Environmental Surveillance and Molecular Epidemiology of Waterborne Pathogen Legionella

Environmental surveillance and molecular epidemiology of waterborne pathogen Legionella pneumophila in health care facilities of Northeastern Greece: a four- year survey

Ioanna G. Alexandropoulou1,*, Spyridon Ntougias2, Theocharis G. Konstantinidis1, Theodoros Parasidis1, Maria Panopoulou3 and Theodoros C. Constantinidis1

1 Laboratory of Hygiene and Environmental Protection, Medical School, Democritus University of Thrace, Campus (Dragana) Building 5, 68100 Alexandroupolis, Greece

2 Department of Environmental Engineering, Democritus University of Thrace, Vas. Sofias 12, 67100 Xanthi, Greece

3Microbiology Laboratory, Medical School, Democritus University of Thrace, Campus (Dragana), 68100 Alexandroupolis, Greece

Corresponding author

* Phone: (+30) 25510 30554, fax: (+30) 25510 30554, email: [email protected] Supplementary Fig. S1. Schematic diagram of water distribution system in Hospital A.

Hospital B: Water distribution System

S S F4th S S F4th

F3rd S S F3rd

F2nd S S F2nd

F1st S S F1st

GF S S GF

S Hospital Boiler GF Entrance

S Water Tank GF

UF: Underground Floor, GF: Ground Floor, :Cold water :Hot water F1st : First Floor, F2nd : Second Floor, S : Sampling site Supplementary Fig. S2. Schematic diagram of water distribution system in Hospital B.

Hospital C: Water distribution System

F3rd S S

F2nd S S

F1st S S

S S Boiler

S Hospital S Cooling Entrance Water Tank GF Tower

UF S F3rd Roof

UF: Underground Floor, GF: Ground Floor :Cold water :Hot water F1st : First Floor, F2nd : First Floor, F3rd : First Floor, F3rd Roof: Third Floor’s roof, S : Sampling site Supplementary Fig. S3. Schematic diagram of water distribution system in Hospital C.

Hospital D: Water distribution System

GF S S F2nd

S S S S F1st S S F2nd S

Hospital S S Entrance GF S F1st

Boiler S

Water Tank S UF

UF: Underground Floor, GF: Ground Floor, :Cold water :Hot water F1st : First Floor, F2nd : Second Floor, S : Sampling site Supplementary Fig. S4. Schematic diagram of water distribution system in Hospital D.

Hospital E: Water distribution System

Hospital S Entrance S F2nd S S

F1st S S S

Boiler Cooling S Tower Water Tank GF S F1st Roof

UF: Underground Floor, GF: Ground Floor, :Cold water :Hot water F1st : First Floor, F2nd : Second Floor, F1st Roof: First Floor’s roof, S : Sampling site Supplementary Fig. S5. Schematic diagram of water distribution system in Hospital E.

0.0020.002

L27isolate L27 Togus1Legionella pneumophila str. Togus 1 Cluster VI: Hospital C (2009) L35isolate L35 AT43290Legionella pneumophila subsp. pneumophila ATCC 43290 PhiladelphLegionella pneumophila subsp. pneumophila str. Philadelphia 1 LegionellaHL06041035 pneumophila subsp. pneumophila str. HL06041035 L6Legionella pneumophila str. Paris L19 L47isolate L1 L52isolate L6 L92isolate L10 L1isolate L11 Cluster V: Hospital A (2008 – 2011) L10isolate L18 L11isolate L19 L18isolate L22 L22isolate L52 Parisisolate L92 L37isolate L37 Cluster IV: Hospital B (2008, 2010) L91isolate L91 LegionellaATCC35289 pneumophila ATCC 35289 Leiden1Legionella pneumophila str. Leiden 1 L50 L56isolate L50 L58isolate L56 L72isolate L58 L77isolate L72 Cluster III: Hospital B (2008 – 2009) & Hospital D (2009 – 2011) L100isolate L77 AlcoyLegionella pneumophila 2300/99 Alcoy isolate L100 L40isolate L40 Cluster II: Hospital E (2010) L14isolate L14 L106isolate L26 L107isolate L28 L104isolate L30 L90isolate L31 L34isolate L34 Cluster I: Hospital C (2008 – 2010) L31isolate L90 L30isolate L104 L28isolate L106 L26isolate L107

(A) Hex2qasdLegionella pneumophila Hextuple_2q, NC_017525 PhiladelphLegionella pneumophila Philadelphia 1, NC_002942 Hex3aLegionella pneumophila Hextuple_3a, NC_017526 iL27solate L27 (Cluster VI) LegionellaThunder pneumophila Thunder Bay, NC_021350 ATCC43290Legionella pneumophila ATCC 43290, NC_016811 LegionellaCP003885 pneumophila LPE509, NC_020521 LegionellaLens pneumophila Lens, NC_006369 LegionellaHL06041035pneumophila HL06041035, NC_018140 ParisLegionella pneumophila Paris, NC_006368 L52isolate L52 (Cluster V) L31isolate L31 (Cluster I) iL58solate L58 (Cluster III) LegionellaLorraine pneumophila Lorraine, NC_018139 LegionellaAlcoy pneumophila 2300/99 Alcoy, NC_014125 LegionellaCorby pneumophila Corby, NC_009494 iL37solate L37 (Cluster IV) L40isolate L40 (Cluster II)

(B) Fig. S6. Unrooted trees based on concatenated sequence analysis of A) asd, mip and mompS genes of representative Legionella pneumophila strains from all RAPD profiles obtained, and B) asd, flaA, mip, mompS, neuA, pilE and proA genes of representative isolates from each

Legionella pneumophila cluster indentified in the abovementioned concatenated sequence analysis. Construction of the trees was conducted by the maximum-likelihood method and scale bar represents substitutions per site.