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.
PurposeAcanthamoeba keratitis (AK) is a painful and potentially blinding corneal infection caused by Acanthamoeba spp. In Madrid, environmental studies have demonstrated a high presence of these free-living amoebae in tap water. Since most of AK cases occur in contact lenses (CL) wearers with inadequate hygiene habits, the presence of Acanthamoeba in discarded CL has been studied and compared with other common etiological agents of keratitis, such as Pseudomonas aeruginosa and Staphylococcus aureus.MethodsOne hundred and seventy-seven healthy individuals from Madrid contributed their discarded CL and answered a questionnaire on hygiene habits. DNA was extracted from the CL solution and analyzed by real-time PCR for Acanthamoeba, Pseudomonas aeruginosa and Staphylococcus aureus. These CL and their solutions were also cultured on non-nutrient agar to isolate Acanthamoeba.ResultsAmong the 177 samples, Acanthamoeba DNA was detected in 87 (49.2%), P. aeruginosa DNA in 14 (7.9%) and S. aureus DNA in 19 (10.7%). Cultivable amoebae, however, were observed in only one sample (0.6%). This isolate was genotyped as T4. The habits reported by this CL owner included some recognized risk factors for AK, but in this study only the practice of “not cleaning the CL case” presented some statistical significant association with Acanthamoeba DNA presence. Detection of the investigated bacterial DNA did not demonstrate statistical significant association with the studied practices, but the presence of P. aeruginosa revealed a possible inhibition of Acanthamoeba in these samples.ConclusionsThe PCR results suggest a high presence of Acanthamoeba spp. in healthy CL wearers from Madrid, but we can assume that CL solutions are properly disinfecting the CL since only 1.1% of the positive PCR samples correspond to viable amoebae and, after four years, only one participant reported stronger ocular problems. Nevertheless, more studies are necessary to corroborate this hypothesis.
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.
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