The environmental isolates were collected based on the protocol according to ISO11731 and the National standard: Hygienic specification of central air conditioning ventilation system in public buildings (WS394-2012). A total of 79 L. pneumophila strains were included in this study: 64 strains were isolated during regular surveillance events which were performed for cooling towers and bath water in Sichuan Province. Thirteen strains were isolated from hot springs through an investigation regarding hot spring recreational areas in 2014, including two cities: Ya'an (10 strains) and Kang ding (3 strains). Ten strains were isolated from city fountains during fountain surveillance for L. pneumophila in 2010 in Chengdu. Lastly, 2 strains were isolated from the sputum of patients, of which one was from the case reported firstly in Sichuan province, in 1989 (Supplementary Table S1 available in www.besjournal.com).
Strain ID Year City Facility Type Source Strain ID Year City Facility Type Source 8931 1989 Chengdu Hospital Human XJ2010017 2010 Chengdu Hotel Cooling Tower 97008 1997 Chengdu Hotel Cooling Tower XJ2010025 2010 Chengdu Hotel Cooling Tower WZL 2000 Chengdu Hospital Human XJ2010041 2010 Chengdu Hotel Cooling Tower 00-02 2000 Chengdu Hotel Cooling Tower XJ2010044 2010 Chengdu Hotel Cooling Tower 00-03 2000 Chengdu Hotel Cooling Tower XJ2010051 2010 Neijiang Hotel Cooling Tower 2007026 2007 Chengdu Hotel Cooling Tower XJ2010098 2010 Chengdu Hotel Cooling Tower 2007091 2007 Chengdu Hotel Cooling Tower XJ20110891 2011 Luzhou Hotel Cooling Tower 2007094 2007 Chengdu Hotel Cooling Tower XJ20110895 2011 Zigong Hotel Cooling Tower 2007101 2007 Chengdu Hotel Cooling Tower XJ20111923 2011 Nanchong Hotel Cooling Tower 2007102 2007 Chengdu Hotel Cooling Tower XJ2011924 2011 Nanchong Hotel Cooling Tower 2007103 2007 Chengdu Hotel Cooling Tower XJ2011925 2011 Nanchong Hotel Cooling Tower 2007104 2007 Chengdu Hotel Cooling Tower XJ20120317 2012 Chengdu Hotel Bath Water 2007106 2007 Chengdu Hotel Cooling Tower XJ20130044 2013 Chengdu Hotel Cooling Tower 2007109 2007 Chengdu Hotel Cooling Tower XJ20130047 2013 Chengdu Hotel Bath Water 2007112 2007 Chengdu Hotel Cooling Tower XJ20130052 2013 Chengdu Hotel Cooling Tower 2008090 2008 Chengdu Hotel Cooling Tower XJ20130055 2013 Chengdu Hotel Cooling Tower 2008422 2008 Chengdu Hotel Cooling Tower XJ20130275 2013 Chengdu Hotel Bath Water 2008423 2008 Chengdu Hotel Cooling Tower XJ20130461 2013 Ya'an Hotel Cooling Tower 2009022 2009 Chengdu Hotel Cooling Tower XJ20140347 2014 Kangding Public Bath Hot Spring 2009029 2009 Chengdu Hotel Cooling Tower XJ20140348 2014 Kangding Public Bath Hot Spring 2009039 2009 Leshan Hotel Cooling Tower XJ20140360 2014 Kangding Public Bath Hot Spring 2009040 2009 Chengdu Hotel Cooling Tower XJ20140374 2014 Ya'an Public Bath Hot Spring 2009042 2009 Mianyang Hotel Cooling Tower XJ20140375 2014 Ya'an Public Bath Hot Spring 2009045 2009 Chengdu Hotel Cooling Tower XJ20140376 2014 Ya'an Public Bath Hot Spring 2009047 2009 Chengdu Hotel Cooling Tower XJ20140378 2014 Ya'an Public Bath Hot Spring 2009058 2009 Chengdu Hotel Cooling Tower XJ20140379 2014 Ya'an Public Bath Hot Spring 2009065 2009 Chengdu Hotel Bath Water XJ20140380 2014 Ya'an Public Bath Hot Spring 2009141 2009 Chengdu Hotel Cooling Tower XJ20140381 2014 Ya'an Public Bath Hot Spring 2009142 2009 Chengdu Hotel Bath Water XJ20140382 2014 Ya'an Public Bath Hot Spring CD1 2010 Chengdu City Center Fountain XJ20140386 2014 Ya'an Public Bath Hot Spring CD2 2010 Chengdu City Center Fountain XJ20140387 2014 Ya'an Public Bath Hot Spring CD3 2010 Chengdu City Center Fountain XJ20140450 2014 Neijiang Hotel Cooling Tower CD4 2010 Chengdu City Center Fountain XJ20140453 2014 Neijiang Hotel Cooling Tower CD5 2010 Chengdu City Center Fountain XJ20140454 2014 Nanchong Hotel Cooling Tower CD6 2010 Chengdu City Center Fountain XJ20140457 2014 Luzhou Hotel Cooling Tower CD7 2010 Chengdu City Center Fountain XJ20140469 2014 Neijiang Hotel Cooling Tower CD8 2010 Chengdu City Center Fountain 1607-GC-019A 2016 Luzhou Hotel Cooling Tower CD9 2010 Chengdu City Center Fountain 1609-GC-021A 2016 Luzhou Hotel Cooling Tower CD10 2010 Chengdu City Center Fountain 1609-GC-023A 2016 Luzhou Hotel Cooling Tower LP8 2010 Chengdu Hotel Cooling Tower
Table Supplementary Table S1. Information of Strains Used in this Study
All isolates were identified using Legionella-specific PCR targeting the 16S rRNA gene to determine the species and serogroup of L. pneumophila were determined by slide agglutination with polyclonal antisera (Tianjin Biochip Corporation).
Seventy-nine strains were lined on buffered charcoal yeast extract (BCYE) supplemented with glycine (3 g/L), vancomycin (1 mg/L), polymyxin B (80, 000 UI/L) and cycloheximide (80 mg/L) (GVPC) agar (BeiJing Land Bridge Technology Co., Ltd.), and the plates were incubated at 37 ℃ with 5% CO2 for 3-4 days, the bacterial colonies were collected to prepare for Genomic DNA. Genomic DNA was extr-acted with a QIAamp DNA minikit (Qiagen, Dusseldorf, Germany) according to the manufacturer's guidelines.
The genotyping was performed by means of the standard SBT method of the European Working Group for Legionella Infections (EWGLI) including 7 genes (flaA, pilE, asd, mip, mompS, proA, and neuA)[22-23]. PCR was performed with a reaction volume of 25 µL including 22 µL of the PCR mixture, 1 µL of each primer (10 µmol/L) (TsingKe Biological Technology Co., Ltd.), and 20 ng of the DNA template. PCRs were performed with an initial denaturation step at 95 ℃ for 5 min; followed by 35 cycles, each consisting of initial denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 45 s; and final extension at 72 ℃ for 10 min. Then, each gene was sequenced by TsingKe Biological Technology Co., Ltd. The online Legionella SBT Quality Tool (http://www.hpa-bioinformatics.org.uk/cgi-bin/legionella/sbt-/seq_assemble_legionella1.cgi) was used to assign individual allele numbers (e.g., 1-4-3-1-1-1-1) for the 7 genes described previously and a sequence type (ST) represented by a number (e.g., ST1).
The BioNumerics software (Applied Maths, Kortrijk, Belgium) was used to create a maximum likelihood tree using the unweighted pair group method with arithmetic means (UPGMA), and this revealed the genetic distance among the 79 identified strains. The clonal complexes were also analyzed with this software to create minimum spanning trees (MST). In the MSTs, the clusters of related STs that descended from a common ancestor were defined as clonal groups (CGs).
In this study, VNTR genotyping of the strains selected were conducted 8-locus MLVA (MLVA-8) including designated Lpms1b, Lpms3, Lpms13, Lpms17, Lpms19b, Lpms33, Lpms34, and Lpms35[24-25] (Supplementary Table S2 available in www.besjournal.com), according to which the primers were designed for PCR amplification of the markers. PCR was divided into three groups (VNTR1, VNTR2, VNTR3) and conducted in a reaction volume of 25 µL, respe-ctively, containing 12.5 µL of 2× PCR mixture, 1 µL of each fluorescent-labeled primer (10 µmol/L) (TsingKe Biological Technology Co., Ltd.), 20 ng of the DNA template, and 5.5 µL (VNTR1, VNTR2) or 7.5 µL (VNTR3) filtered sterile water. The PCR protocol was as follows: initial denaturation at 94 ℃ for 5 min; followed by 35 cycles, each consisting of initial denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, and extension at 72 ℃ for 45 s; and final extension at 72 ℃ for 10 min. PCR products were identified by dye-based capillary electrophoresis (TsingKe Biological Techn-ology Co., Ltd.). The number of repeats in the alleles was estimated by subtracting the invariable flanking region from the amplicon size divided by the repeat unit length, as determined for reference strain Philadelphia-1 according to a method described previously.
Loci Primer Sequences (5' to 3') Repeat Size/bp Panel Dye Lpms1b ACGAGCATATGACAAAGCCTTG 45 VNTR1 Fam CGGATCATCAGGTATTAATCGC Lpms3 CAACCAATGAAGCAAAAGCA 96 VNTR1 Hex AGGGGTTGATGGTCTCAATG Lpms13 CAATAGCATCGGACTGAGCA 24 VNTR1 TAMRA TGCCTGTGTATCTGGAAAAGC Lpms19 GAACTATCAGAAGGAGGCGAT 21 VNTR2 Fam GGAGTTTGACTCGGCTCAGG Lpms17 CAGCTCACCCCGTATCACTT 39 VNTR2 Hex TAACATCAATGACCGCGAAA Lpms33 ACCACAGCAGTTTGAACATAAT 125 VNTR2 TAMRA GGGAGAAGTTATAGATCTATTCG Lpms34 GAAAAGGAATAAGGCGCAGCAC 125 VNTR3 Hex AAACCTCGTTGGCCCCTCGCTT Lpms35 CTGAAACAGTTGAGGATGTGA 18 VNTR3 TAMRA TTATCAACCTCATCATCCCTG
Table Supplementary Table S2. L. pneumophila MLVA-8 Setup for Capillary Electrophoresis
The BioNumerics software was used to create a maximum likelihood tree using UPGMA, which revealed the genetic distance among the 42 strains.
Two virulence genes lvh and rtxA, which were previously proven to have a strong correlation with LD in clinical cases, were chosen for examination in 79 strains. The primers and PCR reaction procedure were the same as those described previously.
Thirty-three L. pneumophila strains were selected to represent different 33 STs, sources, and sero-groups and subjected to an intracellular growth assay repeated three independent times, with the reference strain Philadelphia-1 strain (ATCC33152) as a positive control.
All the strains were added to J774 cells, and together to co-culture for 1 h, 24 h, 48 h, and 72 h, respectively, then the CFUs of intracellular bacteria were counted to determine growth. J774 cells were incubated in RPMI 1640 tissue culture medium containing 10% calf serum at 37 ℃ with 5% CO2. Phosphate-buffered saline (PBS) was used to dilute the bacteria to 1 × 108 CFU/mL as the primary dilution, which was then diluted 10-fold with the culture medium containing the J774 cells (2 × 105 per well) to reach a multiplicity of infection (MOI) of approximately 10. The 24-well plates containing the culture medium and bacteria were incubated at 37 ℃ in 5% CO2 and with saturated humidity. To measure internalization, PBS was used to wash away extracellular bacteria and other potentially interfering substances. Then, 1 mL of sterile distilled water was added to the wells to release internal bacteria from the host cells, and the CFUs were counted by plating dilutions of the medium on BCYE agar plates (Oxoid Microbiological Products, UK).
Bacterial Isolates and Culture Conditions
SBT and Data Analyses
MLVA and Data Analysis
Detection of Virulence Genes
Intracellular Growth Assay
Legionella-specific 16S rRNA gene was detected for identifying the 79 isolates included in the present study, by which all of the strains belonged to Legionella species, of which eleven serogroups were identified. Two serogroups (LP9 and LP14) were simultaneously identified in one strain (XJ20140386) isolated from a hot spring. The major serogroup was LP1 containing 43 strains (54.43%), the following was LP7 of 12 strains (15.19%), LP6 of 6 strains (7.59%), LP11 of 4 strains (5.06%), LP12 of 3 strains (3.80%), and 9 strains belonging to various serogroups: 2 LP3 strains (2.53%), 2 LP9 strains (2.53%), 2 LP13 strains (2.53%), 2 LP15 strains (2.53%), 1 LP5 (1.27%), and LP10 strain respectively (Table 1).
Serogroup Cooling Tower (n = 49) Bath Water (n = 5) Fountain (n = 10) Hot Spring (n = 13) Clinical (n = 2) Total (n = 79) No. Proportion (%) No. Proportion (%) No. Proportion (%) No. Proportion (%) No. Proportion (%) No. Proportion (%) LP1 30 61.22 2 40.00 4 40.00 6 46.15 1 50.00 43 54.43 LP3 0 0.00 0 0.00 0 0.00 2 15.38 0 0.00 2 2.53 LP5 0 0.00 1 20.00 0 0.00 0 0.00 0 0.00 1 1.27 LP6 4 8.16 0 0.00 0 0.00 1 7.69 1 50.00 6 7.59 LP7 10 20.41 0 0.00 2 20.00 0 0.00 0 0.00 12 15.19 LP9 2 4.08 0 0.00 0 0.00 0 0.00 0 0.00 2 2.53 LP10 1 2.04 0 0.00 0 0.00 0 0.00 0 0.00 1 1.27 LP11 0 0.00 0 0.00 3 30.00 1 7.69 0 0.00 4 5.06 LP12 1 2.04 2 40.00 0 0.00 0 0.00 0 0.00 3 3.80 LP13 0 0.00 0 0.00 0 0.00 2 15.38 0 0.00 2 2.53 LP15 1 2.04 0 0.00 1 10.00 0 0.00 0 0.00 2 2.53 LP9*LP14 0 0.00 0 0.00 0 0.00 1 7.69 0 0.00 1 1.27
Table 1. Serogroup Distribution of 79 Strains
For SBT, full 7-allele profile was obtained of 79 isolates, 22 STs could be found in the EWGLI SBT database (http://bioinf-ormatics.phe.org.uk/legionella/legionella_sbt/php/sbt_homepage.php), but the other 11 profiles could not be found in the database. Novel profiles were submitted to the ESGLI SBT database and acquired the STs.
As shown in Figure 1, the 79 strains were analyzed by SBT, and 33 different STs were found, of which 11 (ST2355-ST2365) were identified for the first time. A total of 17 STs were from cooling towers [index of discrimination (IOD), 0.763], 4 were from bath water (IOD, 0.900), 8 were from hot springs (IOD, 0.897), and 5 were from fountains (IOD, 0.756). ST1 have been found in both cooling towers, fountain and bath water. All these isolates had high IODs.
Ten STs (ST1, ST36, ST630, ST2359, ST1777, ST187, ST2363, ST328, ST641, and ST7) contained more than one strain, while the other 23 STs contained only a single strain, respectively, among which ST1 was the predominant type, accounting for 37.97% (30/79), followed by ST630 (7/79, 8.86%), ST2359 (4/79, 5.0%), and ST1777 (3/79, 3.8%). However, the strains with identical ST types belonged to different serogroups. This was observed in the case of ST1, which corresponded to three different serogroups-LP1, LP7, and LP9.
The results of MST analysis (Figure 2) showed that 33 STs were predicted to form five clonal groups, whereas 5 STs, which differed from every other ST in four or more genes, were identified as singletons. Clonal group 1 contained 11 STs, 4 were isolated from the hot spring (ST690, ST2358, ST2359, ST2360), 4 from cooling tower (ST2355, ST1640, ST1562, ST971), 2 from bath water (ST1639, ST2357), and 1 from fountain (ST2365). Clonal group 2 contained 7 STs, 6 STs were isolated from cooling towers (ST1011, ST7, ST630, ST1604, ST1113, and ST1777), 1 ST (ST1) was distributed in cooling towers, bath water, and fountain. Clonal group 3 contained 5 STs in all: 3 isolated from cooling towers (ST579, ST187, and ST242), 1 from a hot spring (ST2361), and 1 from a fountain and clinical patient (ST36). Clonal group 4 had only 3 STs, ST583 and ST1279 from cooling towers and 1 from a hot spring (ST641). Lastly, clonal group 5 had 2 STs, both of which were from the fountain (ST2363 and ST2364). The five singletons were from various sources, S1 (ST1562), S2 (ST2356), S3 (ST1999), and S5 (ST578) contained 1 strain, and were isolated from cooling tower, bath water, patient and hot spring, respectively. S4 contained 2 stains (ST328), and both were isolated from hot spring in the same city (Kang ding).
Figure 2. Minimum spanning tree analysis of the 79 L. pneumophila from Sichuan, China. The sequence types (STs) are displayed as circles. The size of each circle indicates the number of isolates within the particular type, and the ST are shown near the circle. The colors of the halo surrounding the STs denote types that belong to the same clonal group.
The 42 LP1 strains were selected for MLVA, from which 8 MLVA types were obtained. Using these, we created a maximum likelihood tree, demonstrated in Figure 3. Of the 8 MLVA types, M08 was the most principal type, accounting for 47.62% of the LP1 strains (20/42), the other major type was M07 (10/42, 23.81%), then the following was M01 and M04 (6/42, 14.29%; 2/42, 4.76%). Most of the MLVA were source specific. All but one of the 20 strains of the M08 were obtained from cooling towers; the exception was 1 strain from a fountain. Similarly, all 6 strains of the M01 were obtained from hot springs, except 1 that came from a fountain.
As shown in Figure 3 for of the 42 LP1 strains, 12 different STs and 8 different MLVA types were identified. Comparing these results, it seems that the strains of the same ST can could belong type into more than one MLVA type. For example, the strains of ST1 belonged were typed into 2 different MLVA (M07 and M08), as did the strains of ST630. In the same way, the strains of the same MLVA could belong to more than one ST, such as the strains of M01 were typed into three STs (ST2369, ST2360, and ST971).
The virulence genes rtxA and lvh of L. pneumophila were identified in all of 79 strains. Both loci were present in all strains, it was suggested that the strains isolated from Sichuan area might have the potential infectivity and pathogenicity to human being.
The mouse BALB/c macrophage cell line J774 was used for testing the intracellular growth ability of the 33 strains, and Philadelphia-1 (ATCC33152) was used as a positive control. To further evaluate the intracellular growth ability of the strains, plate culture counting was performed. Thirty-one isolates (including ATCC33152) had bacterial concentrations of 104-105 CFU/mL, 105-106 CFU/mL, 106-107 CFU/mL and 107-108 CFU/mL on 1 h, 24 h, 48 h, and 72 h of infection, respectively, and showed high proliferative ability in J774 macrophage cells. However, XJ20140360 (ST578), XJ20140453 (ST1604), and WZL (ST1999) exhibited bacterial concentrations of 1 × 105 CFU/mL, 4 × 103 CFU/mL, 2 × 104 CFU/mL on 48 h, and 3 × 104 CFU/mL, 3 × 103 CFU/mL, 2.2 × 104 CFU/mL on 72 h, respectively, and showed low proliferative ability in J774 macrophage cells (Figure 4).