| Legionella, a group of Gram-negative coccobacilli, are the causative agent of Legionnaires’ disease(LD). Since the nomenclature of the genus Legionella in 1979, fifty-eight species and more than seventy serogroups have been described in the genus Legionella and more than twenty species have been associated with human legionellosis. Among them, Legionella pneumophila caused approximately 90% of all reported Legionnaires’ disease cases. Legionella spp. are ubiquitous in natural aquatic environments(streams, rivers, ponds, lakes, hot spring and underground water) and artificial aquatic environments(cooling tower, tap water, air humidifier and fountain).Since human LD is always transmitted via the inhalation of Legionella-containing aerosols generated from environmental water sources, not by human-to-human transmissions. So it is important to study environmental isolates of Legionella not only for establishing an epidemiological link between suspected artificial environmental sources and clinical isolates, but also for comparison of clinical and environmental distributions of Legionella strains. Up to now, most published studies have focused on clinical isolates and artificial environmental isolates, few researches concentrated on the information of Legionella in natural aquatic environments. However, it is equally important to understand the distribution and genetic diversity of legionella in their natural habitats. Because Legionnaires’ disease is not a notifiable infectious diseases in China and the epidemiological data of Legionnaires’ disease are very scarce. Knowledge of the composition and distribution of Legionella and the gennetic diversity of L. pneumophila in natural water environmental is essential for epidemiological studies and control of Legionnaires’ disease. To meet these purposes, we investigated the occurrence and composition of Legionellaceae bacteria in natural freshwater environments in 31 cities in China and analyzed the population structure determined by sequence-based typing of 140 natural environmental isolates of L. pneumophila from China.Water was sampled from the major rivers and lakes of 31 cities during the period July 2010 to September 2014. Five to eight rivers and/or lakes in each city and one to five different areas in each river or lakes were selected to collect water samples. A total of 550 water samples were collected in 31 cities. Three methods were used to concentrate samples: Filtration enrichment(FE), Centrifugation enrichment(CE) and Heat enrichment(HE). The suspected isolates were characterized by sequencing of mip gene and 16 S r RNA gene. Among the 550 samples cultured, 171 samples were positive when the samples were pretreated with heat enrichment(HE), whereas 54 and 74 samples were positive when the samples were pretreated with filtration enrichment(FE) and centrifugation enrichment(CE), respectively. The sensitivity of HE is significantly higher than which of FE(31.1% vs. 9.8%, P<0.001, Chi-Square test) and CE(31.1% vs. 13.1%, P<0.001, Chi-Square test). The isolation rate of L. pneumophila, L. longbeachae, L. feeleii and L. gormanii can be significantly affected by three methods(Fisher exact test, P<0.05). Significant association was found between isolation rate of Legionella and water temperature. Results of Spearman rank test showed positive correlation of Legionella isolation rate with water temperature(ρ=0.929,P=0.003).A total of 404 unrelated environmental isolates assigned to 23 legionella species were isolated from the 239 positive water samples. Legionella pneumophila predominated in the isolates and constituted 36.1%(146/404) of the isolates. L. longbeachae, L. feeleii and L. gormanii predominated among non-L. pneumophila species and accounted for 15.3%, 12.6% and 12.4%, respectively. Legionella contamination was detected in 29 of 31 sampling cities which revealed ubiquitous occurrence of legionellae strains in the natural aquatic environments in China. L. pneumophila which was isolated in 25 of 29 cities was the specie isolated most frequently. Among non-L. pneumophila species, L. feeleii, L. longbeachae and L. gormanii isolated in 22 cities, 21 cities and 20 cities, respectively. Among 239 positive samples, 75 samples contained two or more than two different Legionella species and 31 samples had three or more than three different Legionella species. In some sample great diversity was observed, 8 samples had four different Legionella species. In conclusion, our results demonstrated a ubiquitous occurrence and high biodiversity of the legionellae strains in the natural aquatic environments in China.At the same time, 140 natural isolates of L. pneumophila were genetyped using sequence-based typing. As a result, the 140 isolates could be divided into 84 sequence types(STs) and the index of diversity(IOD) was 0.988(95% CI: 0.983-0.992). Among the 84 STs, 31 STs could be found in the EWGLI SBT database and 53 STs were new to the EWGLI database. Three STs, ST1417, ST1923 and ST1968, containing 6 isolates were the most common ST. But the distribution of these three STs were different, ST1923 and ST1968 were only found in Haikou and ST1417 could be found in six different cities(Changsha, Haerbin, Haikou, Shanghai, Taiyuan and Urumqi). 20 STs consisted of groups containing two to five isolates and the remaining 61 STs had only one strain. Eleven new alleles( pil E626; mip69, 75; momp S87, 88 and 89; pro A611, 684; neu A611, 679 and 681) were found in this study.The population structure analysis illustrates 10 clonal complexes(CCs) and 47 singletons. With 21 isolates and 10 STs, CC I was the prevalent clonal complex. This CC contained the isolates that were isolated from 9 cities and the predicted founder was ST1670. Among the 10 STs, ST239 and ST1439 were associated with Legionnaires’ disease according to ESGLI SBT database. The most common CC found in artificial water environments in China, ST1 complex, seldom detection in natural water. In addition, 10 strains with complete lag-1 gene were not in any one clonal complex which suggests high pathogenic strains is not dominant in natural water. To investigate intragenic and intergenic recombination, we utilized Sawyer’s test implemented in START2 and RDP4 to analyze the seven gene fragments individually and he concatenated sequences of the seven loci. Sawyer’s test failed to identify any intragenic recombination events. However, twelve putative intergenic recombinant events were identified. Among the 84 STs, recombinant events can be detected in 74 STs and 17 STs have two recombinant events.There were no significant difference in molecular level between different areas and environments, but significant difference could be found between natural isolates and artificial isolates.In conclusion, our results demonstrated a remarkable genetic diversity and frequent recombination of L. pneumophila from natural water in China. There was no significant difference of molecular variance of natural isolates between geographical areas but significant difference of molecular variance could be found between natural isolates and artificial isolates. |
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