What is meant by a water infection

Legionella in drinking water when is it dangerous?

Transcript

1 Medical Faculty Carl Gustav Carus Institute for Medical Microbiology and Hygiene / Institute for Virology Legionella in drinking water when is it dangerous? Fulda, BÄMI, Christian Lück Institute for Medical Microbiology and Hygiene TU Dresden Consulting laboratory for Legionella of the Robert Koch Institute

2 C Lück IMMH TUD Legionella Laboratory Legionella in the Dresden MOPO prefabricated building area

3 Legionella in the Gagfah Sächsische Zeitung 22 / in 248 of 964 systems (25.7%) the technical measure value was exceeded. In one system only / 100ml after thermal disinfection and repairs dropped briefly to 6000 / 100ml Now again / 100ml Gagfah: We do everything necessary and possible to eliminate the legionella problems. We are dependent on the help of the tenants. (Use taps). Otherwise the legionella would remain in the pipes. Gagfah installed shower filters and lifted the shower ban

4 Legionella in the prefabricated building area Legionella in the water No illnesses The tenant wanted to save on rent debt. Technical measures by the housing association reduced the detection rate from around 30% to 1%. Detection limit 10 / 100ml C Lück IMMH TUD Legionella laboratory

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6 Incidence of Legionella in buildings KbE / l> other public. Baths Nursing homes, hospitals, gyms, children's facilities C Lück IMMH TUD Legionella-Labor Hentschel et al. 2006, Federal Ges bl

7 Legionella infestation (retrograde analysis of samples) C Lück IMMH TU Dresden Partners or institutions: IHPH Bonn Dr.Pleischl, Dr.Koch, Prof. Exner University of Münster, Prof. Mathys Lower Saxony LGA; Hanover Dr. Suchewirth Ing.-Büro für Trinkwasserhygiene Waldmann, Rödermark, IBW-H Herr Hentschel; GA Frankfurt, nationwide operating hospital operator Institute for Medical Microbiology and Hygiene Dresden Hygiene Institute of the Ruhr Area Gelsenkirchen (DB Langer) TUD Dr.-Ing. Rühling Faculty of Mechanical Engineering Institute for Energy Technology

8 Legionella infestation (retrograde analysis of samples) TUD Dr.-Ing. Rühling Faculty of Mechanical Engineering Institute for Energy Technology C Lück IMMH TU Dresden

9 Legionella infestation (retrograde analysis of samples) C Lück IMMH TU Dresden TUD Dr.-Ing. Rühling Faculty of Mechanical Engineering Institute for Energy Technology

10 Environmental follow-up status by setting of infection, EU / EEA, 2011 Setting n (environmental status known)% investigation% Legionella found when site investigated Community-acquired 1,196 17% 39% Domestic travel% 63% Travel abroad% 41% Healthcare associated% 86% Other 54 48% 88% Total 1.707 20% 51%

11 Biofilm 1. Entry from drinking water supply 2. Propagation on Biofim in protozoa (complex ecosystem) 3. Release and possible infection of H.Y. Lau and N.J. Ashbolt Journal of Applied Microbiology 2009

12 amoebas are insidious czichos

13 Ecology of Legionella All Legionella must multiply in the environment Amoebas determine the colonization of a terminal strand Environmental resistance C Lück IMMH TU Dresden AbuKwaik described Species pneumophila serogroup 1 MAb 3-1 positive bozemanii micdadei longbeachae

14 Pathogenicity / Virulence With some bacteria the cause of their pathogenicity is quite obvious. Others have not yet disclosed their virulence factors Czichos

15 Distribution of the 10 novel Dot / Icm T4SS effectors in 87 pneumophila isolates analyzed for the presence of homologues of the novel pneumophila 130b effectors ltpa to ltpj by PCR Class 1 formed by the rare effectors ltpb, ltpc, ltpe, ltpf, and ltpj, detected in <15% of strains Class 2 contains the more prevalent effectors ltpa, ltpd, ltpg, ltph, and ltpi, found in 34% to 62% of isolates no correlation between the presence of the novel effectors and the clinical or environmental origin of the strains. C Lück IMMH TUD Legionella Laboratory Schroeder et al. JB 2010

16 Infection dose for Legionella pneumonia in guinea pigs animal model Infection route (CFU) Ref Aerogen (Phil) 12/12 Fever Aerogen LD50 intraperitoneal LD50 strain Corby aerogenic strain Corby 100% lethal, aerogenic strain Phil no lethality, aerogenic strain Corby, intratracheal, LD50 strain Corby , intraperitoneal, LD50 Phil strain, intratracheal, LD50 Phil strain, intraperitoneal, LD nt Berendt, 1980 Jepras, 1985 Lück, unpubl. Bacteria grown extracellularly CFU = number of bacteria 16

17 Legionella diagnostics culture on special agar, sensitivity 10-70% urine antigen detection (only pneumophila Sg 1) Real-time PCR (in respiratory samples) all Legionella species (16s rrna, 5s rrna, mip) pneumophila 16S rrna gene, mip gene Sg1 LPS gene clone specific Paris clone (IS elements) Direct typing from clinical samples RARE: Antigen detection in the respiratory material Antibody detection C Lück IMMH TU Dresden

18 Legionnaires' case definitions RKI (draft 2014) Laboratory diagnostic evidence Isolation of pathogens only from secretions of the respiratory tract (e.g. BAL, tracheal secretions, sputum), lung tissue or pleural fluid. Nucleic acid detection (e.g. PCR) only from resp. Material Antigen detection only in urine (e.g. ELISA, immunochromatography), indirect (serological) detection: Antibody detection using IFT (significant change (titer increase) between two samples) Antibody detection using IFT (one-time significantly increased value only for the detection of Legionella pneumophila serogroup 1). Detection of antigen in secretions of the respiratory tract and detection of IgM and IgG antibodies by means of ELISA (also a combination) are not considered laboratory diagnostic evidence due to insufficient validation. Epidemiological confirmation: Epidemiological connection with laboratory-diagnosed infection in humans. In the case of travel infections: (hotel, campsite, ship, etc.) record the exact travel data (start of illness, travel time, hotel name with address, etc.). Data is transmitted to the European network ELDSnet via the RKI. C Lück IMM Identification of hotels and the like as sources of infection with subsequent monitoring and, if necessary, decontamination measures.

19 Legionella people reported to the RKI (1.4.13) Legionella 2001 Legionella 2002 Legionella 2003 Legionella 2004 Legionella 2005 Legionella 2006 Legionella 2007 Legionella 2008 Legionella 2009 Legionella 2010 Legionella 2011 Legionella 2012 Legionella 2013 Leg Wo Leg Wo Q-Fever 2013 Listeriosis 2013 Ornithosis 2013 C Lück IMMH TU Dresden

20 Sources of infection Legionella pneumonia Often Rarely Very rarely (yet) not: C Lück IMMH TUD Legionella laboratory hot water systems in the home environment, in hotels, baths, in hospitals, etc. Recooling plants with moist cooling Whirl pools Thermal baths, humidifiers in restaurants and shops (Food), inhalers indoor fountains / ornamental fountains, birth tubs gastric irrigation probe / transoesophageal echo probe (tap water) wound infections after bathing, ice machines, dental units, street humidifiers, windshield wipers, garden soil (longbeachae), car washes, greenhouses

21 Serological typing - Legionella isolates (according to origin)% 90% 80% 70% 60% 50% non-pneumophila pneumophila 2-15 pneumophila Sg 1 (MAb 3/1 neg) pneumophila Sg 1 (MAb3 / 1 pos) Mab 3 -1 positive strains are more virulent Molecular basis not fully understood Hydrophobic LPS = better transmission 40% 30% 20% Outbreak strains are MAb2 (3-1) positive 10% 0% Outpatient Travel-associated Nosocomial Routine Water C Lück IMMH TUD Legionella- laboratory

22 Phylogenetic relationships between 122 different ST of pneumophila as population snapshot using eburst (n = 396) Comparison of allele profiles (flaa, pile, asd, mip, momp, proa, neua) 18 clonal complexes 317 isolates belong to a clonal group (70 ver . Sequence types) 79 isolates singletons (52 different sequence types) underlined in red: sequence types unique in Germany C Lück IMMH TUD Legionella-Labor

23 C Lück IMMH TUD Legionella-Labor E-burst patient isolates

24 Distribution of ST (clones) EWGLI Database (Stand) 5071 Clinical / 2706 Water Isolates 1577 ST Water Clinical 45% of diseases caused by 6 clones: Local clones: ST182, 332, 334 Unique clones can cause diseases / outbreaks ST1 3-1 pos ST1 3-1 neg ST8 ST9 ST20 ST23 ST27 ST36 ST37 ST40 ST42 ST47 ST59 ST62 ST79 ST82 ST94 ST146 ST182 ST332 ST334 ST287 C Lück IMMH TUD Legionella laboratory

25 Outbreak GA Soest 160 cases of pneumonia from August 1 to September 6 (confirmed) 2 (3) deaths. Clinical isolates from 7 patients: pneumophila, serogroup 1, Mab subtype Knoxville, ST 345 (rare) This epidemic strain was found 2 RKW from 2 companies sewage treatment plant other environmental sources. Only about 10% of the water isolates. An employee of a company falls ill

26 (1) Recommendations for carrying out a risk analysis in accordance with the Drinking Water Ordinance (UBA) With the new regulation by the Second Ordinance amending the Drinking Water Ordinance, the obligations of the UsI (entrepreneur or other owner) if the technical measure value for Legionella is exceeded are stipulated. 100KbE Legionella / 100ml According to 16 paragraph 7 number 2 TrinkwV 2001, the preparation of a risk analysis is mandatory. For special risk groups or special facilities such as B. Hospitals may require hospital hygiene requirements that go beyond the measures described here to ensure drinking water hygiene. Such additional requirements are not the subject of this recommendation. Drinking Water Ordinance of May 21, 2001 (Federal Law Gazette I p. 959), most recently amended by the second ordinance amending the Drinking Water Ordinance of December 5, 2012 (Federal Law Gazette I p. 2562). C Lück IMMH TUD

27 Evaluation orientating examination systemic examination according to 14 para. 3 of the Drinking Water Ordinance 2001 Legionella (CFU / 100 ml) 1) Evaluation measure Further investigations. 3) Follow-up examination> extremely high level of contamination Direct hazard prevention required (disinfection and restriction of use, e.g. ban on showering) Refurbishment required Immediately 1 week after disinfection or refurbishment> high contamination Refurbishment requirement depends on the result d. Further examinations Immediately -> 100 medium contamination None Within 4 weeks - <100 No / low contamination None None After 1 year (after 3 years) 2) 1) CFU = colony-forming unit 2) Will be carried out at two follow-up examinations. If less than 100 Legionella are detected in 100 ml every year, the examination interval can be extended to a maximum of 3 years. 3) If the preliminary investigation is carried out with a sample size that corresponds to that of a more extensive investigation, the measures specified in Table 1b apply directly. C Lück IMMH TUD Legionella Laboratory

28 C Lück IMMH TUD Legionella laboratory

29 Genotyping results of Strain isolated in the Netherlands Primary prevention: Routine examination Secondary prevention: Search for IQ by disease Conclusion. primary prevention efforts do not include the most important reservoir (s) causing the transmission of pneumophila in the Netherlands Euser et al Eur JCM C Lück IMMH TUD Legionella-Labor

30 domestic environment hospital water samples (patient: Lp1 Olda, ST1) Legionella number in the home environment: 2/2 samples positive: 8 or 100 / 100ml Lp1 MAbtype Olda, ST 1 (1,4,3,1,1,1, 1) Water installation technically outdated and poorly maintained. Legionella number in the KH 1/8 samples positive Lp1 Lp10 4 / 100ml Bellingham ST196 (3,10,1,28,14,9,11) ST: nt in 8/15 cases CAP Legionella CFU in the normal range in the home environment Correct time? ? Correct extraction point? C Lück IMMH TU Dresden

31 C Lück IMMH TUD CRISPR / CAS region Lp1 ST 62 strains ULM L etc.

32 Is the technical measure value validly justified? Hentschel et al. Federation. Ges Bl No evidence of the epidemiological validity of the technical measure value for Legionella of 100KBE / 100ml - No epidemiological data collected. Good technology with a high probability associated with non-detection of Legionella. However, technical deficiencies are associated with legionella contamination. The more technical deficiencies there are, the higher the legionella contamination C Lück IMMH TUD Legionella Laboratory

33 Legionella in drinking water systems: when do we have to fear it? Epidemiology begins with the diagnosis 900 reported versus at least estimated cases Diagnostic methods are better than their reputation Attention Legionella in water Infection - may but must not be Is it more important to detect infections than Legionella in water? C Lück IMMH TU Dresden

34 Legionella in drinking water systems: when do we have to fear it? Virulent legionella also occur without diseases or do we not find the diseases? The number of germs can fluctuate fold. Different virulence ?? Prediction of markers ?? Mab 3-1 good with clinical strains poor with environmental strains In the water focus on virulent clones ?? Mab2, 3-1 positive sequence type Non-pneumophila species Are they relevant? C Lück IMMH TUD Legionella Laboratory

35 Summary Legionella are widespread water bacteria Multiplication in C warm water peripherally Prevented persons are particularly susceptible There is no dose-effect correlation No exact limit values ​​for the amount in the water Virulent clones responsible for the majority of infections Many Legionella are harmless but which ??? Preventive: high heating, chlorination, filter cold water <20 C warm water> 60 C water must flow C Lück IMMH TUD Legionella laboratory

36 IMMH Thank you for your attention, Dr. Jürgen Helbig Sigrid Gäbler Kerstin Lück Susann Menzel Tetyana Koshkolda Markus Petzold Prof. Dr. med. Enno Jacobs RKI: Klaus Heuner Financial support BMBF RKI DFG EU Health Dept. Soest Dr. Brockmann Prof Dr. Martin Exner

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38 C Lück IMMH TUD Legionella laboratory sampling points according to DVGW 551

39 Technical defects C Lück IMMH TUD Legionella-Labor Hentschel et al. Federation. Ges Bl. 2011

40 Search for the source of infection Patient material Water samples 2-3 colonies at least 10 colonies 1. Serological typing in pneumophila: serogroup, MAb subtype or species determination: MALDI-TOF, DNA sequence (mip, 16S rrna) 2. Genomic fingerprint :, SBT, PFGE, AFLP, RAPD-PCR identity> transfer from the water system C Lück IMMH TUD Legionella laboratory

41 Microbiological monitoring for monitoring nosocomial legionnaires' culture of water boiler and distal ends? Culture from water samples positive culture negative detection of serogroup 1 MAB 3-1 positive detection of serogroup 2-14, other species Legionella tests available in cooperation / reference laboratory urine antigen test on site, culture / DNA detection culture / DNA detection Yu, V .: Infect. Control Hosp. Epidemiol. 19:, Allegheny County Health Dept. Directive:

42 C Lück IMMH TUD Legionella laboratory

43 Molecular typing scheme Exchange of types not strains or DNA C Lück IMMH TUD Legionella laboratory adapted from Fry et al. 1999

44 Typing pneumophila Monoclonal antibody (Mab) typing Sequence BasedTyping (SBT) Chip Typing Spoligotyping Fast, reproducible, exchangeable, cheap, good discrimination C Lück IMMH TUD Legionella-Labor

45 Culture BYCE + / blood agar - BYCE- / blood agar ± latex agglutination No Legionella Lp Sg 1 Lp Sg 2-14 species negative Determination of the mab subtype ELISA / IFA Dresden panel Determination of the mab serogroup ELISA / IFA DFA MALDI-TOF Mab3 / 1 pos Philadelphia , Allentown, Benidorm, Knoxville France Mab3 / 1 neg Olda, Oxford, Heysham, Camperdown, Bellingham Denver mip sequencing genotyping SBT, PFGE, ALFP species serogroup possibly mab subtype ST

46 What is a monoclonal (MAb) subtype? Reactivity with a panel of monoclonal antibodies Designation of a type according to a strain with this pattern e.g. B. 1,2,3 for Knoxville 1, 2, 5, 7 for Benidorm Dresden 8/5 3/1 10/6 8/4 20/1 Standard panel Type strain (ATCC) Philadelphia 1 (33152) Allentown 1 (43016 ) Benidorm 030E (43108) Knoxville 1 (33153) France 5811 (43112) OLDA (43109) Oxford 4032E (43110) Heysham 1 (43107) Camperdown 1 (43113) Bellingham 1 (43111) Denver (Stout, 1988) C Lück IMMH TUD Legionella laboratory

47 Typing methods for pneumophila Monoclonal antibody (Mab) typing Sequence BasedTyping (SBT) Chip Typing Spoligotyping Fast, reproducible, exchangeable, cheap, good discrimination C Lück IMMH TUD Legionella-Labor

48 Phylogeny of sequenced pneumophila strains built using 26 housekeeping genes frr, infc, nusa, pgk, rpla, rplb, rplc, rpld, rple, rplk, rpll, rplm, rpln, rplp, rpls, rplt, rpma, rpob, rpsb, rpsc , rpse, rpsi, rpsk, rpsm, smpb, tsf Alcoy ma b ST flaa pile asd mip momp pro A neu A Phil Phil Paris Phil Corby Alcoy Kno x Kno x Wadsw Beni Lens Beni C Lück IMMH TUD Schroeder et al. JB 2010 Legionella Laboratory

49 Typing methods for pneumophila Monoclonal antibody (Mab) typing Sequence BasedTyping (SBT) Chip Typing Spoligotyping Fast, reproducible, exchangeable, cheap, good discrimination C Lück IMMH TUD Legionella-Labor

50 Chip Diagnostic microarray was developed and used to characterize a collection of pneumophila isolates. Genes used as targets were selected from previous studies. Variable genomic element / islands 8 genes Genes associated with the LPS synthesis (Serogroup / monoclonal subgroup) 12 genes Core genome gens 7 genes No antibiotic resistance genes 97 spots in quadruplicate

51 Pathogenicity / genetic islands Trb1 [Corby] 3,500,000 MRR [Corby] TraG [Corby] R33 [Corby] MagA / TraD [Phil] 2,625,000 pneumophila 875,000 F2 [Corby, Lens 298 [Corby] 127 [Corby, Paris] 1,750,000 LvhB10 [Phil , Paris, Lens]

52 Constructing and Testing of several PCR-based systems to detect monoclonal (MAb) subgroups Sg 1 specific primers Strain (MAb subgroup) lag truncated 1 2 lag lag-12-like RC1 (Olda) Görlitz 5432 (Bellingham) Lens (Benidorm) Philadelphia -1 (Philadelphia) 1 2 lag-1 2-like lag lag-12-like Paris (Philadelphia) Corby (Knoxville) Uppsala 3 (Knoxville) Benidorm / Bellingham specific? Intergenic region ORF 6-8 primer bp primer 2 Knoxville specific? Intergenic region ORF 7-9 primer bp primer 2 (Thürmer et al. JMM 2009: 58,

53 Chip 2 Chip Biotin T4SS effector WL T4SS effector L T4SS effector CA IMP-GMP CA IMP-GMP 6 HWPL IMP-GMP HWL IMP-GMP 6 WPCA IMP-GMP 6 LPS 8 Knox LPS 11 HWPL LPS 11 CA r33_1 r33_2 sdbb WL sdbb CA LPP 0109 P LPP 0109 WPLCA LPC_1158 LPS 10 Knox trad_phil 1 neub 01 neub 02 neub 03 neub 04 neub 05 neub 06 neub 07 neub 08 neub 09 neub 10 neub 11 pily 1 pily 2 pily 3 LPS 7 1 LPS 7 2 LPS 7 3 LPS 7 4 LPS 7 5 LPS 7 8 helb_611 helb_612 helb_613 helb_614 helb_615 helc_611 helc_612 helc_613 helc_614 lag1_611 lag1_612 lag1_613 lag1_614 lag1_615 lag1_616 maga_611 maga_612 mrr_611 mrr_612 pacl_611 pacl_612 pacl_613 pacl_614 pacl_615 pacl_616 pile_611 pile_612 pile_613 pile_614 proa_611 proa_612 proa_613 proa_614 proa_615 proa_616 proa_617 proa_618 rnpb_611 rnpb_612 rnpb_613 rnpb_614 rnpb_615 trad_phil 2 trag_corby_611 trag_corby_612 trbi_611 trbi_612 trbi_613 trbi_614 trbl_corby_611 virb10_611 virb10_612 virb10_613 virb10_614 virb10_617 virb10_616. virb10_617. W OLDA W Bell W Bell W Bell W Bell W Denv 9 .. WWW OLDA W OLDA W OLDA W LA W OLDA 1 .. W OLDA 1 .. W OLDA 1 .. W OLDA 1 .. W OLDA 1 .. W Knox 62 .. W Knox 62 .. W Knox 62 .. L Knox 62 .. L Knox 62 .. L Knox 62 .. W Knox 62 .. L Knox 62 .. L Knox 62 .. L Knox 62 .. L Knox 62 .. L Knox 62 .. L Knox 62. C Lück IMMH TUD Legionella laboratory

54 Typing methods for pneumophila Monoclonal antibody (Mab) typing Sequence BasedTyping (SBT) Chip Typing Spoligotyping Fast, reproducible, exchangeable, cheap, good discrimination C Lück IMMH TUD

55 Organization of the CRISPR / CAS system (CassII-B type) of Lp1 strain CAS genes Leader DR Spacer DR f Lpn (38 x) 0 cas9_endonuclease "cas1" cas2 cas4 219 bp 38 bp ~ 34.5 bp (1372 aa) (aa) (99 aa) (197 aa) bp bp PCR specific for strain Ulm: Cas4-Repeat 14 PCR specific for all strains: cas1-cas4 strain Paris Lp1 Phil: 42 repeats strain130b Lp1 Beni: 60 repeats C Lück IMMH TUD

56 2010 Outbreak Ulm Source of infection Epidemiologically no common source Swimming pool Celebration Travel / hotel diffusely distributed over the city >> Recooling plant 9/30 Culture Legionella pos 5x Lp1 1x Mabtyp Knoxville, ST 62 Intermittent operation since summer 2009 Weather situation: Relatively warm Dense cloud layer Euro Surveill. 2010; 15 (4): 1-2 C Lück IMMH TUD

57 Sources of infection for Legionella pneumonia C Lück IMMH TUD Legionella laboratory

58 origin of pneumophila strains from 8 patients and 10 cooling towers (KT) during the outbreak in Ulm, December 2009 / January 2010 Maximum germ count / 100ml serogroup / monoclonal subgroup (N tested) Sequence type (ST) (N tested) fla pil asd mip momp pro New occurrence of these tribal patients (n = 8) 1 Knoxville Almost exclusively in sick people worldwide 1. KT causer Knoxville 1 (n = 19) 62 (n = 4) Almost exclusively in sick people worldwide 1. KT OLDA 2 (n = 1) 1 (n = 1) Worldwide by patient and water 1st CT (n = 1) 908 (n = 1) Only 1st CT (n = 3) NA (n = 2) F 5 Only 1st CT (n = 8) 984 (n = 1) only 1st KT rubrilucens 3 (n = 1) nt 2nd KT (n = 94) NA (n = 2) F only 2nd KT rubrilucens 3 (n = 2) nt 3rd KT Bellingham 2 (n = 1) 334 (n = 1) Rarely in patients and water in D and NL 4th CT (n = 1) 681 (n = 1) Rarely 4th CT (n = 1) 956 (n = 1) Rarely 5.KT 1 1 OLDA 2 (n = 1) 1 (n = 1) Worldwide patient and water 6.KT 1 New species (n = 1) 4 7.KT Bellingham 2 (n = 1) 334 (n = 1) Rarely at Pati. and water in D and NL 8.KT OLDA 2 (n = 2) 595 (n = 1) Rarely 8.KT 6 4 (n = 1) NA (n = 1) F Worldwide patient and water 9.KT 5 1 OLDA 2 (n = 1) 1 (n = 1) Rarely 10.KT 1 1 OLDA 2 (n = 1) 104 (n = 1) Rarely C Lück IMMH TUD

59 CAS-PCR Primer: Lcas4 F2 / Lcas4 R2 SSSWS No.Strain Origin mab-subtype Comment 1 L11_007 Ludwigsburg 1Alle-Fran 0 2 L10_486 Heidelberg 1Knox + 3 L10_571 GA Ulm 1Knox 0 4 L11_154 Viersen 1Alle-Fran + 5 L11_209 Hannover 1Knox 6 W11_1115 Hanover 1Phil 0 7 L04_399 Cologne 1Knox + 8 L03_095 Saarbrücken 1Phil 0 9 L10_033 ULM 1Knox + 10 L10_034 ULM 1Knox + 11 L10_070 ULM 1Knox + 12 L10_071 ULM 1Knox + 15 W10_080 ULM 1Knox + 14 W10_078 ULM 1Knox + 14 W10_078 ULM 1Knox + 14 W10_078 ULM 1Knox + 14 W10_078 ULM 1Knox + 14 W10_078 ULM 1Knox + 14 16 W10_081 ULM 1Knox + 17 L07_375 Dresden 1Knox + 18 L06_457 Dresden 1Knox + no. Strain origin mab-subtype remark 19 W09_454 LUA Trier 1Phil 0 20 W09_456 LUA Trier 1Phil 0 21 L09_451 Ulm LU3 Or 1Phil 0 24 L10_069 ULM 1Knox + 25 W10_055 ULM 1Phil 0 26 L02_521 Bad Langensalza 1Phil 0 27 W12_935 Friedrichshafen 1Alle-Fran 0 28 L12_398 Greifswald 1Alle-Fran 0 29 L10_496 GA Marburg- 1Phil 0 31 Biedenkopf 30 L7 Stuttgart + 113_nox -Fran 0 32 W13_219 joys city ​​1 All-Fran 0

60 C Lück IMMH TUD CRISPR / CAS region Lp1 ST 62 strains from ULM L etc.

61 cases of with date of hospitalization cases of with confirmed date of onset Infection time outbreak recognized Inspection and shutdown of the cooling towers 0 21.Dec. 22.Dec. 23 Dec. 24th dec. 25th Dec. 26 Dec. 27 Dec 28 Dec. 29 Dec 30th dec. 31 Dec 01 Jan Jan 02 Jan 03 Jan 04 Jan 05 Jan 06 Jan 07 Jan 08 Jan 09 Jan Jan 10 Jan 11 Jan Jan 12

62 Epidemic curve ST182 Disinfection riser 1st detection Serogroup 1 Knoxville Installation of terminal filters 1/9 6 2/9 6 1/9 7 2/9 7 1/9 8 2/9 8 1/9 9 2/9 9 1 / 0 0 2/0 0 1/0 1 2/0 1 1/0 2 2/0 2 1/0 3 2/0 3 1/0 4 2/0 4 1/0 5 2/0 5 Serogroup 8 Serogroup 1 Bellingham 1 urine antigen / immunofluorescence serogroup 1 Knoxville 1 (ST182) serogroup 1 (subtype cannot be determined) 10 (83%) patient immunosuppressed (4 bronchial carcinomas, 1 lymphoma, age: years (mean value 60) gender: 4 (33%) female Died: 7 (58%) 2 high-dose corticosteroids, 1 kidney transplant, 2 chemotherapy)

63 Zweibrücken eruption through Lp 1 Allentown / France ST82

64 travel group from NRW index case in Berlin hospital died Kultur Lp1, Mabtyp Benidorm (3-1 positive), ST25 (one-time) 2 further inpatients serologically positive 4 outpatients serologically positive 7 people seronegative All in Hotel B no environmental examinations C Lück IMMH TUD Legionella laboratory

65 I am addressing you with this because I learned yesterday from my property management that the legionella test in my house showed a legionella concentration of 1700 CFU / 100 ml, in the neighboring house even 2000 CFU / 100 ml. As a consequence, the hot water temperature has now been increased, "intensive flushing of the systems should take place at short notice" and "in the next few weeks (!)" The drinking water systems will be "checked in terms of hygiene and technology". With the latter point, I am particularly shocked by the time frame that is given. Furthermore, we are asked (in this case "especially people with a weak immune system") to avoid activities in which aerosols form (steam, mist), to let the old water run off aerosol-free before showering, to clean or replace shower heads and aerators. I am a gynecologist myself and, despite my knowledge that predominantly immunosuppressed people are at risk and an acute risk is only to be feared from values> CFU / 100 ml, I am very concerned and unsure about the recommended behavior. Do you see the recommendations as sufficient and correct? In my bathroom, in which I naturally shower and bathe hot, fumes are generated every time I shower. I can see this clearly, for example, in the cone of light under the ceiling lights. I would therefore recommend that all processes in which hot water is consumed and "aerosols" = vapor and fog are created should be interpreted in such a way that I am not allowed to shower hot. The hygiene officer at my hospital has also specified that it is not enough to let the old water run off briefly before taking a shower, but that all hot water pipes in my apartment should run for 15 minutes each day at temperatures> 55 for renovation. What specific recommendations do you make for a Legionella concentration of 1700 CFU / 100 ml? Are the recommendations that my property manager has distributed in writing correct? And do they only apply to immunosuppressed people or also to me? In addition, I have another specific question: I have had a periurethral wound healing disorder of unclear origin for 5 months, which neither gynecologists nor dermatologists can conclusively explain. After taking a sample in the wound area, I should take hip baths with Tannolact every day and "shower out" the PE area. Since then the wound has tended to get worse. Of course, wound infections with legionella are rare, but I am now of course also very insecure in this regard and am very worried that the measures recommended to me for healing have brought legionella into the wound region, which prevent the wound healing process. A microbiological smear has been taken, gram-negative rods have been detected, but also E. coli and enterococci, the laboratory informed me, however, that Legionella is not looked for, as it "would never" cause wound infections.

66 Eruption GA Soest

67 Urine antigen testing Initial ICT-ELISA 2/31 positive ICT 21/31 positive ELISA total 31/128 positive ELISA 12/128 borderline (concentration necessary) Total 96 urine antigen positive OD Binax-Eia Binax evaluation 4.65 positive pos 5.00 positive pos 1.00 positive neg 0.82 positive neg 0.64 positive neg 0.55 positive neg 0.86 positive neg 0.39 positive neg 0.42 positive neg 0.42 positive neg 0.35 positive neg 2.9 positive neg 1.67 positive neg value Lab local

68 Typing of clinical samples strain SG-Mab ST fla pil asd mip momp pro neu L Knox L Knox L Knox L L Knox L Knox L Knox L Knox L Knox L13-P401 DNA SG1 345? L13-P402 DNA SG1 ??? m L13-P436 DNA SG1 345? m L13-P733 DNA SG1 345?

69 Search for the source of infection Patient material Water samples 2-3 colonies at least 10 colonies 1. Serological typing in pneumophila: serogroup, MAb subtype or species determination: MALDI-TOF, DNA sequence (mip, 16S rrna) 2. Genomic fingerprint: SBT, ( PFGE, AFLP, RAPD-PCR) Identity (indistinguishability)> Transfer from the water system C Lück IMMH TUD Legionella laboratory

70 Infection dose for Legionella pneumonia in guinea pigs animal model Infection route (CFU) Ref Aerogen (Phil) 12/12 fever Aerogen LD50 intraperitoneal LD50 strain Corby aerogenic strain Corby 100% lethal, aerogenic strain Phil no lethality, aerogenic strain Corby, intratracheal, LD50 strain Corby , intraperitoneal, LD50 Phil strain, intratracheal, LD50 Phil strain, intraperitoneal, LD nt Berendt, 1980 Jepras, 1985 Lück, unpubl. Bacteria grown extracellularly CFU = bacterial count 71

71 Thanks to Dr. Christine Steudel Dr. Andrea Steidle Dr. Jürgen Helbig Jutta Paasche Sigrid Gäbler Kerstin Lück Financial support BMBF RKI DFG EU Tetyana Koshkolda Prof. Dr. med. Enno Jacobs

72 typing water samples 238 water samples examined, 17 positive 20 randomly picked 18 Lp Lp10 14 Lp6 12 Lp1 Knox Lp1 Knox RKW Esser 1. RKW Esser 2. Condens Brewery tributaries Esser River Wester Sewage treatment plant Aeration basin Sewage plant Brewery Sewage treatment plant City Air Sewage treatment plant Brewery City retest

73 Different colony variants in the water Lp1 Knox ST 600 Lp2-14 Lp1 Knox ST 345

74 Period until the epidemic strain is detected in 2 suspicious RKW breweries Sampling of the brewery and disinfection of the recooling plant: Result of fine typing with proof of epidemic strain: Esser Sampling by Esser and closure of the recooling plant High contamination: Result of fine typing with proof of epidemic strain:

75W. nic .. chilled cooling plant Esser 20. W. nic .. chilled cooling plant Esser 20. W. nic ... recooling plant Esser 20. W. nic ... recooling plant Esser 20. W. .. recooling plant Esser 20. W. nic ... recooling plant Esser 20. W ... Recooling plant Esser 20. L Kraut, Ursula Sophie; 57J 20. W. nic .. 600 ?. Recooling plant Esser 20. W. You .. 600? Recooling plant Esser 20. W. nic .. 600 ?. Esser pump shaft 20. W. nic .. 600 ?. Sewage treatment plant activated sludge basin 20. W. nic .. 600 ?. Sewage treatment plant aeration basin 20. W. nic .. 600 ?. Recooling plant Esser 20. W .. 600 ?. Esser pump shaft 20. W. nic .. 600 ?. Fa. Esser inlet from the river 20. W. nic .. 600 ?. Sewage treatment plant aeration basin 20. W. nic .. 600 ?. Sewage treatment plant aeration basin 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Esser pump shaft 20. W Recooling plant Esser 20. W .. 600 ?. Esser pump shaft 20. W. ST600 nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Esser pump shaft 20. W .. 600 ?. Sewage treatment plant activated sludge basin 20. W. nic .. 600 ?. Recooling plant Esser 20. W .. 600 ?. Esser inlet from the river 20. W .. 600 ?. Esser, Bach Wästet in front of the inlet pipe W .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Esser inlet from the river 20. W .. 600 ?. 3858, River Wäster 20. W. nic .. 600 ?. Recooling plant Esser 20. W .. 600 ?. 3969, sewage treatment plant city, inflow sewage treatment plant brewery 20. W. nic .. 600 ?. Recooling plant Esser 20. W. nic .. 600 ?. Recooling plant Esser 20. W Recooling plant Esser 20. W .. 600 ?. Recooling plant Esser 20. W. Du .. 345 ?. Recooling plant Esser 20. W. nic .. 345 ?. 3859, River Wäster 20. W. nic .. 345 ?. 3860, River Wäster 20. W .. 345 ?. 3956, brewery sewage treatment plant, Venturi drain 20. W .. 345 ?. 3969, sewage treatment plant city, inflow sewage treatment plant brewery 20. W .. 345 ?. 4008, air germ collection over aeration basin Klä. 20. W .. 345 ?. 3991, sewage treatment plant brewery, bio 1 basin W .. 345 ?. 3991, sewage treatment plant brewery, bio 1 basin W .. 345 ?. 3992, brewery sewage treatment plant, Bio 1 basin L Severin, Brigitte; 60J 20. W .. 345 ?. Esser pump shaft 20. W. nic .. 345 ?. Esser inlet from the river 20. W. nic .. 345 ?. 3861, River Wäster ST W. nic .. 345 ?. 3863, River Wäster 20. W. nic .. 345 ?. 3864, River Wäster 20. W .. 345 ?. HI Geks a "Prod Condensers Brewery 20. W. nic .. 345?. 3867, River Wäster 20. W .. 345 ?. Fa. Esser inlet from the river 20. W .. 345 ?. Sewage treatment plant aeration basin 20. W. nic .. 345?. 3865, River Wäster W. nic .. 345?. 3866, River Wäster 20. W. nic .. 345 ?. HI Geks b "Prod Brewer Capacitors Brewery W .. 345 ?. Esser inlet from the river 20. W .. 345 ?. 3857, River Wäster 20. W .. 345 ?. Recooling plant Esser 20. W .. 345 ?. Esser, raw water intermediate tank L Simar Ursula; 62J 20. W .. 345 ?. 3953, brewery sewage treatment plant, Bio 1 basin L Wintergast Martin; 45J 20. L Kliem Dieter; 59J 20. W .. 345 ?. 3855, River Wäster 20. W .. 345 ?. 3856, River Wäster 20. W .. 345 ?. 3956, brewery sewage treatment plant, Venturi drainage 20. L Engelhardt Axel; 54J 20. L Kliem Dieter; 59J 20. W .. 345 ?. 3986, city sewage treatment plant, activated sludge basin 20. W ... HI Gelks b "Prod condensers brewery .. C Lück IMMH TUD Legionella laboratory

76 ST 345 () EWGLI Data

77 Soest district ST345

78

79

80 Esser company connection road between Belecke and the location of the open recooling plant Raw water from wastewater without treatment or disinfection

81 Course of the sewer system for mixed water sewerage and brewery wastewater Sewerage system brewery in the area of ​​the sidewalk mixed water sewer system in the middle of the street

82 travel ban ?? Citizens: good information situation of the population in case of symptoms directly to the doctor, ensuring immediate diagnosis and adequate therapy a hospital Maria Hilf travelers: information situation ?, when passing through and after leaving during the incubation period, manifestation after 4 10 (20) days, diagnosis and adequate therapy with Risk of increased complication rate and mortality

83 Timeline of Legionnaires' visitors with possible exposure Incidence rate of 0.2%: 400 additional illnesses Lethality 1.2% (): 5 additional deaths Lethality 7.7% (Ulm): 31 additional deaths Recommendation cancellation of the Montgolfiade Montgolfiade

84 Summary Legionella are widespread water bacteria. Multiplication in warm water. Prevented persons are particularly susceptible to legionnaires. Think Diagnostics !! There is no dose-effect correlation. No exact limit values ​​for the amount in the water. Virulent clones responsible for the majority of infections. Many Legionella are harmless, but which ones ??? Preventive: high heating, chlorination, filter water: cold water <20 C warm water> 60 C water must flow C Lück IMMH TUD Legionella laboratory

85 IMMH Thank you for your attention Dr. Jürgen Helbig Sigrid Gäbler Kerstin Lück Susann Menzel Tetyana Koshkolda Markus Petzold Prof. Dr. med. Enno Jacobs RKI: Klaus Heuner Financial support BMBF RKI DFG EU Health Dept. Soest Dr. Brockmann Prof Dr. Martin Exner

86 C Lück IMMH TUD Legionella laboratory

87 Legionella outbreak in 160 pneumonia cases from August 1 to September 6, 89 cases were confirmed as Legionella infection either by urinary antigen detection, culture and / or PCR in respiratory samples. 2 patients needed intensive care treatment. 2 (3) deaths. Clinical isolates from 7 patients: pneumophila, serogroup 1, Mab subtype Knoxville, ST 345. nsbt directly from clinical samples - same strain This epidemic strain was found 2 RKW from 2 companies sewage treatment plant other environmental sources. Only approx. 10% of the isolates No further infections by ST345 after September 6th. 1 isolate from November was pneumophila, Sg 1, Mab subtype Philadelphia, ST 23. C Lück IMMH TUD Legionella laboratory

88 Outbreaks Legionnaires disease in Europe Stafford 101 cases, 28 deaths, 1985 Lp1 Knox ST 27 Barrow-in-Furness 87 cases, 7 deaths, 2002 Lp1 Beni ST 78 Sarpsborg 56 cases, 10 deaths, 2005 Air scrubber Lp1 ?? Gloucester 15 cases, 3 deaths, 1986 Lp1 Phil / Alle ST 47 Lens 86 cases 18 deaths, Lp1, Beni ST 15 Kapellen 93 cases, 43 confirmed 5 deaths, 1999 Lp1 ?? Bovenkarpsel 188 cases, 133 confirmed, 21 deaths, 1999 Lp1 Phil ST cases, 93 confirmed, 2 (3) deaths, 2013 Lp1 Knoxil, ST 345 cruise ship 8 cases, 1 deaths, case 2008 Lp1 Knox ST 35 NRW-Berlin 7 cases, 1 deaths, 1981 Lp1 Beni ST 184 ULM 64 cases, 5 deaths, 2010 Lp1 Knox ST 62 Armavir cases, confirmed, 3 deaths, 1988 Lp1 Phil, ST 36 In all outbreaks SG1 MAb2 (3-1) positive C Lück IMMH TUD Legionella -Labor Murcia 800 cases, 449 confirmed, 4 deaths, 2001 Lp1 Phil, ST 36

89 C Lück IMMH TUD

90 Conclusions Outbreak On the basis of detailed typing, 2 emitters for epidemic strain detected (recooling plant of the brewery and Esser company) both were either disinfected or shut down and brought under control on the 20th /. Other emitters currently not detected. Above the brewery's primary clarification basin (especially organic basin), high legionella concentrations develop with the possibility of drifting into the brewery's recooling plant, as well as entry into the communal sewage treatment plant via the brewery's sewer with the possibility of multiplying in the activated sludge basin and trickling filters of the communal sewage treatment plant. Sewer of the brewery through the city center as an emitter currently safely assessable so far not dealt with in literature further need for clarification. From the inlet of the sewage treatment plant in Wäster, sustained contamination of Wäster and Möhne with high legionella concentrations including epidemic strain up to Möhnesee. As a result, contamination of recooling water from Esser and possible other users of Wäster and Möhne water C Lück IMMH TU Dresden

91 Control measures outbreak - Closure of the recooling plant from Esser - Disinfection of the brewery's recooling plant (continuously using chlorine dioxide in cooling water) - Covering of the brewery's primary clarifier to minimize drift to the recooling plant - Disinfection of the primary clarifier - UV disinfection of the wastewater before it is discharged into the sewer - Cleaning of the sewer and inserting flow mats in the manhole cover to minimize emissions C Lück IMMH TU Dresden

92 To do list for the near future (Prof. Exner) Politics: - BMU: short-term uniform federal regulation for the registration, investigation and operation of recooling plants and air washers Implementation by the federal states Training of maintenance personnel must be regulated in a mandatory manner - initiation of corresponding regulations in the EU Technical rules: - VDI guidelines 2047 and 2050 to be completed at short notice. Importance of fill water and clarifier in the vicinity of recooling plants C Lück IMMH TU Dresden

93 To do list for the near future (Prof. Exner) Disinfection: - Testing and listing of disinfection processes for disinfecting recooling systems and air washers, e.g. In the VAH disinfectant list according to DIN EN only tested and listed disinfectants may be used for recooling systems and air washers in the future. Significance of suitable disinhibitors in microbiological testing of recooling plants to avoid false negative results C Lück IMMH TU Dresden

94 To do list for the near future (Prof. Exner) Investigation program: - Sewage treatment plants with a load profile depending on the wastewater to be treated - Influence of brewery wastewater and other wastewater that promotes Legionella growth - Kinetics of propagation of legionella in different wastewater - Water profile and clarification of causes in the case of increased Legionella loads C Lück IMMH TU Dresden

95 LPS-biosynthesis locus of 12 pneumophila Sg1 strains corresponding monoclonal subgroup (in brackets). direction of transcription is indicated by arrowheads. filled black arrows indicate transposable elements Asterisk in Uppsala 3, Philadelphia 1 and Paris indicates a partial ORF 2 duplication (ORF 2 like) Orfs 7-11 of W130b represent an inversion. C Lück IMMH TU Dresden

96 CRISPR-Associated Gene cas2 of pneumophila Is Required for Intracellular Infection of Amoebae clustered regularly interspaced palindromic repeat (CRISPR) On lvh genomic island have been linked to phage and plasmid immunity cas2 mutants, grew typically in macrophages, significantly impaired for infection of both Hartmannella and Acanthamoeba species. A complemented cas2 mutant infected the amoebae at wildtype levels C Lück IMMH TU Dresden Gunderson and Cianciotto Mbio 2013 doi: / mbio

97 Conclusions Identification of strains was highly reproducible, Discriminating IOD Micro array> SBT epidemiologically concordant. Facilitated rapid and reliable detection of strain-specific determinants Correlation with the current ST scheme Some ST can be subdivided further Can be used for rapid and high-throughput typing of pneumophila serogroup 1.

98 Ulleryd et al. BMC Infectious Diseases 2012, 12: 313

99 Pneumonia ST332 community acquired or nosocomial? Nov y male patient hospitalized for pacemaker implantation 5 days later cardiac arrest ICU of a large hospital - pneumonia culture pneumophila, Sg1, MAbtyp Philadelphia, ST322 Water in the hospital 2/2 positive, KbE / 100ml, pneumophila, Sg1, MAbtyp Philadelphia, ST322 Water in the private household 8/9 positive, KbE / 100ml pneumophila, Sg1, MAbtyp Philadelphia, ST322 Strains were indistinguishable by PFGE, RAPD-PCR, VNTR distance hospital private house approx. 1km Jan J female - nosocomial Direct SBT from clinical sample - ST 332, private home- no Legionella detected C Lück IMMH TUD Legionella-Labor

100 ST 332 nosocomial cluster December April nosocomial cases All patients older 77y Diagnosed by urinary antigen detection no clinical isolates One patient died Water samples from the hospital: Lp1 no further subtyping Water supply was hyperchlorinated C Lück IMMH TUD Legionella-Labor

101 ST 182 Only strains of Knoxville and Denver subtypes Two of the Denver subtype strains have mutations in the lag -1 gene Caused: nosocomial clusters in Berlin and Frankfurt / Oder Single travel associated cases in tourists from Germany, DK and the UK 12/22 unrelated CAP cases in Berlin Single cases in Germany Environmental strains without relation to clinical cases: Dental units in Berlin and Dresden, warm water supplies Berlin 12/26 unrelated strains) One strain from the Netherlands C Lück IMMH TUD Legionella-Labor

102 1. Objective 2. Methods 3. Results and discussion Multigenome Analysis of Legionella pneumophila Cazalet et al., Paris (Genome Research 03/2008) ORF 28 ORF 27 ORF 26 ORF 25 ORF 24 ORF 23 ORF 22 ORF 21 ORF 20 ORF 19 ORF 18 ORF 17 ORF16 ORF 15 ORF 14 ORF 12 ORF 11 ORF 10 ORF 9 ORF 8 ORF 7 ORF 6 ORF 5 ORF 4 ORF 3 ORF 2 ORF Claudia Farhat, Institute for Medical Microbiology and Hygiene, TU Dresden

103 Nosocomial outbreak Frankfurt / O IQ = Warmwater supply Legionella count initially up to 200 / ml (20,000 / 100ml), After superheating up to 15 / ml Warm water pipes close to the cold water supply (up to 28.7 C): > 200 / ml Epidemic strain Lp1 Knoxville ST182 Lp 1 Philadelphia / OLDA ST1 Lp 6 gormanis, anisa Since August 2003 no further cases C Lück IMMH TUD Legionella-Labor

104 Conclusion Strains with defined monoclonal subtype (Mab-type) and sequence type (ST) might be restricted to defined areas They are responsible for single cases, clusters and outbreaks of community aquired, travel associated and nosocomila origin Many thanks to: Carolin Dix Kerstin Lück Susann Menzel Dr. Jürgen Helbig Prof. Dr. Enno Jacobs C Lück IMMH TUD Legionella Laboratory

105 Genetic Characterization of Legionella pneumophila Isolated from a Common Watershed in Comunidad Valenciana, Spain the population structure of these environmental samples results from the joint action of a global, widespread ST-1 along with genetic differentiation at shorter geographic distances, which are in this case related to the common watershed for the BV localities Sances et al. PLOS2013 C Lück IMMH TUD Legionella laboratory

106 Legionella Pneumophila Serogroup 1 strains in Saxonian Hospitals Methods 7-gene sequence based typing: fla, pile, asd, mip, momp, proa, neua Assignment of specific allele number by SBT database or online sequence type checker Allelic profile or SBT: combination of the seven alleles at each of the loci using predetermined order MTZ Reserch Seminar 107 Speaker: Roman Vandré

107 What is a sequence type? DNA-Sequence DNA-Sequence DNA-Sequence DNA-Sequence DNA-Sequence Gen A Gen B Gen C Gen D Gen E GCTATCAGT 1 GCTATAAGT 2 GCGATCAGT 3 GCGATCAGT 3 GTCCGTATG 1 GTCGGTATG 2 GTCCGTATG 1 GTCCGTATG 1 GATCGATT 2CCATGAT GATGC 1 GATAGTATG 1 CAGTCCGTA 1 CAGTCCCTA 2 CAGTCCCTA 2 GGCCGTATG 1 GTCCGTATG 2 GTCCGTATG 2 GTCCGTATG 2 SequenceA - SequenceB - SequenceC - SequenceD - SequenceE Strain ST 1 Strain ST 2 Strain ST 3 Strain ST 3 C Lück IMMH TUD Legionella Laboratory

108 Sources of infection Legionella pneumonia Often Rarely Very rarely (yet) not: C Lück IMMH TUD Legionella laboratory hot water systems in the home environment, in hotels, baths, in hospitals, etc. Recooling plants with moist cooling Whirl pools Thermal baths, humidifiers in restaurants and shops (Food), inhalers indoor fountains / ornamental fountains, birth tubs gastric irrigation probe / transoesophageal echo probe (tap water) wound infections after bathing, ice machines dental units road humidification machines windshield wiper systems, garden soil (longbeachae) greenhouses, car washes,

109 C Lück IMMH TU Dresden

110 Direct sequencing from patients material C Lück IMMH TU Dresden

111 2010 Outbreak Ulm Source of infection Epidemiologically no common source Swimming pool Celebration Travel / hotel diffusely distributed over the city >> Recooling plant 9/30 Culture Legionella pos 5x Lp1 1x Mabtyp Knoxville, ST 62 Intermittent operation since summer 2009 Weather situation: Relatively warm Dense cloud layer C Lück IMMH TUD Legionella Laboratory Von Baum et al. Euro Surveill. 2010; 15 (4): 1-2

112 Nosocomial outbreak Frankfurt / O IQ = (warm) water supply Legionella quantity initially up to 200 / ml (20,000 / 100ml), then up to 15 / ml hot water installation close to cold water (up to 28.7 C):> 200 / ml epidemic strain rarely but Detectable until 2009 Lp1 Knoxville ST182 Lp 1 Philadelphia / OLDA ST1 Lp 6 gormanis, anisa From August 2003 no more diseases C Lück IMMH TUD Legionella laboratory

113 O-acetyltransferase (Lag-1) MAb 3/1 pos MAb 3/1 neg

114 O-acetyltransferase (Lag-1) MAb 3/1 pos MAb 3/1 neg S169L E192G Lück et al Kozak et al. 2009

115 O-acetyltransferase (Lag-1) MAb 3/1 pos MAb 3/1 neg homologous recombination Bernander et al. 2003

116 Tn10 mutants ORF 8 and ORF 11 ~ 1.8 kb insertion with kanamycin resistance cassette 1 2 lag-1 2-like nt153 / nt145 nt139 / nt317 methyltransferase unkno wn (maybe glycosyltransferase)

117 Tn10 mutants ORF 8 and ORF lag-1 2-like

118 Monoclonal antbodies Tn10 mutants switch from Benidorm to Allentown MAb 8/5 3/1 8/4 9/1 10/2 10/6 10/7 10/8 12/2 20/1 26/1 26/2 30/4 39/2 Subgroup Philadelphia (Phil-1, Paris) Allentown (Lorraine) Benidorm (Lens) Knoxville (Corby, Alcoy, Upsalla 3) / 0 + / / / / / 0 ++ ELISA reactivity OLDA (RC1) Camperdow n Heysham Bellingham (Görlitz, HL)