Detection of Legionella pneumophila at High Altitude in Tibetan Plateau

Infection with legionellae is an important cause of pneumonia known as Legionnaires' disease (LD). Legionellae are ubiquitous organisms at low altitudes (Sakamoto et al., 2009). To the best of our knowledge, however, no studies have yet reported the presence of legionellae at high altitudes. Although intense ultraviolet radiation and low temperature may effectively inhibit bacterial growth, legionellae parasitize protozoa, acquiring resistance under harmful conditions if encapsulated in protozoan cysts. To investigate whether legionellae are present at high altitudes, we collected water samples from a hot spring, puddles, streams, and tap water at altitudes from 3000 to 4200 m in Qinghai Province, China. The samples were concentrated through a 0.22-μm-pore-size membrane filter (Corning, Medfield, MA). After filtration, collected bacteria were resuspended in normal saline, mixed with an equal volume of 0.2 M KCl-HCl buffer (Muto Pure Chemicals Co., Tokyo), and inoculated onto WYOα plates (Eiken Chemical Co., Tokyo). The inoculated plates were incubated at 35°C. Smooth colonies showing a grayish white to grey-blue-purple, yellow, or green color were counted as suspicious legionellae and subcultured on Legionella agar (Becton-Dickinson, Mountain View, CA) supplemented with L-cysteine and ferric pyrophosphate without antibiotics. Serotyping was also performed by a slide agglutination test using commercial specific antisera (Denka Seiken, Tokyo) with a detection limit of 150 CFU/L. Real-time PCR with SYBR GreenER (Invitrogen Life Sciences, Carlsbad, CA) was also performed on water samples using the ABI Prism 7000 system (Applied Biosystems, Foster City, CA). The target primer pair was 16S rRNA (Sense primer: 5′-GCT TTCGTGCCTCAGTGTCA-3′; anti-sense primer: 5′-GCGTAGAGATCG GAAGGAAC A-3′; probe: 5′-FAM-CCAGGTAGCCGCCTTCGCCAC- TAMRA-3′) (Sato et al., 2004).

A total of 14 samples were collected, including four samples from a hot spring, four samples from puddles, three samples from streams, and three samples from tap water. Legionella pneumophila was isolated from three of four samples collected from the water (35°–40°C) of a hot spring at an altitude of 3930 m. This finding was not surprising, given the fact that L. pneumophila shows enhanced colonization at temperatures ranging from 25° to 42°C. Bacterial counts of the three samples were 1200 CFU/L, 600 CFU/L, and 300 CFU/L, respectively. Serotyping revealed that the samples contained L. pneumophila serogroups 1 and 3 (i.e., the major etiologic agents of LD) (Table 1).

L. pneumophila was not isolated from puddle, stream, or tap water samples; however, this does not confirm the absence of legionellae, as these species can exist in a viable but nonculturable state depending on the condition, re-entering a culturable state once subjected to an appropriate condition (Hussong et al., 1987). In fact, real-time PCR detected Legionella DNA in 12 of the 14 samples. Our results suggest a possibility that legionellae may be commonly present at high altitudes as well.

Although the presence of legionellae may not be directly linked to occurrence of LD, chronic obstructive pulmonary disease, a significant risk factor for acquiring LD, is highly prevalent among high altitude populations, possibly due to indoor air pollution caused by burning biomass fuels in confined spaces, occupational dusts, and oxidative stress (Roig et al., 1991; Basnyat et al., 2001). In travelers, differentiation between LD and high altitude pulmonary edema can be difficult, given that common symptoms of LD include fever, cough, and tachypnea. These symptoms may be exacerbated under hypoxic conditions. While early treatment with macrolides and quinolones is crucial in patients with LD, specific tests for LD are not available in most places. It is important to keep in mind that LD can occur even at high altitudes.