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence. Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.