Lactobacillus: Implications for the Probiotic Field

Nomenclature Upheaval of the Genus Lactobacillus: Implications for the Probiotic Field

Bruno POT

Singapore, June 5, 14h30 What’s it all about?

• The current genus Lactobacillus • What do the rules say? • Something about the methodology (minimal standards) • The implications for the industry • Conclusions? The genus Lactobacillus

Life On May 1st, 2018: Domain or Empire: Bacteria

• Number of Lactobacillus species: 231 Kingdom: Eubacteria • Number of Lactobacillus subspecies: 29 Phylum or Division: Firnicutes

Class: Bacilli http://www.bacterio.net/lactobacillus.html Sublass - idae Order: Lactobacillales (Ludwig et al. 2010) Suborder - inae Family: Lactobacillaceae (Winslow et al. 1917) Subtribe, tribe, subfamily - inae -eae -oideae Genus: Lactobacillus (Beijerinck 1901)

Species: delbrueckii (Leichmann 1896) Subspecies: delbrueckii

L. delbrueckii = Type Species

→ Heterogeneity is huge The genus Lactobacillus

Species within the genus were originally classified according to carbohydrate metabolism (either the Embden-Meyerhof pathway or the phosphoketolase pathway, more recently also the pentose phosphate pathway): - obligately homofermentative (metabolic group A) - facultatively heterofermentative (metabolic group B) - obligately heterofermentative lactobacilli (metabolic group C)

This difference is depending on specific genes and can be strain dependent.

→ Not really suitable as a taxonomic marker

A revised genome-based re-classification of the genus seems to be the only way out BUT, even then there are several ways to have a look at it MLSA-based (2015)

Homofermentative and heterofermentative lactobacilli reflect major physiological and ecological differences

Phylogenomic analysis and basic features of 174 Lactobacillus and Pediococcus type strains based on the concatenated protein sequences of single-copy core genes (Multi- Locus Sequence Analysis).

The assignment of species to phylogenetic groups shown here confirms previous assignments of species to phylogenetic groups (POT et al., 2014) with the following exceptions or additions: L. kunkeei and Lactobacillus ozensis are now grouped together in a new L. kunkeei group; Lactobacillus selangorensis (previously in the L. perolens group) forms a separate group adjacent to L. sakei; L. amylophilus (previously in the L. delbrueckii group) forms a separate group adjacent to the L. delbrueckii group; Lactobacillus algidus (previously L. salivarius) forms a separate group adjacent to the L. salivarius group. Lactobacillus concavus was assigned to a new group with the reclassified L. dextrinicus. L. mellis and L. mellifer form a separate group.

Zheng J, Ruan L, Sun M, Gänzle M. 2015. A genomic view of lactobacilli and pediococci demonstrates that phylogeny matches ecology and physiology. Appl Environ Microbiol 81:7233–7243. doi:10.1128/AEM.02116-15. Phylogeny (Maximum Likelihood) of 213 Lactobacillus strains based on 73 core gene sequences (2015 situation; slightly more species)

Species already removed from the genus Lactobacillus

Sun et al., 2015. Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. Nat Commun; 6: 8322. Published online 2015 Sep 29. doi: 10.1038/ncomms9322 Phylogeny (Maximum Likelihood) of 213 Lactobacillus strains based on 73 core gene sequences (2015 situation; slightly more species)

Species already removed from the genus Lactobacillus

Sun et al., 2015. Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. Nat Commun; 6: 8322. Published online 2015 Sep 29. doi: 10.1038/ncomms9322 16S rDNA 100 96 97 98 99 82 83 84 85 86 87 88 89 90 91 92 93 94 95 81 Eggerthia “Lactobacillus” rogosae (sp cand; type strain missing) “Lactobacillus “catenaformis (T) “Lactobacillus “vitulinus (T) "Lactobacillus" fructosus (T) "Lactobacillus" kandleri (T) Kandleria and "Lactobacillus" halotolerans (T) from

"Lactobacillus" confusus (T) genus "Lactobacillus" minor (T) "Lactobacillus" viridescens (T) Lactobacillus reuteri (T) Weissella already Lactobacillus panis (T) Lactobacillus antri (T) Lactobacillus oris (T) the Lactobacillus pontis (T) Lactobacillus frumenti (T) Lactobacillus vaginalis (T) Lactobacillus coleohominis (T) Lactobacillus Lactobacillus secaliphilus (T) Lactobacillus alvi (T) renamed

Lactobacillus "thermotolerans" (T) removed Lactobacillus ingluviei (T) Lactobacillus fermentum (T) Species Lactobacillus "cellobiosus" (T) Lactobacillus mucosae (T) Lactobacillus equigenerosi (T) Lactobacillus gastricus (T) Lactobacillus amylophilus (T) Lactobacillus amylotrophicus (T) Lactobacillus iners (T) Lactobacillus johnsonii (T) Lactobacillus gasseri (T) Lactobacillus taiwanensis (T) Lactobacillus psittaci (T) Lactobacillus jensenii (T) Lactobacillus fornicalis (T) Lactobacillus equicursoris (T) Lactobacillus "leichmannii" Lactobacillus delbrueckii subsp. delbrueckii (T) Lactobacillus delbrueckii subsp. lactis (T) Lactobacillus delbrueckii subsp. bulgaricus (T) Lactobacillus delbrueckii subsp. indicus (T) Lactobacillus acetotolerans (T) Lactobacillus acidophilus (T) Lactobacillus "suntoreyus" Lactobacillus gallinarum (T) Lactobacillus helveticus (T) Lactobacillus ultunensis (T) Lactobacillus kefiranofaciens subsp. kefiranofaciens (T) Lactobacillus kefiranofaciens subsp. kefirgranum (T) Lactobacillus crispatus (T) Lactobacillus amylovorus (T) Lactobacillus kitasatonis (T) Lactobacillus gigeriorum (T) Lactobacillus amylolyticus (T) Lactobacillus hamsteri (T) Lactobacillus kalixensis (T) Lactobacillus intestinalis (T) based Lactobacillus siliginis (T) Lactobacillus rossiae (T) Lactobacillus oligofermentans (T) Lactobacillus suebicus (T) Lactobacillus vaccinostercus (T) Lactobacillus "durianis" (T) Lactobacillus brantae (T) Lactobacillus fuchuensis (T) Lactobacillus "bavaricus" (T) Lactobacillus sakei subsp. sakei (T) Lactobacillus sakei subsp. carnosus (T) Lactobacillus curvatus (T) Lactobacillus graminis (T) Lactobacillus sharpeae (T) Lactobacillus thailandensis (T)

) Lactobacillus pantheris (T) Lactobacillus camelliae (T) Lactobacillus manihotivorans (T) Lactobacillus nasuensis (T) Lactobacillus saniviri (T) Lactobacillus casei (T) Lactobacillus rhamnosus (T) Lactobacillus zeae (T) Lactobacillus paracasei subsp. tolerans (T) Lactobacillus paracasei subsp. paracasei (T) Lactobacillus paucivorans (T) similarities Lactobacillus spicheri (T) Lactobacillus namurensis (T) Lactobacillus zymae (T) Lactobacillus acidifarinae (T) Lactobacillus Lactobacillus brevis (T) Lactobacillus koreensis (T) species Lactobacillus parabrevis (T) Lactobacillus senmaizukei (T) Lactobacillus hammesii (T) Lactobacillus malefermentans (T) Lactobacillus kimchicus (T) Lactobacillus similis (T) Lactobacillus odoratitofui (T) ) of Lactobacillus "pasteurianus" (T) Lactobacillus collinoides (T)

less Lactobacillus paracollinoides (T) Lactobacillus xiangfrangensis (T) Lactobacillus fabifermentans (T) Lactobacillus paraplantarum (T) Lactobacillus plantarum subsp. argentoratensis (T) Lactobacillus plantarum subsp. plantarum (T) Lactobacillus "arizonensis" (T)

sequence Lactobacillus pentosus (T) Lactobacillus alimentarius (T) Lactobacillus bobalius (T) Lactobacillus paralimentarius (T) Lactobacillus kimchii (T) Lactobacillus nantensis (T)

Joining Lactobacillus mindensis (T) Lactobacillus futsaii (T) Lactobacillus farciminis (T) Lactobacillus crustorum (T) Lactobacillus versmoldensis (T) Lactobacillus nodensis (T) Lactobacillus tucceti (T) Lactobacillus kunkeei (T) rDNA Lactobacillus ozensis (T) Lactobacillus "heterohiochii" (T) situation; situation; Lactobacillus homohiochii (T) Lactobacillus fructivorans (T) Lactobacillus florum (T) Lactobacillus sanfranciscensis (T) Lactobacillus lindneri (T) Lactobacillus senioris (T) Lactobacillus parafarraginis (T)

16S Lactobacillus hilgardii (T) Lactobacillus farraginis (T) Lactobacillus diolivorans (T) Lactobacillus kisonensis (T) Lactobacillus rapi (T)

2014 Lactobacillus buchneri (T) Lactobacillus parakefiri (T)

( Lactobacillus kefiri (T)

Neighbour Lactobacillus parabuchneri subsp. parabuchneri (T) Lactobacillus parabuchneri subsp. ferintoshensis (T)

( Lactobacillus otakiensis (T)

only Lactobacillus sunkii (T) Lactobacillus concavus (T) Lactobacillus dextrinicus (T) Lactobacillus selangorensis (T) Lactobacillus harbinensis (T) Lactobacillus perolens (T) Lactobacillus algidus (T)

on Lactobacillus saerimneri (T) Lactobacillus ceti (T) Lactobacillus vini (T) Lactobacillus nagelii (T) Lactobacillus ghanensis (T) Lactobacillus satsumensis (T) Lactobacillus cacaonum (T)

Lactobacillus mali subsp. yamanashiensis (T) Lactobacillus Lactobacillus mali subsp. mali (T) Lactobacillus hordei (T) Lactobacillus capillatus (T) Lactobacillus sucicola (T) Lactobacillus uvarum (T) Lactobacillus aquaticus (T)

Lactobacillus apodemi (T) genus Lactobacillus animalis (T)

Phylogeny Lactobacillus murinus (T) Lactobacillus ruminis (T) Lactobacillus agilis (T) Lactobacillus equi (T) Lactobacillus pobuzihii (T) Lactobacillus acidipiscis (T) Lactobacillus "cypricasei" (T)

Lactobacillus aviarius subsp. araffinosus (T) The 19; Lactobacillus aviarius subsp. aviarius (T) Lactobacillus hayakitensis (T) Lactobacillus salivarius "subsp. salicinius" (T) Lactobacillus salivarius "subsp. salivarius" (T) Lactobacillus floricola (T)

Lactobacillus composti (T)

Lactobacillus rennini (T)

Lactobacillus "backi" (T) Lactobacillus bifermentans (T)

Editor(s): W.H. Holzapfel & B.J.B. Wood & B.J.B. Holzapfel W.H. Editor(s): |https://doi.org/10.1002/9781118655252.ch19 Chapter Lactobacillus coryniformis subsp. coryniformis (T) 2014al., et Pot Lactobacillus coryniformis subsp. torquens (T) 16S rDNA 100 96 97 98 99 82 83 84 85 86 87 88 89 90 91 92 93 94 95 81

“Lactobacillus” rogosae (sp cand; type strain missing) “Lactobacillus “catenaformis (T) Eggerthia “Lactobacillus “vitulinus (T) Kandleria "Lactobacillus" fructosus (T) "Lactobacillus" kandleri (T) "Lactobacillus" halotolerans (T) Weissella "Lactobacillus" confusus (T) "Lactobacillus" minor (T) "Lactobacillus" viridescens (T) Lactobacillus reuteri (T) Lactobacillus panis (T) Lactobacillus antri (T) Lactobacillus oris (T) Lactobacillus pontis (T) Lactobacillus frumenti (T) Lactobacillus vaginalis (T) Lactobacillus coleohominis (T) Lactobacillus secaliphilus (T) Lactobacillus alvi (T) Lactobacillus "thermotolerans" (T) Lactobacillus ingluviei (T) Lactobacillus fermentum (T) Lactobacillus "cellobiosus" (T) Lactobacillus mucosae (T) Lactobacillus equigenerosi (T) Lactobacillus gastricus (T) Lactobacillus amylophilus (T) Lactobacillus amylotrophicus (T) Lactobacillus iners (T) Lactobacillus johnsonii (T) Lactobacillus gasseri (T) Lactobacillus taiwanensis (T) Lactobacillus psittaci (T) Lactobacillus jensenii (T) Lactobacillus fornicalis (T) Lactobacillus equicursoris (T) Lactobacillus "leichmannii" Lactobacillus delbrueckii subsp. delbrueckii (T) Lactobacillus delbrueckii subsp. lactis (T) Lactobacillus delbrueckii subsp. bulgaricus (T) Lactobacillus delbrueckii subsp. indicus (T) Lactobacillus acetotolerans (T) Lactobacillus acidophilus (T) Lactobacillus "suntoreyus" Lactobacillus gallinarum (T) Lactobacillus helveticus (T) Lactobacillus ultunensis (T) Lactobacillus kefiranofaciens subsp. kefiranofaciens (T) Lactobacillus kefiranofaciens subsp. kefirgranum (T) Lactobacillus crispatus (T) Lactobacillus amylovorus (T) Lactobacillus kitasatonis (T) Lactobacillus gigeriorum (T) Lactobacillus amylolyticus (T) Lactobacillus hamsteri (T) Lactobacillus kalixensis (T) Lactobacillus intestinalis (T) based Lactobacillus siliginis (T) Lactobacillus rossiae (T) clusters clusters Lactobacillus oligofermentans (T) Lactobacillus suebicus (T)

Lactobacillus vaccinostercus (T) 16 15 4 Lactobacillus "durianis" (T) Lactobacillus brantae (T) Lactobacillus fuchuensis (T) Lactobacillus "bavaricus" (T) Lactobacillus sakei subsp. sakei (T) Lactobacillus sakei subsp. carnosus (T) Lactobacillus curvatus (T) Lactobacillus graminis (T) Lactobacillus sharpeae (T) Lactobacillus thailandensis (T)

) Lactobacillus pantheris (T) Lactobacillus camelliae (T) Lactobacillus manihotivorans (T) Lactobacillus nasuensis (T) Lactobacillus saniviri (T) Lactobacillus casei (T) Lactobacillus rhamnosus (T) Lactobacillus zeae (T) Lactobacillus paracasei subsp. tolerans (T) defined Lactobacillus paracasei subsp. paracasei (T) - Lactobacillus paucivorans (T) similarities Lactobacillus spicheri (T) Lactobacillus namurensis (T) Lactobacillus zymae (T) Lactobacillus acidifarinae (T) Lactobacillus Lactobacillus brevis (T) Lactobacillus koreensis (T) species Lactobacillus parabrevis (T) Lactobacillus senmaizukei (T) Lactobacillus hammesii (T)

Lactobacillus malefermentans (T) well Lactobacillus kimchicus (T) Lactobacillus Lactobacillus similis (T) Lactobacillus odoratitofui (T) ) of Lactobacillus "pasteurianus" (T) Lactobacillus collinoides (T)

less Lactobacillus paracollinoides (T)

Lactobacillus xiangfrangensis (T) of Lactobacillus fabifermentans (T) Lactobacillus paraplantarum (T) Lactobacillus plantarum subsp. argentoratensis (T) Lactobacillus plantarum subsp. plantarum (T) Lactobacillus "arizonensis" (T)

sequence Lactobacillus pentosus (T) Lactobacillus alimentarius (T) Lactobacillus bobalius (T) Lactobacillus paralimentarius (T) Lactobacillus kimchii (T)

Lactobacillus nantensis (T) genus

Lactobacillus "heterohiochii" (T) the situation; situation; Lactobacillus homohiochii (T) number Lactobacillus fructivorans (T) Lactobacillus florum (T) Lactobacillus sanfranciscensis (T) Lactobacillus lindneri (T) Lactobacillus senioris (T) Lactobacillus parafarraginis (T)

16S Lactobacillus hilgardii (T) Lactobacillus farraginis (T) Lactobacillus diolivorans (T) Lactobacillus kisonensis (T) Lactobacillus rapi (T)

2014 Lactobacillus buchneri (T) Lactobacillus parakefiri (T)

( Lactobacillus kefiri (T)

Neighbour Lactobacillus parabuchneri subsp. parabuchneri (T) Lactobacillus parabuchneri subsp. ferintoshensis (T) within ( Lactobacillus otakiensis (T)

on Lactobacillus saerimneri (T) Lactobacillus ceti (T) Lactobacillus vini (T) Lactobacillus nagelii (T) Lactobacillus ghanensis (T) Lactobacillus satsumensis (T) Lactobacillus cacaonum (T)

Lactobacillus mali subsp. yamanashiensis (T) Lactobacillus Lactobacillus mali subsp. mali (T)

Lactobacillus hordei (T) considerable Lactobacillus capillatus (T) Lactobacillus sucicola (T) Lactobacillus uvarum (T) Lactobacillus aquaticus (T) a

Lactobacillus apodemi (T) genus Lactobacillus animalis (T)

Phylogeny Lactobacillus murinus (T) Lactobacillus ruminis (T) Lactobacillus agilis (T) Lactobacillus equi (T) Lactobacillus pobuzihii (T) Lactobacillus acidipiscis (T) Lactobacillus "cypricasei" (T)

Lactobacillus floricola (T)

Lactobacillus composti (T)

Lactobacillus rennini (T)

Lactobacillus "backi" (T)

Lactobacillus bifermentans (T) 1 2 3 4 5 6 7 8 9 10 11 12 13 1 13 12 11 10 9 8 7 6 5 4 3 2 1

Editor(s): W.H. Holzapfel & B.J.B. Wood & B.J.B. Holzapfel W.H. Editor(s): |https://doi.org/10.1002/9781118655252.ch19 Chapter Lactobacillus coryniformis subsp. coryniformis (T) 2014al., et Pot Lactobacillus coryniformis subsp. torquens (T) Heterogenity revealed by 16S rRNA sequencing already led to

nomencaltural changes in the past…

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

100

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

100

85 86 87 88 89 90 91 92 93 94 95 96 97 98

99 16S rDNA sequence similarity scaling bar 100 Lactobacillus amylophilus 100 Lactobacillus amylotrophicus Lactobacillus iners 87 100 Lactobacillus hominis 98 100 Lactobacillus floricola Lactobacillus siliginis 100 Lactobacillus johnsonii 83 Lactobacillus composti 100 Lactobacillus rossiae 70 Lactobacillus gasseri 19 Lactobacillus taiwanensis 100 Lactobacillus rennini 59 Lactobacillus suebicus 100 56 "Lactobacillus backi" 98 Lactobacillus psittaci 2 Lactobacillus bifermentans 100 Lactobacillus vaccinostercus 94 47 100 Lactobacillus durianis 100 Lactobacillus fornicalis 100 Lactobacillus coryniformis subsp. coryniformis Lactobacillus jensenii Lactobacillus coryniformis subsp. torquens 59 Lactobacillus oligofermentans "Lactobacillus hokkaidonensis" 96 Lactobacillus acetotolerans 65 Pediococcus ethanolidurans Lactobacillus equicursoris 54 Pediococcus siamensis Lactobacillus reuteri 100 100 Lactobacillus delbrueckii subsp. sunkii Pediococcus cellicola 97 95 Lactobacillus panis 69 81 37 Lactobacillus leichmannii (partial sequence) Pediococcus parvulus 97 Lactobacillus antri 100 47 Lactobacillus delbrueckii subsp. delbrueckii 94 72 Pediococcus damnosus 100 Lactobacillus oris 70 Lactobacillus delbrueckii subsp. lactis Pediococcus inopinatus Lactobacillus pontis 45 93 Pediococcus claussenii 54 Lactobacillus delbrueckii subsp. bulgaricus 83 Lactobacillus frumenti 51 Lactobacillus delbrueckii subsp. indicus Pediococcus argentinicus 100 45 97 Pediococcus lolii Lactobacillus vaginalis 99 Lactobacillus pasteurii 95 Pediococcus acidilactici Lactobacillus mucosae Lactobacillus gigeriorum 38 69 Pediococcus pentosaceus 99 Lactobacillus equigenerosi 91 Lactobacillus amylolyticus Pediococcus stilesii Lactobacillus gastricus 90 Lactobacillus hamsteri Lactobacillus paucivorans 63 40 100 Lactobacillus fermentum 44 Lactobacillus kalixensis 89 Lactobacillus spicheri 100 Lactobacillus namurensis Lactobacillus cellobiosus 88 Lactobacillus intestinalis 70 43 100 Lactobacillus zymae 14 “Lactobacillus alvi “ Lactobacillus acidophilus 38 99 Lactobacillus kefiranofaciens subsp. kefiranofaciens Lactobacillus acidifarinae 100 Lactobacillus ingluviei 82 100 21 Lactobacillus brevis 55 Lactobacillus thermotolerans Lactobacillus kefiranofaciens subsp. kefirgranum 74 Lactobacillus koreensis Lactobacillus ultunensis 41 73 93 Lactobacillus coleohominis 99 87 Lactobacillus parabrevis Lactobacillus secaliphilus 86 Lactobacillus sobrius * 69 Lactobacillus senmaizukei 41 95 Lactobacillus amylovorus Lactobacillus hammesii 100 Eggerthia catenaformis 83 Kandleria vitulina Lactobacillus kitasatonis 100 Lactobacillus malefermentans 87 37 Lactobacillus crispatus "Lactobacillus oryzae" Fructobacillus fructosus 99 91 76 Lactobacillus gallinarum Lactobacillus kimchicus 100 Weissella kandleri 100 Lactobacillus similis 41 Lactobacillus helveticus 100 38 Weissella halotolerans Lactobacillus suntoreyus 100 Lactobacillus odoratitofui Weissella confusa 100 Lactobacillus collinoides 59 Lactobacillus sharpeae 100 Weissella minor 98 Lactobacillus thailandensis 53 Lactobacillus paracollinoides 100 19 “Lactobacillus pastorianus” Weissella viridescens 61 Lactobacillus pantheris "Lactobacillus leiae" Lactobacillus camelliae 57 Lactobacillus senioris 98 Lactobacillus nasuensis 85 97 34 Lactobacillus parafarraginis 87 100 Lactobacillus manihotivorans 70 Lactobacillus hilgardii "Lactobacillus porcinae" 97 Lactobacillus farraginis 16S rDNA gene sequence based phylogenetic tree showing Lactobacillus saniviri Lactobacillus diolivorans 100 Lactobacillus brantae 40 Lactobacillus kisonensis 79 43 Lactobacillus rapi the phylogenetic relationships among species of the genera 100 Lactobacillus casei 33 91 Lactobacillus buchneri 92 Lactobacillus rhamnosus Lactobacillus parakefiri 78 Lactobacillus zeae 67 83 Lactobacillus kefiri Lactobacillus, Pediococcus and the species Paralactobacillus Lactobacillus paracasei subsp. tolerans 54 100 97 Lactobacillus parabuchneri Lactobacillus paracasei subsp. paracasei 58 Lactobacillus ferintoshensis Lactobacillus algidus 93 Lactobacillus otakiensis selangorensis (now Lactobacillus selangorensis). Phylogenetic 41 Lactobacillus saerimneri Lactobacillus sunkii Lactobacillus ceti 100 Lactobacillus kunkeei Lactobacillus apodemi Lactobacillus ozensis groups are named after the first species from the group 63 100 Lactobacillus heterohiochii * 95 Lactobacillus animalis 55 68 Lactobacillus murinus 0 63 100 Lactobacillus homohiochii Lactobacillus ruminis 100 Lactobacillus fructivorans validly described (indicated in bold) and are marked by a color 52 77 Lactobacillus florum 87 89 Lactobacillus agilis 98 Lactobacillus sanfranciscensis Lactobacillus equi 71 Lactobacillus lindneri coding. 44 Lactobacillus pobuzihii 100 Lactobacillus fuchuensis Lactobacillus acidipiscis 78 Lactobacillus graminis 100 Lactobacillus cypricasei 54 77 60 Lactobacillus sakei subsp. carnosus Distance between taxa is given by the sum of the length of the Lactobacillus aviarius subsp. araffinosus * 72 Lactobacillus bavaricus * 100 42 18 Lactobacillus aviarius subsp. aviarius Lactobacillus sakei subsp. sakei 93 Lactobacillus hayakitensis Lactobacillus curvatus horizontal branches. The percent scale on top of the figure 70 16 100 Lactobacillus xiangfrangensis 99 Lactobacillus salivarius subsp. salicinius 4 Lactobacillus salivarius subsp. salivarius 70 Lactobacillus fabifermentans Lactobacillus vini 97 Lactobacillus paraplantarum can be used to estimate distances. 68 Lactobacillus plantarum subsp. argentoratensis 100 Lactobacillus nagelii 80 Lactobacillus plantarum subsp. plantarum 36 Lactobacillus ghanensis 71 73 Lactobacillus arizonensis The Neighour Joining tree was obtained using the 78 89 Lactobacillus satsumensis 45 Lactobacillus pentosus Lactobacillus oeni 93 Lactobacillus versmoldensis Lactobacillus cacaonum "Lactobacillus ginsenosidimutans" 88 100 BioNumerics software version 6.6 (Applied Maths, Sint- 97 97 Lactobacillus mali subsp. yamanashiensis * 87 Lactobacillus nodensis 71 Lactobacillus mali subsp. mali Lactobacillus tucceti 100 Lactobacillus alimentarius 94 Lactobacillus hordei 71 Martens-Latem, Belgium) with the following parameters: 61 100 Lactobacillus bobalius 94 Lactobacillus capillatus 98 Lactobacillus paralimentarius 83 Lactobacillus sucicola 100 Lactobacillus kimchii Multiple alignment based (alignment was manually edited). 96 Lactobacillus uvarum Lactobacillus kimchiensis Lactobacillus aquaticus 36 Lactobacillus nantensis Lactobacillus concavus 39 Lactobacillus mindensis 100 42 Lactobacillus futsaii Default cost table, with unknown bases discarded and no gap 38 Lactobacillus dextrinicus 51 Lactobacillus selangorensis 81 Lactobacillus farciminis 81 Lactobacillus crustorum 100 Lactobacillus perolens penalties were used. A Jukes & Cantor correction for back 87 "Lactobacillus shenzhenensis" 100 Lactobacillus siliginis 12 78 Lactobacillus harbinensis Lactobacillus rossiae Lactobacillus floricola 59 98 mutations was also applied. Branch qualities are indicated per 83 Lactobacillus composti 100 19 100 branching point (expressed in per cent) and were calculated from 1000 bootstrap simulations. Pot et al., 2014 Names between double quotes have not yet been formally Chapter 19; The genus Lactobacillus published, but appear in the literature. Some species names Editor(s): W.H. Holzapfel & B.J.B. Wood are no longer in use. |https://doi.org/10.1002/9781118655252.ch19 *, partial 16S sequences only available. Phylogeny inferred from a 73 core gene dataset (Manually curated) (213 genomes)

Weissella Oenococus Leuconostoc Fructobacillus

Carnobacterium Atopobium Sun et al, 2015; NATURE Pediococcus COMMUNICATIONS | Lactococcus 6:8322 | DOI: 10.1038 Olsenella Genome wide heterogenity (213 genomes)

Sun et al, 2015; NATURE COMMUNICATIONS | 6:8322 | DOI: 10.1038 Based on sequences of 187 Lactobacillus draft genomes and 26 genomes from 8 other genera historically associated with or grouped within the lactobacilli.

Lactobacillus: a genus more diverse than a family

• Genome size: 1.23Mb (L. sanfranciscensis) - 4.91 Mb (L. parakefiri)

• GC content : 31.93 - 57.02 % G+C

• Lactobacillus pan genome: large open genome with 44,668 gene families (in 2015) and still straight…. Nucleotide Identity distributions compared

Modified from: Sun et al, 2015; NATURE COMMUNICATIONS | 6:8322 | DOI: 10.1038

Total Nucleotide Identity Average Nucleotide Identity (pairwise matched nucleotide sequences ) (pairwise comparison of homologous sequences)

F a L m a i c t l o y b a c il l u s ANI distribution based on all the analyzed genome sequences of the genera Lactobacillus and Pediococcus. An ANI of 95 to 96% corresponds to 98.65% 16S rRNA gene sequence similarity (97-98 % defines a species)

The ANI threshold of 67.5 to 68% - confirms the 16S rRNA based phylogenetic groups (Pot et al. 2014) - groups bacterial species that are related with respect to their ecology and metabolic potential These levels of ANI normally separate phylogentic groups at the genus level….

The L. salivarius and L. delbrueckii groups are most heterogenous with respect to ANI and physiological properties, but the cluster analysis justifies maintaining these diverse groups.

Zheng J, Ruan L, Sun M, Gänzle M. 2015. A genomic view of lactobacilli and pediococci demonstrates that phylogeny matches ecology and physiology. Appl Environ Microbiol 81:7233–7243. doi:10.1128/AEM.02116-15. Relationship between ANI and 16S rRNA gene sequence similarity.

Species level

Modified from: Mincheol Kim, Hyun-Seok Oh, Sang-Cheol Park, Jongsik Chun. 2014. Towards a taxonomic coherence between L average nucleotide identity and 16S rRNA a Genus c gene sequence similarity for species t o demarcation of prokaryotes. b IJSEM 64; DOI: 10.1099/ijs.0.064931-0 a level c il l u s Intrageneric comparison of the genome signature (already in 2006: 5 Lb. species only) Modified from: van Passel et al; BMC Evolutionary Biology 2006: 6:84 doi:10.1186/1471-2148-6-84

genome-specific relative frequencies of dinucleotides

The average genomic dissimilarity between different species of the same genus Heterogeneity is a fact!

And it is growing, rapidly growing The current number of Lactobacillus species

• The increased description of novel species since the 90 per 10 years year 2000 has made the genus grow to one of the 80 biggest in the bacterial taxonomy. 70 60 50 40 Number of species, including synonyms: 231 30 Number of subspecies, including synonyms: 29 20 10 0

Number of new species descriptions in the genus Lactobacillus

16 Number is expected 14 to grow further, given 12 the discovery of new 10 species via metagen- 8 omic analyses of complex 6 ecosystems. 4 2 0 What’s it all about?

• The current genus Lactobacillus • What do the rules say? • Something about the methodology (minimal standards) • The implications for the industry • Conlusions? Classification (?) of bacteria

• There is no official classification of bacteria, but, there is a valid nomenclature. • Nomenclature is therefore a vital component of systematic bacteriology and determined by the International Code of Nomenclature of Bacteria* (See also https://www.ncbi.nlm.nih.gov/books/NBK8807/). • The code to be used today is the 1990 Revision of the Bacteriological Code (Lapage et al. 1992); it describes the rules to change or create names for bacteria. Importantly: • The starting point of the official nomenclature is the “APPROVED List of valid Bacterial Names”, published in the International Journal of Systematic Bacteriology (IJSB) from 1980, which retroactively validated all previous lists. • Since then, new names and name changes (VALIDATION LISTS) are validly published only in the official journal of the ICSP and the Bacteriology and Applied Microbiology Division of the International Union of Microbiological Societies: the International Journal of Systematic and Evolutionary Microbiology (IJSEM; www.ijsem.org), the former International Journal of Systematic Bacteriology, IJSB), published by the Microbiology Society.

* Official publication of the International Committee on Systematics of Prokaryotes (ICSP; http://icsp.org/), formerly the International Committee on Systematic Bacteriology (ICSB).

Further reading: • J. P. Euzeby, International Journal of Systematic Bacteriology, 1997, p. 590-592 • B. J. Tindall, International Journal of Systematic and Evolutionary Microbiology, 2015), p. 3226–3227 http://www.bacterio.net/ https://www.ncbi.nlm.nih.gov/books/NBK8810/ https://www.ncbi.nlm.nih.gov/books/NBK8808/ Also: a name does never dissapear…

• According to the Code, a name, once described, can never dissapear or be re-used for another description (Rule 23). • Names can therefore be of the type • nomen conservandum: a name which must be used instead of all earlier synonyms and homonyms • nomen rejiciendum: rejected name, a name which must not be used to designate any taxon • Only the Judicial Commission can conserve or reject names (Rule 56a, b) • There are also other types of names: • legitimate—in accordance with the Rules; • illegitimate—contrary to the Rules; • effectively published—in printed matter made generally available to the scientific community (Rule 25); • validly published—effectively published and accompanied by a description of the taxon or a reference to a description and certain other requirements (Rules 27–32); • correct—the name which must be adopted for a taxon under the Rules • And there are • objective synonyms: more than one name has been associated with the same type • subjective synonyms: different names have been associated with different types that in the opinion of the bacteriologist concerned belong to the same taxon) • senior synonyms: the synonym first published • junior synonyms: the later synonyms General philosophy

• Each species should fit in a genus → is already the case • Species should be assigned only if supported by sufficient data → not a problem here • Lumping into larger taxonomical units is undesirable if these units exceed ‘normal diversity’ →already the case in Lactobacillus, as illustrated • An adapted and appropriate nomenclature is needed → so, re-naming should be preferred Renamings are part of an ongoing process

Weissella Oenococus Leuconostoc Fructobacillus

Carnobacterium Atopobium Sun et al, 2015; NATURE Pediococcus COMMUNICATIONS | Lactococcus 6:8322 | DOI: 10.1038 Olsenella Two ways to deal with what is left in Lactobacillus

KEEP Lactobacillus SPLIT Lactobacillus

• Maintain the genus designation Lactobacillus for • The diversity of phylogenetic groups in the all groups (as suggested before) genus Lactobacillus justifies their formal recognition as genera, as exemplified by ▪ Hammes WP, Hertel C. 2006; the genus Pediococcus, Leuconostoc, ▪ Salvetti E, Torriani S, Felis GE. 2012; Weissella, Paralactobacillus, Atopobium, ▪ Pot B et al. 2014; etc… ▪ Zheng J et al. 2015; ▪ Sun Z. et al., 2015 • It will solve the taxonomic (naming) • Propose the terms “Lactobacillus Genus problem now, and prevent that additional species names will be added to the genus Complex” (Sun et al.) or “Lactobacillus sensu Lactobacillus that might anyhow need to be lato” (Zheng et al.) which include Lactobacillus renamed in the (near) future. spp. and Pediococcus spp. or define Lactobacillus subgroups. BUT • Phylogenomic analysis can then guide the assignment of new species to phylogenetic • There will be a large number of new genera groups in the larger genus Lactobacillus that will need to be described (at least 12) • The split has considerable (one-time) BUT consequences for the industry and for the • This doesn’t fit the current nomenclatural rules medical and research community. • The genus will continue to grow and might finally need to be split. What’s it all about?

• The current genus Lactobacillus • What do the rules say? • Something about the methodology (minimal standards) • The implications for the industry • Conlusions? What do the ‘Minimal Standards’ say?

Minimal Standards focus on SPECIES

Minimal standards for the description of new cultivable strains that represent novel genera and species belonging to the genera Bifidobacterium, Lactobacillus and related genera are proposed in accordance with Recommendation 30b of the Bacteriological Code (1990 Revision): the description of novel species should be based on phenotypic, genotypic and ecological characteristics to ensure a rich polyphasic characterization.

Concerning genotypic characterization, in addition to DNA G+C content (mol%) data, the description should be based on DNA–DNA hybridization (DDH), 16S rRNA gene sequence similarities and There are no specific rules for the description at least two housekeeping genes (e.g. hsp60 and of a genus within the family Lactobacillaceae recA). Genotypic as well as phenotypic data are needed What do the ‘Minimal Standards’ say?

Furthermore: It can be expected that genome sequences will play an increasing role in understanding the phylogeny of and in the definition of species in the future.

Regarding complete bacterial genomes, sets of multiple housekeeping genes have been established that can accurately predict genome relatedness and improve the accuracy of species identification (Coenye et al., 2005).

The selection of genes useful for species discrimination should follow some basic rules:

(i) genes should be present in one or few copies in most bacterial genomes;

(ii) they should possess a higher rate of evolution when compared with rRNA genes;

(iii) they should not easily recombine;

(iv) they should possess enough variability to allow discrimination of species in a given genus (Zeigler, 2003). Methodology that has been used to delineate possible new genera

• Calculate a core gene sequence based phylogenetic tree (Maximum likelihood tree, based on probability) • Compare with the current phylogeny (containing 16 phylogroups; Salvetti et al., 2012; Pot et al, 2014) • Perform a recurrent Group Violation test: Determine the frequency distribution of branch distances within and between the phylogroup -members

Branch length distribution and TNI value distribution (1-TNI). A current phylogrouping of the Lactobacillus Genus complex based on the maximum likelihood tree of 73 core genes.

Ideally minimize the overlap… • Calculate the frequency distribution of whole genome-wide genetic distance that is measured by the 1-TNI value

Modified from Sun et al, 2015; Based on the 16S rRNA sequence tree of Salvetti et al., 2012. NATURE COMMUNICATIONS | 6:8322 | DOI: 10.1038 Methodology that has been used to delineate possible new genera

• Manually edit the phylogroup membership to reduce the overlap

• After this editing, an improved separation of branch length distribution was obtained.

• Reducing the overlap more will be difficult due to unequal clock rates or speciation rates of the tree members. Modified from Sun et al, 2015; NATURE COMMUNICATIONS | 6:8322 | DOI: 10.1038 New genus 11 Uncertain 3

(Maximum Likelihood Tree Future Lactobacillus genus based on 73 core genes )

Uncertain 1 & 2 New genus 10 New genus 9 New genus 8 New genus 7

Result after Manual New genus 6 Editing

New genus 5

Existing genera: Weissella, Oenococcus, Leuconostoc and Fructobacillus

New genus 4

New genus 3

New genus 2

New genus 1

Existing genus: Pediococcus Sun et al, 2015; NATURE COMMUNICATIONS | 6:8322 | DOI: 10.1038 An altermative method to consider?

A Proposed Genus Boundary for the Prokaryotes Based on Genomic Insights

Qi-Long Qin,Bin-Bin Xie, Xi-Ying Zhang,Xiu-Lan Chen,Bai-Cheng Zhou,Jizhong Zhou, Aharon Oren,Yu-Zhong Zhang

2014 Journal of Bacteriology p 2210–2215

Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values.

A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%.

Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation.

Remains to be evaluated for the genus / genera within Lactobacillus. What’s it all about?

• The current genus Lactobacillus • What do the rules say? • Something about the methodology (minimal standards) • The implications for the industry • Conlusions? What is left in Lactobacillus?

Indicative distance scale; the real distance between species equals the sum of horizontal distances

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Lactobacillus amylophilus 100 Lactobacillus amylotrophicus Only the species in the phylogenetic Lactobacillus iners group of Lactobacillus delbrueckii, 100 Lactobacillus hominis 98 100 100 Lactobacillus johnsonii the type species of the genus, will Lactobacillus gasseri 70 Lactobacillus taiwanensis remain in the reduced genus 100 Lactobacillus psittaci 94 Lactobacillus fornicalis 100 Lactobacillus (+/- 30 species). Lactobacillus jensenii Lactobacillus acetotolerans 96 Lactobacillus equicursoris Lactobacillus delbrueckii subsp. sunkii 100 Many of them have substantiated Lactobacillus leichmannii * 81 37 Lactobacillus delbrueckii subsp. delbrueckii 100 47 industrial importance 70 Lactobacillus delbrueckii subsp. lactis 45 Lactobacillus delbrueckii subsp. bulgaricus 51 Lactobacillus delbrueckii subsp. indicus Lactobacillus pasteurii 99 Consequence: Lactobacillus gigeriorum Lactobacillus amylolyticus 91 90 Lactobacillus hamsteri 63 Lactobacillus kalixensis 44 Many traditional ‘probiotic’ species Lactobacillus intestinalis 88 Lactobacillus acidophilus like L. casei / L. paracasei, L. Lactobacillus kefiranofaciens subsp. kefiranofaciens 100 82 Lactobacillus kefiranofaciens subsp. kefirgranum plantarum, L. salivarius, L. reuteri, L. Lactobacillus ultunensis 99 Lactobacillus sobrius * rhamnosus, etc… will no longer be 86 41 95 Lactobacillus amylovorus 83 Lactobacillus kitasatonis called ‘Lactobacillus’. Lactobacillus crispatus 37 Lactobacillus gallinarum 76 Lactobacillus helveticus 41 Lactobacillus suntoreyus + potentially a number of species described since 2014… Many traditional ‘Lactobacillus’ starter cultures will also have to be From Pot et al. 2014 renamed. Based on 16S rDNA sequences What is left in Lactobacillus?

Separate position may be considered

Species left in the genus Lactobacillus according to Sun et al. 2015 (Based on genome sequence analysis) New genus 11 + Probiotics Uncertain 3

Lactobacillus + Probiotics Commercial strains are everywhere

New genus 10 New genus 9 + Probiotics

New genus 8 + Probiotics New genus 7

New genus 6 + Probiotics

New genus 5 +Probiotics

New genus 4 + Probiotics

New genus 3 + Probiotics

New genus 2 + Probiotics New genus 1

+ Probiotics Modified from Sun et al, 2015; NATURE COMMUNICATIONS | 6:8322 | DOI: 10.1038 Communication adaptations necessary / consequences for

• Clinicians and Health Care Professionals • Magazines and scientific journals • Course materials • Websites • E-learnings • Review articles, meta-analyses… • Consumers • Magazines and journals • Radio and TV commercials • Websites • Brochures • Packaging materials • Recipes and ingredient lists, etc. • Lawyers • Official legislation • Patents • Journalists, etc… • More complicated literature searches to obtain background information • Estimate number of papers on a topic Consequences for

• Regulators • QPS regulation • GRAS notifications • Safety Classes • Biosafety levels & Risk Group definitions • International level • https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=grasnotices&id=429 • http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf • National level • https://www.biosafety.be/content/contained-use-international-classifications-schemes-micro- organisms-based-their-biological • Academic level • https://academic.oup.com/cid/article/46/Supplement_2/S104/276784 • Organisations level • http://2016.igem.org/Safety/Risk_Groups • Official documents • Manuals QPS regulation in Europe

• LABIP is organising a stakeholder meeting in Verona on this later this year, including the authors that are preparing the name change and including representatives from EFSA-QPS working group

• There was an EFSA QPS work group meeting in Parma on May 15th where the issue was discussed on the request of LABIP

• Feedback recieved from the chair of the working group: • Efforts will be made to adapt QPS lists as soon as name changes have been formally published • BUT: name changes on the GENUS level have never been dealth with before Ingredient lists: GRAS status

https://www.fda.gov/Food/IngredientsPackagingLabeling/ GRAS/MicroorganismsMicrobialDerivedIngredients/default .htm Ingredient lists and recipees Become aware of some ‘bad’ experiences from the past

Misidentification can lead to inappropriate patient care. Therefore, laboratory personnel and physicians (at least infectious disease specialists) must remain current regarding changes in taxonomy and the recognition of new species.

https://www.ncbi.nlm.nih.gov/books/NBK8406/

Error in patent, involving confusion of bacterial names in reference to the patent: US 4,210,672 The abstract contains the phrase: Yogurt is prepared with a mixture of milk powder and novel Lactobacillus thermophilus (also known as Bacillus coagulans) spores that have specific characteristics ...

How does this error affect the validity of the patent claims?

https://patents.stackexchange.com/questions/13357/error-in-patent-involving-confusion-of-bacterial-names But there will be benefits as well

• Due to their clinical importance, de taxonomy of the Enterobacteriaceae has been developed to an extreme fine level, allowing proper and reliable communication on the disease risks of bacteria such as Escherichia, Shigella, Salmonella (2500 serotypes), etc. • As a consequence, a genus in the family of the Enterobacteriaceae stands at the level of species in most other Gram negative genera. • In the genus Lactobacillus the opposite has happened and as a consequence we current have no ‘discriminatory’ power to name observed (important) differences (e.g. related to applications, fermentation properties, health benefits, etc.) within the genus. • Refining the taxonomy will therefore improve future communication on these industrially important organisms • Refining the taxonomy may also reduce the number of future duplicate descriptions, quite frequently encountered in Lactobacillus, probably due to the large number of species Some ‘bad’ experiences from the past

https://www.ncbi.nlm.nih.gov/books/NBK8406/

How does this error affect the validity of the patent claims?

https://patents.stackexchange.com/questions/13357/error-in-patent-involving-confusion-of-bacterial-names I. Agnarsson et al. 2007 Taxonomy in a Changing World: Seeking Solutions for a Science in Crisis Systematic Biology, (56):531–539, https://doi.org/10.1080/10635150701424546 Widen the discussion I. Agnarsson et al. 2007 Taxonomy in a Changing World: Seeking Solutions for a Science in Crisis Systematic Biology, (56):531–539, https://doi.org/10.1080/10635150701424546 Widen the discussion

A science in crisis • Most top-ranking evolutionary journals do not consider taxonomic revisions • Lower ranking journals reject taxonomic descriptions unless in a paper on a broader subject • Journals allow species descriptions in exceptional cases of certain high- profile fossils and mammals (e.g., Jones et al., 2005; Gess et al., 2006) • Journals focusing on taxonomy typically have low measured impact

Overcoming this “taxonomic impediment” (Rodman and Cody, 2003) is the primary goal of the NSF PEET initiative (Partnerships for Enhancing Expertise in Taxonomy) (NSF, 1994), which has enjoyed much success in training a new generation of taxonomists (Rodman and Cody, 2003).

Probably nobody in this room ever heard about this initiative. Widen the discussion?

Extracted from: E. Munson & K.C. Carroll What’s in a Name? New Bacterial Species and Changes to Taxonomic Status from 2012 through 2015.

2016, doi: 10.1128/JCM.01379-16 J. Clin. Microbiol. 2017; vol. 55(1): 24-42 Tracking a name change is important!

In the medical community…

Since 2014, laboratories subscribing to the College of American Pathologists accreditation program have encountered checklist standard MIC.11375 in the context of biennial inspection exercises.

The standard requires laboratories to assimilate

“taxonomic changes that potentially affect the choice of appropriate antimicrobials to report and/or the interpretative breakpoints to use.”

College of American Pathologists. 2014. Microbiology accreditation checklist. College of American Pathologists, Northfield, IL.

In the food community…

Should we consider a central list of taxonomic changes that affect(ed) recipees and ingredient lists, safety lists, etc., and make it accessible at any time for anyone that has doubts on names? A system (the CATE project) is proposed in which consensus or consolidated taxonomies are presented in the form of Web-based revisions. The workflow is designed to allow the community to offer, online, additions and taxonomic changes (‘proposals’) to the consolidated taxonomies (e.g. new species and synonymies).

A means of quality control in the form of online peer review as part of the editorial process is also included in the workflow. The CATE system rests on taxonomic expertise and judgement, rather than using aggregation technology to accumulate taxonomic information from across the Web. The CATE application and its system and architecture are described in the context of the wider aims and purpose of the project. I. Agnarsson et al. 2007 Taxonomy in a Changing World: Seeking Solutions for a Science in Crisis Systematic Biology, (56):531–539, https://doi.org/10.1080/10635150701424546 Widen the discussion

I dn’t think anybody in this room ever heard about this initiative. What’s it all about?

• The current genus Lactobacillus • What do the rules say? • Something about the methodology (minimal standards) • The implications for the industry • Conclusions? Conclusions

• Name changes in the genus Lactobacillus have occured frequently in the past without too many negative consequences, • However, renaming has never been so massively and never touched species with that considerable economical importance

• Name changes always induce a risk of confusion: ‘old commercial’ and ‘correct scientific’ names are being used simultaneously for the same thing

• Therefore it is important to prepare for a clear communication towards the consumer, the regulator and the authorities

• Since naming will remain crucial, it might be an occasion to develop a tool for the community to maintain an overview on the changes in taxonomy which are relevant for the Food Industry (role for ISAPP?) Thank you for listening!

(some) questions welcome!