Klebsiella Huaxiensis Sp. Nov., Recovered from Human Urine

bioRxiv preprint doi: https://doi.org/10.1101/335075; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Klebsiella huaxiensis sp. nov., recovered from human urine

Yiyi Hu1,2,3#, Li Wei4#, Yu Feng1,2,3, Yi Xie5, Zhiyong Zong1, 2,3,4§

1Center of Infectious Diseases, West China Hospital, Sichuan University,

Chengdu, China. 2Division of Infectious Diseases, State Key Laboratory of

Biotherapy, Chengdu, China. 3Center for Pathogen Research, West China

Hospital, Sichuan University, Chengdu, China. 4Department of Infection

Control, West China Hospital, Sichuan University, Chengdu, China.

5Laboratory of Clinical Microbiology, Department of Laboratory Medicine, West

China Hospital, Sichuan University, Chengdu, China.

Running title: Klebsiella huaxiensis

Keywords: Klebsiella huaxiensis; Klebsiella; Klebsiella oxytoca; taxonomy;

genome.

Note: The DDBJ/EMBL/GenBank accession numbers for the draft whole

genome sequence and partial 16S rRNA, gyrA and rpoB gene sequences of

strain WCHKl090001T are QAJT00000000, MH179329, MH190069, and

MH190071, respectively.

#Both authors contribute equally.

§Corresponding author. Mailing address: Center of Infectious Diseases, West

China Hospital (Huaxi), Guoxuexiang 37, Chengdu 610041, China. Phone:

86-28-8542-2637. Fax: 86-28-8542-3212. E-mail: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/335075; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Abstract

A Klebsiella strain, WCHKl090001, was recovered from a human urine sample

in China in 2017. Phylogenetic analysis based on gyrA and rpoB

housekeeping genes revealed that the strain was distinct from any previously

described species of the genus Klebsiella though it was clustered with the

Klebsiella oxytoca phylogroup including Klebsiella grimontii, Klebsiella

michiganensis, and Klebsiella oxytoca. The whole genome sequence of strain

WCHKl090001 has an up to 87.18% average nucleotide identity with those of

type strains of all known Klebsiella species. In silico DNA-DNA hybridization

(isDDH) values between strain WCHKl090001 and type strains of all known

Klebsiella species range from 22.3 to 35.2%. Strain WCHKl090001 could be

distinguished from species of the Klebsiella oxytoca phylogroup by its negative

Voges-Proskauer reaction. Genotypic and phenotypic characteristics from this

study indicate that strain WCHKl090001 should be considered to represent a

novel species of the genus Klebsiella, for which the name Klebsiella

huaxiensis sp. nov. is proposed. The type strain is WCHKl090001T

(=GDMCC1.1379T = CCTCC AB 2018106 T).

Klebsiella is a genus of Gram-negative, non-spore-forming bacteria within the

family Enterobacteriaceae. Klebsiella strains are widely distributed in nature

and some Klebsiella species, in particular, Klebsiella pneumoniae is a

common human and animal pathogen causing a variety of infections such as

bacteremia, pneumonia, meningitis, urinary tract infection and intra-abdominal

infection. In addition to the well-known pathogen K. pneumoniae, the genus of

Klebsiella currently comprises Klebsiella aerogenes (also known as Klebsiella

mirabilis and Enterobacter aerogenes) [1], Klebsiella granulomatis [2],

Klebsiella grimontii [3], Klebsiella michiganensis [4], Klebsiella oxytoca,

Klebsiella quasipneumoniae [5], Klebsiella quasivariicola [6] and Klebsiella

variicola [7]. Raoultella ornithinolytica, Raoultella planticola and Raoultella

terrigena were used to belong to the genus Klebsiella but have been

transferred to the genus Raoultella [8]. During our clinical works, we found that

a Klebsiella clinical strain, WCHKl090001, is distinct from all hitherto known

species and therefore may represent a novel species of the genus Klebsiella.

Strain WCHKl090001 was recovered from the urine of a patient at West China

Hospital of Sichuan University, Chengdu, China, in November 2017. The 16S

rRNA gene sequence of strain WCHKl090001 was obtained by PCR using the

universal primers 27F and 1492R [9] and Sanger sequencing. The

nearly-complete 16S rRNA sequence of strain WCHKl090001 was closest

(98.5% identity) to NBRC105695T, the type strain of K. oxytoca. However, the

16S rRNA sequence within Klebsiella is highly conserved and is unable to bioRxiv preprint doi: https://doi.org/10.1101/335075; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

assign Klebsiella strains to the species level due to the limited phylogenetic

resolution [3, 10, 11].

Strain WCHKl090001 was subjected to whole genome sequencing with 150 ×

coverage using the HiSeq X10 Sequencer (Illumina, San Diego, CA), which

generated 1.49 Gb clean bases. Reads were trimmed using Trimmomatic [12]

and were assembled to 149 contigs with a 53.3% GC content using SPAdes

v3.11.1 [13]. Whole genome sequences are available for type strains of the

genera Klebsiella and Raoultella except K. granulomatis, due to the fact that

this species has not yet been cultured axenically [2]. The 383-bp internal

sequence fragment of the gyrA gene (encoding DNA gyrase subunit A) and

501-bp of the rpoB gene (encoding RNA polymerase β-subunit) of type strains

of the genera Klebsiella were retrieved from their whole genome sequences

(Table 1). R. ornithinolytica, R. planticola and R. terrigena were also included

in comparison as they were closely related to Klebsiella species. The

concatenated sequences were aligned by MEGA 7.0 [14] to infer a

maximum-likelihood tree. Strain WCHKl090001 is clustered with type strains of

species of the Klebsiella oxytoca phylogroup including K. grimontii, K.

michiganensis and K. oxytoca (Figure 1).

To further investigate the taxonomy of strain WCHKl090001, both the pair-wise

average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) bioRxiv preprint doi: https://doi.org/10.1101/335075; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

between strain WCHKl090001 and type strains of the genera Klebsiella and

Raoultella were determined. ANI was determined using the JSpecies web

program based on BLAST [15]. Strain WCHKl090001 shared only 76.86 to

87.18% ANI with type strains of all known Klebsiella and Raoultella species

(Table 1), which were well below the ≥95% ANI cutoff to define a bacterial

species [16]. isDDH was performed using GGDC (formula 2) [17]. The isDDH

relatedness between strain WCHKl090001 and any of the known Klebsiella

and Raoultella species ranges from 22.3 to 35.2% (Table 1), much lower than

the ≥70% cutoff to define a bacterial species. The ANI and isDDH analyses

suggest that strain WCHKl090001 represents a new species of the genus

Klebsiella.

Biochemical property of strain WCHKl090001 was determined using the API

20E kit and API 50CH kit according to the manufacturer’s instructions

(bioMerieux, Lyon, France). All tests were carried out by incubating at 35 °C

unless indicated otherwise. Biochemical characteristics of strain

WCHKl090001 were compared with type strains of Klebsiella and Raoultella

species. Strain WCHKl090001 was non-motile by microscopy. Strain

WCHKl090001 was positive for indole, lysine decarboxylase, lactose, mannitol,

and the ONPG test, reduced nitrate to nitrite but was negative for

Voges-Proskauer test, malonate, urease and ornithine decarboxylase. The

negative for Voges-Proskauer test could distinguish strain WCHKl090001 from bioRxiv preprint doi: https://doi.org/10.1101/335075; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

other species of the Klebsiella oxytoca phylogroup.

Genotypic and phenotypic characteristics and the genome sequence of strain

WCHKl090001 lend the support that the strain should be considered to

represent a novel species of the genus Klebsiella, for which the name

Klebsiella huaxiensis sp. nov. is proposed. The type strain is WCHKl090001T.

Description of Klebsiella huaxiensis sp. nov.

Klebsiella huaxiensis (hua.xi.en’sis. N.L. masc. adj. huaxiensis belonging to

West China [Huaxi in Chinese] Hospital, Chengdu, Sichuan Province, China,

where the type strain was recovered).

Cells are Gram-negative, non-motile, gas-producing, and capable of growing

on media such as TSA (Oxoid, Hampshire, UK), LB agar, BHI agar and MH

agar (all from Hopebio, Qingdao, China). Colonies on BHI agar after 24 h of

incubation at 37 °C are light yellow, circular, smooth, convex, glistening, with

entire margins.

K. huaxiensis belongs to the Klebsiella oxytoca phylogroup including K.

grimontii, K. michiganensis, and K. oxytoca. The phenotypic characteristics of

K. huaxiensis strain WCHKl090001T are generally consistent with those for the

K. oxytoca phylogroup. Positive for indole, lysine decarboxylase indole, lysine bioRxiv preprint doi: https://doi.org/10.1101/335075; this version posted May 30, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

decarboxylase, lactose, mannitol, and the ONPG test, reduced nitrate to nitrite

but negative for malonate, urease and ornithine decarboxylase. Negative

reaction for Voges-Proskauer test could distinguish K. huaxiensis from other

species of the Klebsiella oxytoca phylogroup. The G+C content is 53.3%.

The type strain is WCHKl090001T, recovered from a urine culture of a patient

at West China Hospital of Sichuan University, Chengdu, China in November

2017. It is Voges-Proskauer test negative. The GenBank/ EMBL/DDBJ

accession numbers of the gyrA, rpoB and rrs (coding for 16S rRNA) genes are

MH190069, MH190071 and MH179329, respectively. The genome sequence

accession number is QAJT00000000.

Strain WCHKl090001 has been deposited into China Center for Type Culture

Collection as CCTCC AB 2018106 and into Guangdong Microbiology Culture

Center as GDMCC 1.1379.

Funding Information

The work was supported by a grant from the National Natural Science

Foundation of China (project no. 81772233) and a joint grant from the National

Natural Science Foundation of China (project no. 81661130159) and the

Newton Advanced Fellowship, Royal Society, UK (NA150363).

Conflicts of interest

There is no conflict of interest for all authors.

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Table 1. ANI and isDDH values between strain WCHKl090001T and the type

strains of Klebsiella species.

Species Strain Accession no. ANI DDH

(%) (%)

K. aerogenes KCTC 2190T CP002824 81.40 26.0

K. grimontii 06D021T FZTC01000000 87.09 35.0

K. michiganensis H1gT AYMI01000000 87.18 35.2

K. oxytoca NBRC105695T BCZK01000000 86.74 33.8

K. pneumoniae subsp. ozaenae ATCC 11296T CDJH01000000 81.41 25.6

K. pneumoniae subsp. pneumoniae ATCC 13883T JOOW01000000 81.27 25.5

K. pneumoniae subsp. rhinoscleromatis ATCC 13884T CDOT01000000 81.24 25.4

K. quasipneumoniae subsp. quasipneumoniae 01A030T CCDF01000000 81.44 25.8

K. quasipneumoniae subsp. similipneumoniae 07A044T CBZR01000000 81.48 25.6

K. quasivariicola KPN 1705T CP022823 81.47 25.7

K. variicola DSM 15968T CP010523 81.40 25.7

R. planticola B43T BADH01000000 76.86 22.3

R. ornithinolytica ATCC 31898T NC_021066 82.42 27.0

R. terrigena ATCC 33257T LANE01000000 82.49 27.3

Figure legend

Figure 1. Neighbour-joining tree based on the concatenated sequences

of the gyrA and rpoB genes of strain WCHKl090001T and type strains of

Klebsiella and Raoultella species. E. coli DSM30083T (Accession no.

AGSE01000004.1) was used as an outgroup. Bootstrap values > 50% (based

on 1,000 resamplings) are shown. Bar, 0.005 substitutions per nucleotide

position.

K. oxytoca ATCC 13182T (AJ871858.1) K. huaxiensis WCHKl090001T (MH179329) K. grimontii 06D021T (AJ871861) K. michiganensis W14T (JQ070300) R. terrigena ATCC 33257T (NR_037085) R. planticola ATCC 33531T (Y17659.1) R. ornithinolytica ATCC 31898T (AJ251467) K. aerogenes KCTC 2190T (AB004750) K. quasipneumoniae subsp. similipneumoniae 07A044T (HG933295) K. quasivariicola KPN1705T (NZ_CP022823.1) K. pneumonia subsp. rhinoscleromatis CIP 52-210T (CDOT01000001.1) K. quasipneumoniae subsp. quasipneumoniae 01A030T (HG933296) K. variicola F2R9T (NR_025635.1) K. pneumonia subsp. pneumonia ATCC 13883T (AF130981) K. granulomatis K22-14T (EU333881.1) K. pneumonia subsp. ozaenae ATCC 11296T (CDJH01000001.1) E. coli DSM 30083T (AGSE01000004.1)