Joseph Palmer in the environment What is Clostridium?

• > > > Clostridales > > Clostridium

• Contains many species of medical and biotechnological importance

• Highly pleomorphic, with heterogeneous phenotype and genotype

• Genus subject to recent taxonomic reshuffling 99 Clostridium estertheticum subsp. laramiense strain DSM 14864 Clostridium estertheticum subsp. laramiense strain DSM 14864 99 Clostridium92 99 estertheticum Clostridium frigoris subsp. strain laramiense D-1/D-an/II strain DSM 14864 92 92 Clostridium47 Clostridium frigoris Clostridium strainalgoriphilum D-1/D-an/II frigoris strain strain 14D1 D-1/D-an/II 47 10047 Clostridium algoriphilum Clostridium strain bowmanii algoriphilum 14D1 strain DSMstrain 14206 14D1 100 100 Clostridium Clostridium bowmanii Clostridium tagluense strain bowmaniiDSM strain 14206 A121 strain DSM 14206 Clostridium Clostridium tagluense tunisiense Clostridium strain strain tagluense A121 TJ strain A121

Clostridium76 tunisiense Clostridium strain tunisiense TJ strain TJ 86 Clostridium sulfidigenes strain MCM B 937 76 76 86 86 Clostridium 100sulfidigenes Clostridium Clostridium strain sulfidigenesthiosulfatireducens MCM B 937 strain strain MCM LUP B 93721 100 100 Clostridium thiosulfatireducens Clostridium100 thiosulfatireducens Clostridiumstrain LUP amazonense 21 strain LUPstrain 21NE08V

100 100 Clostridium amazonense Clostridium Clostridium senegalense strain amazonense NE08V strain JC122strain NE08V 30 48 Clostridium Clostridium senegalense Clostridium subterminale strain senegalense isolate JC122 DSM strain2636 JC122 30 30 48 48 Clostridium100 subterminale Clostridium Clostridium isolate subterminale argentinense DSM 2636 strain isolate ATCC DSM 27322 2636 99 100 Clostridium100 argentinense Clostridium Clostridium schirmacherense strain argentinense ATCC strain27322 strain AP15 ATCC 27322 99 Clostridium99 schirmacherense Clostridium schirmacherense strainClostridium AP15 collagenovorans strain AP15 strain SG

100 Clostridium collagenovorans Clostridium Clostridium collagenovorans strain acetobutylicum SG strain DSM SG1731 100 Clostridium aurantibutyricum strain DSM 793 100 100 Clostridium acetobutylicum Clostridium acetobutylicumDSM 1731 DSM 1731 17 100 Clostridium100 aurantibutyricum Clostridium94 aurantibutyricumClostridium strain DSM botulinum 793 strain BKT015925 DSM 793 17 17 100 Clostridium haemolyticum strain JCM 1402 94 Clostridium94 botulinum Clostridium BKT015925 botulinum BKT015925 99 100 Clostridium100 Clostridium haemolyticum NTstrain haemolyticum JCM 1402 strain JCM 1402 99 23 99 Clostridium novyi Clostridium Clostridium NT ganghwense novyi NT strain HY-42-06 Clostridium oceanicum strain DSM 1290T 23 23 Clostridium strain ganghwense HY-42-06 strain HY-42-06 99 Clostridium oceanicumClostridium strain oceanicum DSM A str. 1290T ATCC strain 19397 DSM 1290T 100 Clostridium botulinum Clostridium Clostridium A sporogenes str. botulinum ATCC strain 19397 A DSM str. 795ATCC 19397 5 99 99 Clostridium pascui strain DSM 10365 100 100 Clostridium100 sporogenes Clostridium strain sporogenes DSM 795 strain DSM 795 5 5 Clostridium peptidivorans strain TMC4 100 100 Clostridium pascui Clostridium strain DSM pascui 10365 strain DSM 10365 Clostridium acetireducens strain 30A 48 Clostridium peptidivorans Clostridium strain peptidivorans TMC4 strain TMC4 strain DSM 17049 99 Clostridium acetireducens Clostridium strain acetireducens 30A strain 30A 48 48 41 Clostridium tetanomorphum strain DSM 4474 99 Clostridium99 lundense Clostridium strain lundenseDSM 17049 strain DSM 17049 45 Clostridium malenominatum 41 41 Clostridium tetanomorphum Clostridium tetanomorphumstrain DSM 4474 strain DSM 4474 61 Clostridium cochlearium strain JCM 1396 45 45 Clostridium malenominatum Clostridium malenominatum 100 E88 61 61 Clostridium cochlearium Clostridium strain cochlearium JCM 1396 strain JCM 1396 Clostridium swellfunianum strain S11-3-10 Clostridium estertheticum subsp. laramiense strain DSM 14864 100 Clostridium100 tetani Clostridium E88 tetani E88 99 Clostridium cellulovorans 743B 92 Clostridium frigoris strain D-1/D-an/II Clostridium swellfunianum Clostridium strain swellfunianum S11-3-10 strain S11-3-10 100 Clostridium algidicarnis strain NCFB 2931 33 47 Clostridium algoriphilum strain 14D1 Clostridium cellulovorans Clostridium 743B cellulovorans 743B 39 Clostridium putrefaciens strain DSM 1291 100 100 Clostridium100 algidicarnis Clostridium strain algidicarnis NCFB 2931 strain NCFB 2931 Clostridium bowmanii strain DSM 3314206 33 46 8139 39 Clostridium putrefaciens Clostridium strain putrefaciens DSM 1291 strain DSM 1291 Clostridium tagluense strain A121 Clostridium amylolyticum strain SW408 46 46 Clostridium intestinale Clostridium intestinale Clostridium tunisiense strain TJ 81 81 100 Clostridium polynesiense strain MS1T Clostridium amylolyticum Clostridium strain amylolyticum SW408 strain SW408 76 86 Clostridium sulfidigenes strain MCM B 937 82 Clostridium bovipellis strain B30 100 Clostridium100 polynesiense Clostridium strain polynesiense MS1T strain MS1T 100 Clostridium thiosulfatireducens strain LUP 21 SM101 82 82 Clostridium bovipellis Clostridium strain bovipellisB30 strain B30 100 Clostridium amazonense strain NE08V 100 strain JCM 1409 34 Clostridium perfringens Clostridium SM101 perfringens SM101 Clostridium senegalense strain JC122 90 Clostridium sardiniense strain KZ 1544 Clostridium baratii Clostridium strain JCM baratii 1409 strain JCM 1409 30 100 100 strain AB32 48 Clostridium subterminale isolate DSM 2636 34 34 44 9092 90 Clostridium sardiniense Clostridium strain sardiniense KZ 1544 strain KZ 1544 strain ATCC 27322 Clostridium isatidis isolate MV1 100 Clostridium colicanis strain AB32 44 44 Clostridium colicanis Clostridium strain AB32 chauvoei strain 2585 99 Clostridium schirmacherense strain AP15 92 92 96 Clostridium99 isatidis Clostridium Clostridiumisolate MV1isatidis septicum isolate strain MV1 Pasteur III Clostridium collagenovorans strain SG 61 96 96 Clostridium Clostridium botulinum strain Bchauvoei2585 str. Eklund strain 17B(NRP) 2585 100 Clostridium acetobutylicum DSM 1731 99 Clostridium99 Clostridium neonatale strain septicum Pasteur16S III strain Pasteur III 100 Clostridium aurantibutyricum strain DSM 793 61 61 Clostridium Clostridium botulinum butyricum Clostridium B strain str. botulinum EklundJKY6D1 17B(NRP) B str. Eklund 17B(NRP) 17 94 94 Clostridium botulinum BKT015925 Clostridium Clostridium corinoforum neonatale Clostridium strain 16SDSM neonatale5906 16S 100 Clostridium haemolyticum strain JCM 1402 63 94 94 Clostridium Clostridium saccharobutylicum strain butyricum JKY6D1 DSM 13864strain JKY6D1 99 Clostridium novyi NT Clostridium Clostridiumcorinoforum strain corinoforum DSM strain 5906 NCIMB strain 14988 DSM 5906 63 63 23 Clostridium ganghwense strain HY-42-06 Clostridium72 Clostridium saccharobutylicum Clostridium saccharoperbutylacetonicum saccharobutylicum DSM 13864 N1-4(HMT) DSM 13864 Clostridium oceanicum strain DSM 1290T Clostridium55 Clostridium beijerinckii strain strain beijerinckii NCIMB DSM 5430 14988 strain NCIMB 14988 72 72 99 Clostridium botulinum A str. ATCC 19397 Clostridium100 saccharoperbutylacetonicumClostridium Clostridium roseum saccharoperbutylacetonicum strain N36 N1-4(HMT) N1-4(HMT) 100 strain DSM 795 80 Clostridium55 acidisoli Clostridium strain CK7455 diolis Clostridium strain DSM diolis 5430 strain DSM 5430 5 100 Clostridium pascui strain DSM 10365 100 Clostridium100 pasteurianum Clostridium100 BC1 roseum Clostridium strain N36 roseum strain N36

Clostridium peptidivorans strain TMC4 80 Clostridium80 acidisoli ClostridiumClostridium strain akagii CK74acidisoli strain strain CK58 CK74 Clostridium acetireducens strain 30A 100 100 Clostridium48 Clostridium carboxidivorans BC1 pasteurianum P7 BC1 48 Clostridium100 akagiiClostridium Clostridium strain drakei CK58 akagii strain SL1strain CK58 99 Clostridium lundense strain DSM 17049 44 41 94 strain ATCC 25775 Clostridium tetanomorphum strain DSM 4474 48 Clostridium 48carboxidivorans Clostridium carboxidivoransP7 P7 Clostridium malenominatum 100 Clostridium Clostridium100 drakei Clostridium magnumstrain SL1 strain drakei FM5 strain SL1 45 44 44 94 94 64 Clostridium scatologenes Clostridium strain scatologenes ATCC 25775 strain ATCC 25775 NBRC 12016 61 Clostridium cochlearium strain JCM 1396 Clostridium algifaecis strain MB9-9 100 Clostridium tetani E88 Clostridium magnum Clostridium strain FM5magnum strain82 FM5 99 Clostridium tyrobutyricum Clostridium swellfunianum strain S11-3-1064 64 Clostridium kluyveri Clostridium NBRC 12016kluyveri NBRC 12016 63 Clostridium Clostridium autoethanogenum algifaecis DSM strain 10061 MB9-9 Clostridium cellulovorans 743B 82 82 Clostridium algifaecis strain MB9-9 99 99 100 DSM 13528 Clostridium tyrobutyricum Clostridium tyrobutyricum 100 Clostridium algidicarnis strain NCFB 2931 33 63 63 Clostridium autoethanogenum Clostridium autoethanogenum DSM 10061 DSM 10061 39 Clostridium putrefaciens strain DSM 1291 0.02 100 Clostridium100 ljungdahlii Clostridium DSM ljungdahlii13528 DSM 13528 46 Clostridium intestinale 81 Clostridium amylolyticum strain SW408 100 Clostridium polynesiense strain MS1T0.02 0.02

82 Clostridium bovipellis strain B30 Clostridium perfringens SM101

100 Clostridium baratii strain JCM 1409 34 90 Clostridium sardiniense strain KZ 1544

44 Clostridium colicanis strain AB32 92 Clostridium isatidis isolate MV1

96 Clostridium chauvoei strain 2585 99 Clostridium septicum strain Pasteur III 61 Clostridium botulinum B str. Eklund 17B(NRP) Clostridium neonatale 16S

94 Clostridium butyricum strain JKY6D1 Clostridium corinoforum strain DSM 5906 63 Clostridium saccharobutylicum DSM 13864 Clostridium beijerinckii strain NCIMB 14988 72 Clostridium saccharoperbutylacetonicum N1-4(HMT)

55 Clostridium diolis strain DSM 5430 100 Clostridium roseum strain N36

80 strain CK74 100 Clostridium pasteurianum BC1 Clostridium akagii strain CK58

48 Clostridium carboxidivorans P7 100 strain SL1 44 94 Clostridium scatologenes strain ATCC 25775 Clostridium magnum strain FM5

64 Clostridium kluyveri NBRC 12016

82 Clostridium algifaecis strain MB9-9 99 Clostridium tyrobutyricum 63 Clostridium autoethanogenum DSM 10061 100 Clostridium ljungdahlii DSM 13528

0.02 Pathogenesis

C. botulinum ……….., Botox, EGS

C. tetani ……………… tetanus

C. perfringens …….. gas gangrene / food poisoning

C.* difficile ………….. antibiotic-associated pseudomembranous colitis

• C. septicum, C. chauvoei, C. baratii, C. argentinense, C. haemolyticum, C. intestinalis, C. noyvi, C. subterminale and others… • * C. difficile has recently been reclassified as Peptoclostridium difficile Clostridium in the Environment

• Ubiquitous in many natural environments

• Spores- soils, marine & estuarine sediments

• Natural intestinal microbiota- faeces, sewage & organic amendments Soil Growth Factors

Palmer et al. (unpublished work) What is required?

• More prevalence data on multiple pathogenic spp. across environments • Key growth data on Clostridium pathogens in the soil

• A molecular tool for multiple Clostridium pathogen screening and differentiation • Determination of what species are truly Clostridium

1. Taxonomic Revision

• Previous Clostridial based on phenotypic/ 16S rDNA • Recent research reassigned 78 previous Clostridium spp. to other genera1 – C. difficile  Peptoclostridium difficile • Nomenclature of some spp. differs between resources  imperative to deduce true phylogeny

1 Yutin and Galperin (2013). Environ. Microbiol. v15, 10 Method

• Utilised 101 complete genome sequences on NCBI

• Assessed 206 housekeeping genes for their homology amongst clostridial representatives

• 50 suitable genes were aligned and trimmed

• Created 50 housekeeping gene concatenation

• Used NJ, ML & BI models to create phylogeny using CIPRES platform 100 Clostridium botulinum Group I (13 strains) 100 Clostridium botulinum B str. Osaka05 Clostridium botulinum Prevot 594 97 100 Clostridium sporogenes str. NCIMB 10696 91 100 Clostridium sporogenes str. DSM 795 100 Clostridium scatologenes str. ATCC 25775 Clostridium carboxidivorans P7 100 Clostridium ljungdahlii DSM 13528 100 100 Clostridium autoethanogenum DSM 10061 100 Clostridium kluyveri NBRC 12016 DNA 100 Clostridium kluyveri DSM 555

100 Clostridium tetani 12124569 100 Clostridium tetani E88 100 Clostridium botulinum BKT015925 Clostridium novyi NT 100 Clostridium pasteurianum DSM 525 100 100 Clostridium pasteurianum ATCC 6013 100 Clostridium pasteurianum BC1 100 Clostridium acetobutylicum EA 2018 Clostridium acetobutylicum ATCC 824 100 Clostridium acetobutylicum DSM 1731 100 Clostridium cellulovorans 743B Clostridium borminense M2/40 Clostridum sensu stricto Clostridium algidicarnis strain B3 100 Clostridium intestinale URNW 76 Clostridium perfringens F262 99 91 Clostridium perfringens str. FORC 003 100 Clostridium perfringens ATCC 13124 100 Clostridium perfringens str. 13 100 Clostridium colicanis 209318 Clostridium baratii str. Sullivan 99 Clostridium botulinum str. NCTC 8550 100 100 Clostridium botulinum E3 str. Alaska E43 100 Clostridium botulinum str. NCTC 8266 100 100 Clostridium botulinum E1 str. BoNT E Beluga Clostridium botulinum 202F

100 Clostridium botulinum B str. Eklund 17B(NRP) 100 100 Clostridium botulinum B str. Eklund 17B Clostridium saccharobutylicum DSM 13864 Clostridium butyricum str. HM 68 100 Clostridium saccharoperbutylacetonicum N1-4(HMT) 72 Clostridium beijerinckii str. NCIMB 14988 100 Clostridium pasteurianum NRRL B 598 100 91 Clostridium beijerinckii ATCC 35702 100 Clostridium beijerinckii NCIMB 8052 100 [Clostridium] ultunense Esp Gottschalkia acidurici 9a

100 Clostridium aceticum str. DSM 1496 Peptoclostridium sticklandii str. DSM 519 92 100 Peptoclostridium sordellii ATCC9714 100 Peptoclostridium sordellii JGS6382 Peptoclostridium 100 Peptoclostridium mangenotii LM2 100 Peptoclostridium difficile (22 strains) 100 Ruminiclostridium cellulosi DG5 100 Ruminiclostridium stercorarium subsp stercorarium DSM 8532 100 Ruminiclostridium stercorarium subsp stercorarium DSM 8532 100 Ruminiclostridium josui JCM 17888 99 Ruminiclostridium Ruminiclostridium cellulolyticum H10 100 Ruminiclostridium clariflavum DSM 19732 100 Ruminiclostridium thermocellum DSM 1313 100 Ruminiclostridium thermocellum ATCC 27405 Cellulosilyticum lentocellum DSM 5427 complete genome 100 Lachnoclostridium phytofermentans KNHs212 100 Lachnoclostridium phytofermentans ISDg Lachnoclostridium hathewayi WAL 18680 100 Lachnoclostridium Lachnoclostridium bolteae 90A9 100 100 Lachnoclostridium indolis DSM 755 100 Lachnoclostridium saccharolyticum WM1

0.15 100 Clostridium botulinum Group I (13 strains) 100 Clostridium botulinum B str. Osaka05 Clostridium botulinum Prevot 594 97 100 Clostridium sporogenes str. NCIMB 10696 91 100 Clostridium sporogenes str. DSM 795 100 Clostridium scatologenes str. ATCC 25775 Clostridium carboxidivorans P7 100 Clostridium ljungdahlii DSM 13528 100 100 Clostridium autoethanogenum DSM 10061 100 Clostridium kluyveri NBRC 12016 DNA 100 Clostridium kluyveri DSM 555

100 Clostridium tetani 12124569 100 Clostridium tetani E88 100 Clostridium botulinum BKT015925 Clostridium novyi NT 100 Clostridium pasteurianum DSM 525 100 100 Clostridium pasteurianum ATCC 6013 100 Clostridium pasteurianum BC1 100 Clostridium acetobutylicum EA 2018 Clostridium acetobutylicum ATCC 824 100 Clostridium acetobutylicum DSM 1731 100 Clostridium cellulovorans 743B Clostridium borminense M2/40 Clostridum sensu stricto Clostridium algidicarnis strain B3 100 Clostridium intestinale URNW 76 Clostridium perfringens F262 99 91 Clostridium perfringens str. FORC 003 100 Clostridium perfringens ATCC 13124 100 Clostridium perfringens str. 13 100 Clostridium colicanis 209318 Clostridium baratii str. Sullivan 99 Clostridium botulinum str. NCTC 8550 100 100 Clostridium botulinum E3 str. Alaska E43 100 Clostridium botulinum str. NCTC 8266 100 100 Clostridium botulinum E1 str. BoNT E Beluga Clostridium botulinum 202F

100 Clostridium botulinum B str. Eklund 17B(NRP) 100 100 Clostridium botulinum B str. Eklund 17B Clostridium saccharobutylicum DSM 13864 Clostridium butyricum str. HM 68 100 Clostridium saccharoperbutylacetonicum N1-4(HMT) 72 Clostridium beijerinckii str. NCIMB 14988 100 Clostridium pasteurianum NRRL B 598 100 91 Clostridium beijerinckii ATCC 35702 100 Clostridium beijerinckii NCIMB 8052 100 [Clostridium] ultunense Esp Gottschalkia acidurici 9a

100 Clostridium aceticum str. DSM 1496 Peptoclostridium sticklandii str. DSM 519 92 100 Peptoclostridium sordellii ATCC9714 100 Peptoclostridium sordellii JGS6382 Peptoclostridium 100 Peptoclostridium mangenotii LM2 100 Peptoclostridium difficile (22 strains) 100 Ruminiclostridium cellulosi DG5 100 Ruminiclostridium stercorarium subsp stercorarium DSM 8532 100 Ruminiclostridium stercorarium subsp stercorarium DSM 8532 100 Ruminiclostridium josui JCM 17888 99 Ruminiclostridium Ruminiclostridium cellulolyticum H10 100 Ruminiclostridium clariflavum DSM 19732 100 Ruminiclostridium thermocellum DSM 1313 100 Ruminiclostridium thermocellum ATCC 27405 Cellulosilyticum lentocellum DSM 5427 complete genome 100 Lachnoclostridium phytofermentans KNHs212 100 Lachnoclostridium phytofermentans ISDg Lachnoclostridium hathewayi WAL 18680 100 Lachnoclostridium Lachnoclostridium bolteae 90A9 100 100 Lachnoclostridium indolis DSM 755 100 Lachnoclostridium saccharolyticum WM1

0.15 2. Protocol Development

• Require a rapid, high-throughput molecular method for multi-pathogen detection & enumeration

• 16S rRNA gene too conserved- can’t be specific to Clostridium & discriminate between species

• Evaluated alternate genes to develop PCR-TRFLP assay to screen for target pathogens

• Pathogens present in environmental samples can be individual quantified using qPCR

Forward primer: 5’ AGATAYAGAAAAGAAGARACTA 3’ Reverse primer: 5’ CTTTWCCTTCTAARAATTCWAR 3’ In-silico analysis

• Gene analysis of 70+ housekeeping genes: PGK gene (Phosphoglycerate kinase) was selected

• A range of F/R primer combination tested to give best specificity & PCR conditions

• HIGHLY SPECIFIC : Primer set developed that targets major 10 pathogen groups

• Tested on 8 Clostridum/Peptoclostridium target pathogens

Pathogen differentiation

• Used in-silico restriction digestion software to develop Clostridium baratii 707 89 Clostridium botulinum Group I 227 39 a T-RFLP protocol to Clostridium sporogenes 227 39 differentiate between Clostridium botulinum Group III 491 155 pathogen groups Clostridium haemolyticum 491 155 Clostridium botulinum Group II 458 292 Clostridium colicanis 458 89 • 799 BP amplicon can be Clostridium novyi 440 359 digested with restriction Clostridium perfringens 195 310 Clostridium tetani 12124569 85 591 enzymes SspI and SfcI to give Clostridium tetani E88 113 39 a unique combination of Peptoclostridium difficile 64 211 peaks for each group (using Peptoclostridium sordellii 64 85 Peptolostridium bifermentans 64 39 labelled primers)

24, 223 Sporogenes (50ng)

79, 60 Sordellii (50ng)

Bacillus Neg.

79, 306 60, 191, 447 Mix. (100ng)

24, 223 Sporogenes (25ng)

79, 207, 301, Mix (200ng) 60, 191, 223

Septicum (25ng)

306, 191 Perfringens (50ng) 207, 60 Difficile (50ng)

306, 191 Perfringens (25ng)

207, 60 Difficile (25ng)

Bifermentans (50ng) 23, 60 Results & Refinement

• PCR: highly specific, only targets pathogens of interest. Works on 5 species tested to date • T-RFLP: On pure DNA, produced unique peak profile depending on pathogen species • Further strains to be tested, sensitivity analysis to be performed

Species forward reverse P. bifermentans 39 23 64 60 P. difficile 211 207 64 60 P. sordellii 85 79 64 60 C. perfringens 310 306 195 191 C. sporogenes 39 24 227 223 Environmental sample Screening

• Screen subset of NSIS soil DNA for pathogens using protocol • Screen estuarine sediments DNA extracts and digestates • If present, increase the subset size. Quantify pathogen abundance with qPCR • Running a year long field trial at farms to determine pathogen survival Microcosm studies

• Started microcosm studies to manipulate key soil/ environmental parameters and the effect on multiple pathogen species survival

• First one investigate the effect of water concentration and soil type

• Use the growth data to identify potential hotspots on agricultural land Conclusions

• Developed a robust analytical pipeline for phylogenetic assignment of strains and spp. • Identified the incorrect taxonomy of some strains and spp. • Developed a simple PCR-TRFLP protocol that detects multiple significant pathogens Acknowledgments

• Supervisors Dr Lisa Avery Dr Rupert Hough Dr Helen West