Bacteria Associated with Acute Oak Decline: Where Did They Come From? We Know Where They Go.

Acute oak decline is a high-impact disease causing necrotic lesions on the trunk, crown thinning and the eventual death of oak. Four bacterial species are associated with the lesions-Brenneria goodwinii, Gibbsiella quercinecans, Rahnella victoriana and Lonsdalea Britannica-although an epi-/endophytic lifestyle has also been suggested for these bacteria. However, little is known about their environmental reservoirs or their pathway to endophytic colonisation. This work aimed to investigate the ability of the four AOD-associated bacterial species to survive for prolonged periods within rhizosphere soil, leaves and acorns in vitro, and to design an appropriate method for their recovery. This method was trialled on field samples related to healthy and symptomatic oaks. The in vitro study showed that the majority of these species could survive for at least six weeks within each sample type. Results from the field samples demonstrated that R. victoriana and G. quercinecans appear environmentally widespread, indicating multiple routes of endophytic colonisation might be plausible. B. goodwinii and L. britannica were only identified from acorns from healthy and symptomatic trees, indicating they may be inherited members of the endophytic seed microbiome and, despite their ability to survive outside of the host, their environmental occurrence is limited. Future research should focus on preventative measures targeting the abiotic factors of AOD, how endophytic bacteria shift to a pathogenic cycle and the identification of resilient seed stock that is less susceptible to AOD.

Description of Dryocola gen. nov. and two novel species, Dryocola boscaweniae sp. nov. and Dryocola clanedunensis sp. nov. isolated from the rhizosphere of native British oaks.

While investigating the role of the rhizosphere in the development of Acute Oak Decline, bacterial strains belonging to the family Enterobacteriaceae were isolated from rhizosphere soil following enrichment for the Enterobacterales. Partial sequencing of several housekeeping genes showed that these strains could not be assigned to an existing genus. Overall, 16 strains were investigated using a polyphasic approach to determine their taxonomic status. This involved phenotypic testing and fatty acid analysis paired with phylogenetic analyses of 16S rRNA and housekeeping gene sequences, as well as phylogenomic analysis of whole genome sequences. Phylogenomic and phylogenetic analyses consistently demonstrated that the 16 isolates could be separated into two distinct clusters in a monophyletic clade situated between the genera Cedecea and Buttiauxella. The two clusters could be genotypically and phenotypically differentiated from each other and from their closest neighbours. As such we propose the description of Dryocola boscaweniae gen. nov. sp. nov. (type strain H6W4T = CCUG 76177T = LMG 32610T) and Dryocola clanedunesis sp. nov. (type strain H11S18T = CCUG 76181T = LMG 32611T).

Description of a novel species of Leclercia, Leclercia tamurae sp. nov. and proposal of a novel genus Silvania gen. nov. containing two novel species Silvania hatchlandensis sp. nov. and Silvania confinis sp. nov. isolated from the rhizosphere of oak.

BACKGROUND: Acute Oak Decline (AOD) is a decline disease first reported on native oaks in the UK, but in recent years reports from further afield such as Europe and the Middle East, indicate that the distribution and host range is increasing at an alarming rate. The stem weeping symptoms of the disease partially develop due to polymicrobial-host interaction, caused by several members of the order Enterobacterales. While investigating the rhizosphere soil of AOD-unaffected trees, termed 'healthy' trees, and diseased oaks suffering from Acute Oak Decline (AOD), an enrichment method designed for enhanced recovery of Enterobacterales led to the recovery of several isolates that could not be classified as any existing species. These isolates showed a close relationship to the genus Leclercia, of which both species are of clinical importance, but the type species Leclercia adecarboxylata also displays plant growth-promoting properties in the rhizosphere. RESULTS: Partial sequencing of four housekeeping genes revealed similarity to the genus Leclercia with varying degrees of relatedness. As such a complete polyphasic approach was used to determine the true taxonomic position of these isolates. This involved whole genome sequencing, phylogenomic analysis, phylogenetic analysis of both the 16S rRNA and four housekeeping gene sequences, combined with phenotypic testing and fatty acid analysis. Both the phylogenomic and phylogenetic analyses separated the isolates into four clusters, two of which were contained in the Leclercia clade. The remaining two clusters formed a separate lineage far removed from any currently defined species. Further investigation into the role of the isolates as plant growth-promoting bacteria as well as plant pathogens was investigated computationally, revealing a number of plant growth-promoting traits as well as virulence genes related to motility, adhesion and immune modulation. CONCLUSION: Based on the genotypic and phenotypic data presented here, these isolates could be differentiated from each other and their closest neighbours. As such we propose the description of Leclercia tamurae sp. nov. (type strain H6S3T = LMG 32609T = CCUG 76176T), Silvania gen. nov., Silvania hatchlandensis sp. nov. (type strain H19S6T = LMG 32608T = CCUG 76185T) and Silvania confinis sp. nov. (type strain H4N4T = LMG 32607T = CCUG 76175T). Due to their interesting protein annotations and alignments, these species warrant further investigation for their role in relation to plant health.

Transfer of Erwinia toletana and Erwinia iniecta to a novel genus Winslowiella gen. nov. as Winslowiella toletana comb. nov. and Winslowiella iniecta comb. nov. and description of Winslowiella arboricola sp. nov., isolated from bleeding cankers on broadleaf hosts.

Following a screening campaign of bleeding cankers of broadleaf hosts in Great Britain, numerous bacterial strains were isolated, identified by 16S rRNA and protein-coding gene sequencing and ultimately classified. During the course of the study, several Gram-negative, facultatively anaerobic strains were isolated from bleeding Platanus x acerifolia (London plane) and Tilia x europaea (common lime) cankers that could not be assigned to an existing species. Partial 16S rRNA gene sequencing placed these strains in the genus Erwinia, as a close phylogenetic relative of Erwinia toletana. In an effort to determine the taxonomic position of the strains, a polyphasic approach was followed including genotypic, genomic, phenotypic, and chemotaxonomic assays. Multilocus sequence analysis based on four protein-coding genes (gyrB, rpoB, infB, and atpD) confirmed the phylogenetic position of the strains as a novel taxon of subgroup 3 of the genus Erwinia, along with E. toletana and E. iniecta, and furthermore, provided support for their reclassification in a novel genus. Whole genome comparisons allowed the delimitation of the novel species and also supported the proposed transfer of subgroup 3 species to a novel genus in the Erwiniaeae. Phenotypically the novel species could be differentiated from E. toletana and E. iniecta, and the novel genus could be differentiated from the closely related genera Erwinia and Mixta. Therefore, we propose (1) the reclassification of E. toletana and E. iniecta in a novel genus, Winslowiella gen. nov., as Winslowiella toletana comb. nov. and Winslowiella iniecta comb. nov., with W. toletana comb. nov. as the type species (type strain A37T = CFBP 6631T = ATCC 700880T = CECT 5263T), and (2) classification of the novel strains as Winslowiella arboricola sp. nov. (type strain BAC 15a-03bT = LMG 32576T = NCPPB 4696T).

Brenneria tiliae sp. nov., isolated from symptomatic Tilia × moltkei and Tilia × europaea trees in the UK.

Several strains of a previously undescribed bacterial species were isolated from mature Tilia hybrid trees suffering from bleeding cankers at various geographic locations in the UK. The strains were Gram-negative, facultatively anaerobic, and partial sequencing of the gyrB gene revealed that the strains belong to the genus Brenneria with the closest phylogenetic neighbours being Brenneria corticis and Brenneria nigrifluens. Further investigation using a polyphasic approach was undertaken to determine the taxonomic position of the novel species. Phylogenies based on the 16S rRNA gene and multilocus sequence analysis of partial housekeeping gene sequences of gyrB, rpoB, infB and atpD revealed that the strains formed an independent cluster within the genus Brenneria. The phenotypic and chemotaxonomic assays demonstrated that the strains could be differentiated from the closest relatives. Genome analysis of representative strains revealed in silico DNA-DNA hybridization values below the threshold for species delimitation, although the average nucleotide identity values obtained when compared to B. corticis (95.9-96%) were slightly higher than the suggested cut-off value of 95%. However, as all other data suggests that the strains belong to a novel taxon that can be differentiated from the closest relatives, we propose that the strains represent a novel species in the genus Brenneria, Brenneria tiliae sp. nov. (type strain WC1b.1T=LMG 32575T=NCPPB 4697T).

Description of three novel species of Scandinavium: Scandinavium hiltneri sp. nov., Scandinavium manionii sp. nov. and Scandinavium tedordense sp. nov., isolated from the oak rhizosphere and bleeding cankers of broadleaf hosts.

While investigating the bacterial populations of environmental samples taken from a mix of healthy and Acute Oak Decline afflicted Quercus robur (pedunculate or English oak) rhizosphere soil samples and swabs of bleeding lesions on Tilia spp. (lime) and Quercus rubra (red oak) trees, several strains belonging to the order Enterobacterales were isolated using selective media and enrichment broth. Seven strains from the Q. robur rhizosphere, three strains from Tilia spp. and one from Q. rubra were investigated, with their taxonomic status determined via a polyphasic taxonomic approach. Initially stains were identified as potential members of the recently described genus Scandinavium, based on the partial sequencing of three housekeeping genes. Further analysis of phenotypic traits, including fatty acid profiles, coupled with 16S rRNA gene and phylogenomic analysis of whole genome sequences were applied to a subset of the strains. Phylogenetic and phylogenomic analysis repeatedly placed the isolates in a monophyletic clade within Scandinavium, with four distinct clusters observed, one of which corresponded to Scandinavium goeteborgense, the type species of the genus. The remaining three clusters could be phenotypically and genotypically differentiated from each other and S. goeteborgense. As such, we describe three novel species of the genus, for which we propose the names Scandinavium hiltneri sp. nov. (type strain H11S7T = LMG 32612T = CCUG 76179T), Scandinavium manionii sp. nov. (type strain H17S15T = LMG 32613T = CCUG 76183T) and Scandinavium tedordense sp. nov. (type strain TWS1aT = LMG 32614T = CCUG 76188T). Additionally, the descriptions of the genus Scandinavium and the type species, S. goeteborgense, are emended.

Rahnella perminowiae sp. nov., Rahnella bonaserana sp. nov., Rahnella rivi sp. nov. and Rahnella ecdela sp. nov., isolated from diverse environmental sources, and emended description of the genus Rahnella.

Bacteria isolated from onion bulbs suffering from bacterial decay in the United States and Norway were previously shown to belong to the genus Rahnella based on partial housekeeping gene sequences and/or fatty acid analysis. However, many strains could not be assigned to any existing Rahnella species. Additionally, strains isolated from creek water and oak as well as a strain with bioremediation properties were assigned to Rahnella based on partial housekeeping gene sequences. The taxonomic status of these 21 strains was investigated using multilocus sequence analysis, whole genome analyses, phenotypic assays and fatty acid analysis. Phylogenetic and phylogenomic analyses separated the strains into five clusters, one of which corresponded to Rahnella aceris. The remaining four clusters could be differentiated both genotypically and phenotypically from each other and existing Rahnella species. Based on these results, we propose the description of four novel species: Rahnella perminowiae sp. nov. (type strain SL6T=LMG 32257T=DSM 112609T), Rahnella bonaserana sp. nov. (H11bT=LMG 32256T=DSM 112610T), Rahnella rivi sp. nov. (FC061912-KT=LMG 32259T=DSM 112611T) and Rahnella ecdela sp. nov. (FRB 231T=LMG 32255T=DSM 112612T).

Brenneria goodwinii growth in vitro is improved by competitive interactions with other bacterial species associated with Acute Oak Decline.

Brenneria goodwinii, Rahnella victoriana and Gibbsiella quercinecans are three bacterial species frequently isolated together from oak displaying symptoms of Acute Oak Decline (AOD), which include weeping patches on trunks. All three bacterial species play a role in lesion formation in the current episode of AOD in Britain, although B. goodwinii is the most dominant. The ongoing research into stem lesion formation characteristic of this polybacterial syndrome has been focussed primarily on the pathogenicity, identification and taxonomy of these bacteria. As all three species were newly classified within the past ten years, there are many unanswered questions regarding their ecology and interactions with each other. To determine the effect of bacterial interactions on fitness in vitro, we examined pairwise (diculture) and multispecies (triculture) interactions between B. goodwinii, R. victoriana and G. quercinecans in oak leaf media microcosms. Additionally, the effect of co-culturing on the evolution of these species was determined and the evolved B. goodwinii strains were examined further by whole genome sequencing. Our results indicate that B. goodwinii thrived in monoculture with significantly higher viable cell counts than the other two species. Additionally, B. goodwinii performed well in pairwise culture with mutually competitive interactions observed between B. goodwinii and R. victoriana, and between B. goodwinii and G. quercinecans. In the multispecies triculture, B. goodwinii and R. victoriana appeared to exhibit co-ordinated behaviour to outcompete G. quercinecans. After four weeks B. goodwinii grown in co-culture with the other two species developed greater evolved fitness than the strain grown in monoculture as reflected by the increased viable cell counts. The competitive interactions taking place between the threes species indicated evolving improved fitness of B. goodwinii in vitro, that gave it a growth advantage over both R. victoriana and G. quercinecans which showed no significant changes in fitness. Overall, B. goodwinii gains greater benefit in terms of fitness from in vitro competitive interaction with the other two species.

Roadmap for naming uncultivated Archaea and Bacteria.

The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.

A Grain Rot of Rice in Iran Caused by a Xanthomonas Strain Closely Related to X. sacchari.

Rice grain rot disease was detected for the first time in Mazandaran Province, Iran. The bacteria isolated from infected rice plants showed grains rotted and darkening. A Xanthomonas strain closely connected to X. sacchari was identified using molecular and whole genome sequencing approaches confirmed as the causal agent by fulfilling Koch's postulates.

Pseudomonas kirkiae sp. nov., a novel species isolated from oak in the United Kingdom, and phylogenetic considerations of the genera Pseudomonas, Azotobacter and Azomonas.

As the current episode of Acute Oak Decline (AOD) continues to affect native British oak in the United Kingdom, ongoing isolations from symptomatic and healthy oak have yielded a large Pseudomonas species population. These strains could be divided into taxa representing three potential novel species. Recently, two of these taxa were described as novel Pseudomonas species in the Pseudomonas fluorescens lineage. Here, we demonstrate using a polyphasic approach that the third taxon represents another novel Pseudomonas species. The 16S rRNA gene sequencing assigned the strains to the Pseudomonas aeruginosa lineage, while multilocus sequence analysis (based on partial gyrB, rpoB and rpoD sequences) placed the 13 strains in a single cluster on the border of the Pseudomonas stutzeri group. Whole genome intra-species comparisons (based on average nucleotide identity and in silico DNA-DNA hybridization) confirmed that the strains belong to a single taxon, while the inter-species comparisons with closest phylogenetic relatives yielded similarity values below the accepted species threshold. Therefore, we propose these strains as a novel species, namely Pseudomonas kirkiae sp. nov., with the type strain FRB 229T (P4CT=LMG 31089T=NCPPB 4674T). The phylogenetic analyses performed in this study highlighted the difficulties in assigning novel species to the genus Pseudomonas due to its polyphyletic nature and close relationship to the genus Azotobacter. We further propose that a thorough taxonomic re-evaluation of the genus Pseudomonas is essential and should be performed in the near future.

Pseudomonas daroniae sp. nov. and Pseudomonas dryadis sp. nov., isolated from pedunculate oak affected by acute oak decline in the UK.

Twenty-two cream-coloured bacterial strains were isolated from oak trees affected by acute oak decline (AOD) in Southern England. Isolates were Gram-negative, motile, slightly curved rods, aerobic, non-spore-forming, catalase positive and oxidase positive. 16S rRNA gene sequence analysis placed the strains in two separate phylogenetic clusters in the Pseudomonas straminea group, with Pseudomonas flavescens as the closest phylogenetic relative. Multilocus sequence analyses of the gyrB, rpoD and rpoB genes supported the delineation of the strains into two separate taxa, which could be differentiated phenotypically and chemotaxonomically from each other, and their closest relatives. Average nucleotide identity and in silico DNA-DNA hybridization values revealed percentages of genome similarity below the species threshold (95 and 70 %, respectively) between the two taxa and the closest relatives, confirming their novel species status. Therefore, on the basis of this polyphasic approach we propose two novel Pseudomonas species, Pseudomonasdaroniae sp. nov. (type strain FRB 228T=LMG 31087T=NCPPB 4672T) and Pseudomonasdryadis sp. nov. (type strain FRB 230T=LMG 31087T=NCPPB 4673T).

Taxonomy and identification of bacteria associated with acute oak decline.

Acute oak decline (AOD) is a relatively newly described disorder affecting native oak species in Britain. Symptomatic trees are characterised by stem bleeds from vertical fissures, necrotic lesions in the live tissue beneath and larval galleries of the two spotted oak buprestid (Agrilus biguttatus). Several abiotic and biotic factors can be responsible for tree death, however the tissue necrosis and stem weeping is thought to be caused by a combination of bacterial species. Following investigations of the current episode of AOD which began in 2008, numerous strains belonging to several different bacteria in the family Enterobacteriaceae have been consistently isolated from symptomatic tissue. The majority of these enterobacteria were found to be novel species, subspecies and even genera, which have now been formally classified. The most frequently isolated species from symptomatic oak are Gibbsiella quercinecans, Brenneria goodwinii and Rahnella victoriana. Identification of these bacteria is difficult due to similarities in colony morphology, phenotypic profile and 16S rRNA gene sequences. Current identification relies heavily on gyrB gene amplification and sequencing, which is time consuming and laborious. However, newer techniques based on detection of single nucleotide polymorphisms show greater promise for rapid and reliable identification of the bacteria associated with AOD.

Gibbsiella papilionis Kim et al. 2013 is a later heterotypic synonym of Gibbsiella dentisursi Saito et al. 2013.

Synonymy of Gibbsiella dentisursi DSM 23818T ( = NUM 1720T) and Gibbsiella papilionis JCM 18389T ( = LEN33T) was suspected following multilocus sequence analysis (MLSA) of both type strains in a previous classification study, where they were found to share >99.6 % gene sequence similarity. The taxonomic relationship between these two strains was re-examined here using a polyphasic approach. A DNA-DNA hybridization value of 98 % confirmed that the two type strains belong to a single taxon, while the phenotypic profiles were found to be nearly identical. Therefore we propose Gibbsiella papilionis as a later heterotypic synonym of Gibbsiella dentisursi, with the type strain as NUM 1720T ( = DSM 23818T = JCM 17201T).

Rahnella victoriana sp. nov., Rahnella bruchi sp. nov., Rahnella woolbedingensis sp. nov., classification of Rahnella genomospecies 2 and 3 as Rahnella variigena sp. nov. and Rahnella inusitata sp. nov., respectively and emended description of the genus Rahnella.

Isolations from oak symptomatic of Acute Oak Decline, alder and walnut log tissue, and buprestid beetles in 2009-2012 yielded 32 Gram-negative bacterial strains showing highest gyrB sequence similarity to Rahnella aquatilis and Ewingella americana. Multilocus sequence analysis (using partial gyrB, rpoB, infB and atpD gene sequences) delineated the strains into six MLSA groups. Two MLSA groups contained reference strains of Rahnella genomospecies 2 and 3, three groups clustered within the Rahnella clade with no known type or reference strains and the last group contained the type strain of E. americana. DNA-DNA relatedness assays using both the microplate and fluorometric methods, confirmed that each of the five Rahnella MLSA groups formed separate taxa. Rahnella genomospecies 2 and 3 were previously not formally described due to a lack of distinguishing phenotypic characteristics. In the present study, all five Rahnella MLSA groups were phenotypically differentiated from each other and from R. aquatilis. Therefore we propose to classify the strains from symptomatic oak, alder and walnut and buprestid beetles as: Rahnella victoriana sp. nov. (type strain FRB 225(T)=LMG 27717(T)=DSM 27397(T)), Rahnella variigena sp. nov. (previously Rahnella genomosp. 2, type strain CIP 105588(T)=LMG 27711(T)), Rahnella inusitata sp. nov. (previously Rahnella genomosp. 3, type strain DSM 30078(T)=LMG 2640(T)), Rahnella bruchi sp. nov. (type strain FRB 226(T)=LMG 27718(T)=DSM 27398(T)) and Rahnella woolbedingensis sp. nov. (type strain FRB 227(T)=LMG 27719(T)=DSM 27399(T)).

Gibbsiella greigii sp. nov., a novel species associated with oak decline in the USA.

In 2010, cream-coloured, Gram-negative staining, facultatively anaerobic enterobacteria were isolated from a single black oak tree (Quercus kelloggii) exhibiting decline symptoms in southern California, USA. These 12 isolates were tentatively identified as Gibbsiella quercinecans based on partial gyrB sequencing. Closer examination of the strains using multilocus sequence analysis, based on partial sequences of gyrB, rpoB, infB and atpD genes, and almost complete 16S rRNA gene sequencing suggested that the isolates belong to a novel taxon within the genus Gibbsiella with G. quercinecans as their closest phylogenetic relative. DNA-DNA relatedness studies confirmed that the strains belong to a single taxon in Gibbsiella, which can be differentiated from other members of the genus by several phenotypic traits. Therefore, the name Gibbsiella greigii sp. nov. is proposed for this novel species isolated from symptomatic Q. kelloggii in the USA with FRB 224(T) (=LMG 27716(T)=NCPPB 4583(T)) as the type strain.

Description of Brenneria roseae sp. nov. and two subspecies, Brenneria roseae subspecies roseae ssp. nov and Brenneria roseae subspecies americana ssp. nov. isolated from symptomatic oak.

Gram-negative, facultatively anaerobic bacteria were isolated from symptomatic oak tissue in the UK and USA. Partial gyrB sequencing placed ten strains in the genus Brenneria, with B. goodwinii as the closest phylogenetic relative. The strains were investigated further using a polyphasic approach including MLSA (based on partial gyrB, rpoB, infB and atpD gene sequences), 16S rRNA gene sequencing, DNA-DNA relatedness studies and both phenotypic and chemotaxonomic assays. The MLSA and 16S rRNA gene analyses separated the strains into two groups based on origin, suggesting that they belong to Brenneria as two novel species. However, the DNA-DNA relatedness values revealed a closer relationship between the groups and indicated that they should belong to the same species. As the two groups of strains from the UK and USA can be differentiated from each other phenotypically and by ERIC PCR fingerprints, it is proposed to classify them as novel subspecies of a novel Brenneria species. The name Brenneria roseae sp. nov. (FRB 222(T)=LMG 27714(T)=NCPPB 4581(T)) is proposed, with Brenneria roseae subsp. roseae ssp. nov. (FRB 222(T)=LMG 27714(T)=NCPPB 4581(T)) for the strains from the UK and Brenneria roseae subsp. americana ssp. nov. (FRB 223(T)=LMG 27715(T)=NCPPB 4582(T)) for the strains from the USA.

Taxonomic evaluation of the genus Enterobacter based on multilocus sequence analysis (MLSA): proposal to reclassify E. nimipressuralis and E. amnigenus into Lelliottia gen. nov. as Lelliottia nimipressuralis comb. nov. and Lelliottia amnigena comb. nov., respectively, E. gergoviae and E. pyrinus into Pluralibacter gen. nov. as Pluralibacter gergoviae comb. nov. and Pluralibacter pyrinus comb. nov., respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., respectively, and E. turicensis, E. helveticus and E. pulveris into Cronobacter as Cronobacter zurichensis nom. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov., respectively, and emended description of the genera Enterobacter and Cronobacter.

The taxonomy of Enterobacter has a complicated history, with several species transferred to and from this genus. Classification of strains is difficult owing to its polyphyletic nature, based on 16S rRNA gene sequences. It has been previously acknowledged that Enterobacter contains species which should be transferred to other genera. In an attempt to resolve the taxonomy of Enterobacter, MLSA based on partial sequencing of protein-encoding genes (gyrB, rpoB, infB and atpD) was performed on the type strains and reference strains of Enterobacter, Cronobacter and Serratia species, as well as members of the closely related genera Citrobacter, Klebsiella, Kluyvera, Leclercia, Mangrovibacter, Raoultella and Yokenella. Phylogenetic analyses of the concatenated nucleotide sequences revealed that Enterobacter can be divided into five strongly supported MLSA groups, suggesting that the species should be reclassified into five different genera. Further support for this was provided by a concatenated amino acid tree, phenotypic characteristics and fatty acid profiles, enabling differentiation of the MLSA groups. Three novel genera are proposed: Lelliottia gen. nov., Pluralibacter gen. nov. and Kosakonia gen. nov. and the following new combinations: Lelliottia nimipressuralis comb. nov., Lelliottia amnigena comb. nov., Pluralibacter gergoviae comb. nov., Pluralibacter pyrinus comb. nov., Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov., Kosakonia arachidis comb. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov. Additionally, the novel epithet Cronobacter zurichensis nom. nov. is proposed for the reclassification of Enterobacter turicensis into the genus Cronobacter, as Cronobacter turicensis (Iversen et al., 2008) is already in use.