Legionella feeleii: Ubiquitous Pathogen in the Environment and Causative Agent of Pneumonia.

L. feeleii is one of the most frequent Legionella species isolated from natural pools of the central region of Spain. This study aimed to evaluate its ecology and to identify this Legionella species as a respiratory pathogen. A PCR assay for detecting the L. feeleii mip gene was developed to identify it in clinical and environmental samples. Culture and PCR were performed in environmental samples from four drinking water treatment plants (DWTPs). Free L. feeleii was only detected in raw water samples (3.4%), while L. feeleii as an Acanthamoeba endosymbiont was found in 30.7% of raw water, 11.5% of decanter biofilm, and 32% of finished water samples. Therefore, Acanthamoeba spp. plays an essential role in the multiplication, persistence, and spread of Legionella species in the environment. The first case of Legionnaires' disease caused by L. feeleii in Spain is described in this study. The case was diagnosed in an older woman through PCR and sequencing from urine and sputum samples. A respiratory infection could be linked with health care procedures, and the patient presented several risk factors (age, insulin-dependent diabetes, and heart disease). The detection of non-L. pneumophila, such as L. feeleii, is a factor that must be considered when establishing or reviewing measures for the control and prevention of legionellosis.

Presence and interaction of free-living amoebae and amoeba-resisting bacteria in water from drinking water treatment plants.

Free-living amoebae (FLA) are ubiquitous and many isolates have been shown to be infected with amoeba-resisting bacteria, as the example of Acanthamoeba and Legionella interaction. Due to the high environmental prevalence of Acanthamoeba. in the Castilian Plateau (Spain), the aims of this work were to investigate the occurrence of Acanthamoeba and other FLA in water from several sampling points from four Drinking Water Treatment Plants (DWTP) and to investigate the presence of Legionella spp. and other amoeba-resisting bacteria in biofilms in raw and finished water, taking into account that no legislation exists for this protozoa control. Acanthamoeba was detected at different sampling points, and sand filters seemed to contribute to amoebic enrichment. After ozonation, a temporary decrease in viable amoebae was observed. The genotypes detected were T3, T4, and T5, revealing the first report of genotype T5 in waters from this region. Moreover, Balamuthia mandrillaris, Vermamoeba vermiformis and Paravahlkampfia sp. were detected. Regarding Legionella, PCR detection in raw and finished water was higher than by agar culture, but even higher after Acanthamoeba co-culture. Also, Legionella's presence was higher in raw water than in finished water. The decrease of free Legionella observed from raw (27.5%, by PCR) to finished water (3.4% by PCR) contrasted with the increase of Legionella-infected FLA from raw (30.7%) to finished water (52%). At biofilm, free Legionella was not detected, and the percentage of infected FLA was low (3.8%). Legionella species identified in these samples were L. drozanskii, L. donaldsonii and L. feeleii. Additionally, Acanthamoeba co-culture led to the isolation of Pseudomonas aeruginosa, P. stutzeri, P. fluorecens, Achromobacter xylosoxidans and Stenotrophomonas maltophilia. The highly disseminated presence of Acanthamoeba and the detection of amoeba-resisting bacteria inside amoebae highlight the importance of developing methods for controlling FLA in order to limit human pathogenic amoeba-resisting bacteria survival to the water purification processes.

The Influence of Acanthamoeba-Legionella Interaction in the Virulence of Two Different Legionella Species.

The genus Legionella comprises more than 60 species, and about half are associated with infection. Legionella pneumophila is the most commonly associated with these infections and by far the most studied, but L. non-pneumophila species, such as L. feeleii, L. anisa, etc., may also present clinical importance. Free-living amoebae are their preferred environmental host, where these bacteria not only survive but also succeed in multiplying, and this relationship can lead to an increase in bacterial virulence. The goal of this study was to evaluate the alterations of Legionella pathogenicity due to its interaction with Acanthamoeba. For this, the expression of protein effectors SdhA, LegK2, and SidK were evaluated in L. pneumophila and L. feeleii, before and after infecting Acanthamoeba. Additionally, the host response was evaluated by measuring the production of IL-6, IL-8, and IFN-γ in infected macrophages. Regarding the virulence factors, an increase in SdhA expression was observed after these bacteria infected Acanthamoeba, with a higher increase in the macrophage cultures infected with L. feeleii. Also, an increase in the expression of LegK2 was observed after infecting Acanthamoeba, but it was more intense in the cultures infected with L. pneumophila. With regard to SidK, it was increased in L. feeleii after infecting Acanthamoeba, however the same effect was not observed for L. pneumophila. In cytokine production, the effect on IL-6 and IL-8 was similar for both cytokines, increasing their concentration, but higher production was observed in the cultures infected with L. feeleii, even though it demonstrated slightly lower production with the inoculum obtained from Acanthamoeba. Concerning IFN-γ, induction was observed in both species but higher in the infection by L. pneumophila. Nevertheless, it is not known if this induction is enough to promote an efficient immune response against either L. pneumophila or L. feeleii. Altogether, these alterations seem to increase L. feeleii virulence after infecting Acanthamoeba. However, this increase does not seem to turn L. feeleii as virulent as L. pneumophila. More studies are necessary to understand the aspects influenced in these bacteria by their interaction with Acanthamoeba and, thus, identify targets to be used in future therapeutic approaches.

Can artificial tears prevent Acanthamoeba keratitis? An in vitro approach.

BACKGROUND: The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation. METHODS: Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry. RESULTS: Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane. CONCLUSIONS: The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out.

Correction: First Case of Legionnaire's Disease Caused by Legionella anisa in Spain and the Limitations on the Diagnosis of Legionella non-pneumophila Infections.

[This corrects the article DOI: 10.1371/journal.pone.0159726.].

First Case of Legionnaire's Disease Caused by Legionella anisa in Spain and the Limitations on the Diagnosis of Legionella non-pneumophila Infections.

Legionnaires' disease is a severe form of pneumonia, with worldwide relevance, caused by Legionella spp. Approximately 90% of all cases of legionellosis are caused by Legionella pneumophila, but other species can also be responsible for this infection. These bacteria are transmitted by inhalation of aerosols or aspiration of contaminated water. In Spain, environmental studies have demonstrated the presence of Legionella non-pneumophila species in drinking water treatment plants and water distribution networks. Aware that this evidence indicates a risk factor and the lack of routine assays designed to detect simultaneously diverse Legionella species, we analyzed 210 urine samples from patients presenting clinical manifestations of pneumonia using a semi-nested PCR for partial amplification of the 16S rDNA gene of Legionella and a diagnostic method used in hospitals for Legionella antigen detection. In this study, we detected a total of 15 cases of legionellosis (7.1%) and the first case of Legionnaires' disease caused by L. anisa in Spain. While the conventional method used in hospitals could only detect four cases (1.9%) produced by L. pneumophila serogroup 1, using PCR, the following species were identified: Legionella spp. (10/15), L. pneumophila (4/15) and L. anisa (1/15). These results suggest the need to change hospital diagnostic strategies regarding the identification of Legionella species associated with this disease. Therefore, the detection of Legionella DNA by PCR in urine samples seems to be a suitable alternative method for a sensitive, accurate and rapid diagnosis of Legionella pneumonia, caused by L. pneumophila and also for L. non-pneumophila species.