Supplementary Information ISME J: submitted 12.08.2015
Legionella species diversity and dynamics from surface reservoir to tap water: from cold adaptation to thermophily
Running title: Dynamics of Legionella species in drinking water
SupplementaryInformation
revised August 12, 2015
Authors: René Lesnik, Ingrid Brettar and Manfred G. Höfle#
Department of Vaccinology and Applied Microbiology,
Helmholtz Centre for Infection Research (HZI),
Inhoffenstrasse 7,
D-38124 Braunschweig, Germany
# Author for correspondence: Manfred G. Höfle, ; Phone: 49-531-6181-4234
Supplementary Tables
Table S1 All Legionella phylotypes observed in raw water and along the treatment process (PTRs) are listed together with their closest isolated species. Note that sequence similarity does not always reflect phylogenetic clustering (compare Figure 2). Source abbreviations are referring to the Grane and Ecker reservoir before and after chlorination treatment (GR, ER and GT, ET, respectively), the storage containers Lewerberg (Lb) and Lindenberg (Li) and finished cold drinking water from the tap (TW). Non-Legionella species are labelled (*).
Phylotype / Sources / Closest isolated species; accession no. (16S rRNA gene sequence similarity) / Accession no. of representative sequencePTR-01 / GR, GT, ER, ET, Li, TW / Legionella dumoffii (T); Z32637 (98.0%) / KP822848
PTR-02 / GR, ER / Legionella dumoffii (T); Z32637 (98.3%) / KP822889
PTR-03 / GR, ER, Li, TW / Legionella longbeachae (T); ATCC 33462; AY444740 (98.8%) / KP822915
PTR-04 / GR, GT, Li / Legionella dresdenensis (T); type strain: W03-356; AM747393 (98.8%) / KP822856
PTR-05 / GR, ER, Li / Legionella longbeachae (T); ATCC 33462; AY444740 (98.8%) / KP822906
PTR-06 / GR, Lb, TW / Legionella dresdenensis (T); type strain: W03-356; AM747393 (98.8%) / KP822921
PTR-07 / ER / Legionella erythra (T); Z32638 (97.1%) / KP822868
PTR-08 / Reference / Legionella pneumophila (T); Philadelphia 1; AE017354 (100.0%) / -
PTR-09 / GR / Legionella tucsonensis (T); Z32644 (98.8%) / KP822840
PTR-10* / Lb, Li / Methylocaldum tepidum (T); LK6; U89297 (95.4%) / KP822901
PTR-11 / GR / Legionella impletisoli (T); OA1-1; AB233209 (95.4%) / KP822824
PTR-12 / GR / Legionella pneumophila (T); Philadelphia 1; AE017354 (98.0%) / KP822829
PTR-13 / GR / Legionella erythra (T); Z32638 (98.3%) / KP822830
PTR-14 / GR / Legionella tucsonensis (T); Z32644 (98.8%) / KP822832
PTR-15 / GR / Legionella dresdenensis (T); type strain: W03-356; AM747393 (98.8%) / KP822834
PTR-16 / GR / Legionella pneumophila (T); Los Angelos-1; HQ287902 (97.7%) / KP822838
PTR-17 / GR / Legionella dresdenensis (T); type strain: W03-356; AM747393 (97.7%) / KP822843
PTR-18 / GR / Legionella drancourtii (T); type strain: LLAP12; X97366 (98.6%) / KP822844
PTR-19 / GR / Legionella dresdenensis (T); type strain: W03-356; AM747393 (97.7%) / KP822845
PTR-20 / GR / Legionella pneumophila (T); U8W; ATCC 33737; AF122885 (98.3%) / KP822847
PTR-21 / GR / Legionella dresdenensis (T); type strain: W03-356; AM747393 (98.3%) / KP822852
PTR-22 / GR / Legionella pneumophila (T); Philadelphia 1; AE017354 (97.7%) / KP822854
PTR-23 / GR / Legionella pneumophila (T); U8W; ATCC 33737; AF122885 (98.0%) / KP822855
PTR-24 / GR / Legionella pneumophila (T); Philadelphia 1; AE017354 (98.6%) / KP822859
PTR-25 / ER / Legionella birminghamensis (T); NCTC12437; Z49717 (95.7%) / KP822865
PTR-26 / ER / Legionella nautarum (T); NCTC 12375 = ATCC 49506; Z49728 (96.0%) / KP822866
PTR-27 / ER / Legionella longbeachae (T); ATCC 33462; AY444740 (97.7%) / KP822869
PTR-28 / ER / Legionella waltersii (T); 2074-AUS-E; ATCC 51914; AF122886 (97.4%) / KP822870
PTR-29 / ER / Legionella rowbothamii (T); LLAP6; X97359 (97.4%) / KP822873
PTR-30 / ER / Legionella rowbothamii (T); LLAP6; X97359 (97.7%) / KP822875
PTR-31 / ER / Legionella dresdenensis (T); type strain: W03-356; AM747393 (95.7%) / KP822876
PTR-32 / ER / Legionella dresdenensis (T); type strain: W03-356; AM747393 (96.3%) / KP822877
PTR-33 / ER / Legionella pneumophila (T); U8W; ATCC 33737; AF122885 (95.4%) / KP822878
PTR-34 / ER / Legionella longbeachae (T); ATCC 33462; AY444740 (97.7%) / KP822882
PTR-35 / ER / Legionella feeleii (T); ATCC 35072; X73395 (97.7%) / KP822890
PTR-36 / ER / Legionella tucsonensis (T); Z32644 (98.8%) / KP822891
PTR-37 / ER / Legionella tucsonensis (T); Z32644 (98.3%) / KP822892
PTR-38 / Li / Legionella dresdenensis (T); type strain: W03-356; AM747393 (95.9%) / KP822900
PTR-39 / TW / Legionella dresdenensis (T); type strain: W03-356; AM747393 (96.8%) / KP822910
PTR-40 / TW / Legionella worsleiensis (T); nctc 12377; Z49739 (98.6%) / KP822911
PTR-41 / TW / Legionella drancourtii (T); type strain: LLAP12; X97366 (99.1%) / KP822913
PTR-42* / TW / Methylocaldum szegediense (T); OR2; U89300 (95.0%) / KP822917
Table S2The Legionella phylotypes of cold (CW) and hot (HW) drinking water (PTLs) are listed together with their closest isolated species. Labelled source acronyms(*) indicate that relative abundance wasalways below 1.0 %.
PTL-01 / HW, CW / Legionella longbeachae (T); ATCC 33462; AY444740 (98.9%) / KP863810
PTL-02 / HW, CW* / Legionella pneumophila (T); Philadelphia 1; AE017354 (99.7%) / KP863816
PTL-03 / HW, CW / Legionella worsleiensis (T); nctc 12377; Z49739 (99.4%) / KP863825
PTL-04 / HW, CW / Legionella anisa (T); Z32635 (98.3%) / KP863804
PTL-05 / HW, CW / Legionella dresdenensis (T); type strain: W03-356; AM747393 (94.8%) / KP863822
PTL-06 / HW*, CW / Legionella dumoffii (T); Z32637 (97.7%) / KP863811
PTL-07 / HW / Legionella impletisoli (T); OA1-1; AB233209 (95.1%) / KP863839
PTL-08 / HW / Legionella pneumophila (T); U8W; ATCC 33737; AF122885 (98.3%) / KP863845
PTL-09 / HW, CW / Legionella quateirensis (T); nctc 12370; Z49732 (98.6%) / KP863854
PTL-10 / HW, CW / Legionella pneumophila (T); U8W; ATCC 33737; AF122885 (97.4%) / KP863829
PTL-11 / CW / Legionella rowbothamii (T); LLAP6; X97359 (96.3%) / KP863835
PTL-12 / CW / Legionella pneumophila (T); Philadelphia 1; AE017354 (97.4%) / KP863831
PTL-13 / CW / Legionella quateirensis (T); nctc 12370; Z49732 (99.7%) / KP863833
PTL-14 / HW* / Legionella pneumophila (T); Philadelphia 1; AE017354 (98.6%) / KP863827
PTL-15 / HW / Legionella rubrilucens (T); Z32643 (97.1%) / KP863846
PTL-16 / HW* / Legionella pneumophila (T); Philadelphia 1; AE017354 (99.1%) / KP863847
PTL-17 / HW / Legionella rubrilucens (T); Z32643 (97.7%) / KP863828
PTL-18 / HW* / Legionella pneumophila (T); Philadelphia 1; AE017354 (97.1%) / KP863848
PTL-19 / HW / Legionella worsleiensis (T); nctc 12377; Z49739 (99.4%) / KP863843
PTL-20 / HW / Legionella anisa (T); Z32635 (98.3%) / KP863849
PTL-21 / HW / Legionella quateirensis (T); nctc 12370; Z49732 (99.4%) / KP863850
PTL-22 / HW / Legionella tucsonensis (T); Z32644 (98.0%) / KP863837
PTL-23 / HW / Legionella tucsonensis (T); Z32644 (98.6%) / KP863852
PTL-24 / HW / Legionella erythra (T); Z32638 (98.3%) / KP863853
PTL-25 / HW / Legionella pneumophila (T); U8W; ATCC 33737; AF122885 (98.0%) / KP863855
PTL-26 / HW / Legionella worsleiensis (T); nctc 12377; Z49739 (98.6%) / KP863856
PTL-27 / HW / Legionella worsleiensis (T); nctc 12377; Z49739 (98.9%) / KP863841
PTL-28 / CW / Legionella quateirensis (T); nctc 12370; Z49732 (99.4%) / KP863857
PTL-29 / CW / Legionella worsleiensis (T); nctc 12377; Z49739 (98.6%) / KP863858
PTL-30 / CW / Legionella quateirensis (T); nctc 12370; Z49732 (99.4%) / KP863844
PTL-31 / CW* / Legionella yabuuchiae (T); OA1-2; AB233210 (95.1%) / KP863859
PTL-32 / CW / Legionella brunensis (T); Z32636 (96.3%) / KP863860
PTL-33 / CW / Legionella dumoffii (T); Z32637 (99.1%) / KP863861
PTL-34 / CW / Legionella pneumophila (T); U8W; ATCC 33737; AF122885 (96.8%) / KP863838
PTL-35 / CW / Legionella drozanskii (T); LLAP1; X97355 (98.0%) / KP863862
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Supplementary Information ISME J: submitted 12.08.2015
Table S3 Summary of abundances of the major phylotypes of Legionella (PTL) occurring in cold and hot drinking water. Relative abundances were obtained from SSCP gels and cell numbers were calculated from those and Legionella spp. cell numbers as described in Material and Methods. PTLs are sorted according to decreasing average abundance (Mean) and their range is given by lowest and highest values (Minimum and Maximum, respectively). Note that hot water PTLs (PTL-01, -02) show lower variation and that the two major hot water PTLs show a significant correlation with temperature at significance levels of 0.01 (**) and 0.05 (*), respectively.
OTU / Temperature correlation / Cell numbers [cells L-1] / Rel. abundance [%]Trend (R2) / Mean / SD / Min. / Max. / Mean / SD / Min. / Max.
Cold water (n=17)
All / positive (0.10) / 1.65E+06 / (+) / 2.0E+06 / 3.14E+05 / 8.99E+06 / 100.00
PTL-01 / positive (0.18) / 3.44E+05 / (+) / 5.5E+05 / 1.73E+04 / 2.28E+06 / 17.52 / (+) / 11.0 / 4.38 / 41.00
PTL-30 / negative (0.01) / 1.85E+05 / (+) / 1.3E+05 / 1.02E+04 / 5.21E+05 / 15.96 / (+) / 10.9 / 1.64 / 46.46
PTL-06 / positive (0.11) / 2.29E+05 / (+) / 2.7E+05 / 3.42E+04 / 1.17E+06 / 15.50 / (+) / 9.5 / 3.28 / 36.03
PTL-09 / positive (0.04) / 2.67E+05 / (+) / 3.8E+05 / 0.00E+00 / 1.68E+06 / 13.45 / (+) / 7.8 / 0.00 / 24.73
PTL-04 / positive (0.13) / 2.11E+05 / (+) / 3.7E+05 / 2.76E+04 / 1.63E+06 / 9.64 / (+) / 5.7 / 0.00 / 18.10
Hot water (n=12)
All / positive (0.73)** / 3.75E+06 / (+) / 1.7E+06 / 8.63E+05 / 6.28E+06 / 100.00
PTL-01 / positive (0.66)** / 1.36E+06 / (+) / 6.6E+05 / 2.86E+05 / 2.13E+06 / 37.48 / (+) / 5.3 / 24.17 / 44.70
PTL-02 / positive (0.44)* / 8.87E+05 / (+) / 5.4E+05 / 1.56E+05 / 1.74E+06 / 22.74 / (+) / 8.8 / 8.45 / 37.97
PTL-03 / positive (0.14) / 3.36E+05 / (+) / 1.9E+05 / 1.82E+04 / 6.29E+05 / 9.47 / (+) / 5.2 / 1.93 / 19.33
PTL-19 / positive (0.08) / 1.37E+05 / (+) / 1.5E+05 / 0.00E+00 / 4.18E+05 / 5.06 / (+) / 5.9 / 0.00 / 17.54
PTL-20 / positive (0.25) / 1.66E+05 / (+) / 1.9E+05 / 0.00E+00 / 7.43E+05 / 4.35 / (+) / 3.7 / 0.00 / 11.83
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Supplementary Information ISME J: submitted 12.08.2015
Supplementary Figures
Figure S1Seasonal dynamics of total bacterial cell counts (open circles, dashed line) in finished drinking water and water temperature by the time of sampling (filled diamonds, solid line) of cold (A) and hot (B) drinking water. The solid vertical line indicates the turn of the year.
Figure S2Effect of treatment (flocculation and sand filtration) on total bacterial cell counts of the bulk water. Dashed bars represent values from raw water, black bars values after treatment, grey bars values after chlorination and checked bars values of finished drinking water. Sampling sites labelled GR and ER were from the Grane and Ecker reservoir, samples for processed raw water are called GR2 and ER2, GT1 and ET1 were after chlorination, respectively. Lb = Lewerberg and Li = Lindenberg were from storage containers. TW = Tap water at the date indicated. All samples were taken on March 2nd, 2004, except the last which was taken 3 days later (*).
FigureS3Legionella-specific SSCP fingerprints of seasonal DNA samples from drinking water. Distinct fingerprints and dynamics of Legionella species are observed in cold (A) and hot (B) drinking water. These banding patterns are the basis for the cluster analyses given in Fig. 4A and B. Their resulting clusters for cold (CA – CF) and hot water (HA – HC) are indicated.
Figure S4Cluster analysis of Legionella-specific SSCP fingerprints (Figure S4) from cold (A) and hot (B) drinking water. A similarity threshold of 70% (indicated by the dotted line) was applied in both cluster analyses for grouping of the individual community profiles.
Figure S5Comparison of the rank abundance curves of Legionella phylotypes (PTLs)in cold (black diamonds) and hot (grey triangles) drinking water.Patterns of the Legionella rank abundance curves show that the composition structures of both communities are very similar (slope of the regression curves). Variation is high as the coefficient of determination of both regression curves is indicating: R2 = 0.88 and R2 = 0.83 for cold and hot water, respectively.
Figure S6Comparison of mean values and standard deviation of total cell counts of bacteria with cell counts of Legionella spp. in cold and hot drinking water determined by real-time PCR as described in Material and Methods. Although bacterial cells were generally more abundant in cold water (black boxes), the load of Legionella spp. cells per litre was significantly higher in hot water (grey boxes). Width of the boxes is drawn to scale of the number of observations for each type of water, namely 17 and 12 for cold and hot water, respectively. * P value < 0.05; ** P value < 0.01.
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