Abstract:

Objective:

To compare the genetic determinants involved in plant colonization or virulence in the reported genomes of K. variicola, K. quasipneumoniae and K. pneumoniae.

Materials and methods:

In silico comparisons and Jaccard analysis of genomic data were used. Fimbrial genes were detected by PCR. Biological assays were performed with plant and clinical isolates.

Results:

Plant colonization genes such as cellulases, catalases and hemagglutinins were mainly present in K. variicola genomes. Chromosomal β-lactamases were characteristic of this species and had been previously misclassified. K. variicola and K. pneumoniae isolates produced plant hormones.

Conclusions:

A mosaic distribution of different virulence- and plant-associated genes was found in K. variicola and in K. quasipneumoniae genomes. Some plant colonizing genes were found mainly in K. variicola genomes. The term plantanosis is proposed for plant-borne human infections.

Keywords:
bacterial infections; Gram-negative bacterial infections; Enterobacteriaceae infections

Resumen:

Objetivo:

Comparar genes de colonización de plantas o de virulencia en los genomas reportados de K. variicola, K. quasipneumoniae y K. pneumoniae.

Material y métodos:

Se utilizaron análisis in silico y de Jaccard. Por PCR se detectaron genes de fimbrias. Se realizaron ensayos biológicos con aislados de plantas y clínicos.

Resultados:

Los genes de colonización de plantas como celulasas, catalasas y hemaglutininas se encontraron principalmente en genomas de K. variicola. Las β-lactamasas cromosómicas son características de la especie y en algunos casos estaban mal clasificadas. K. variicola y K. pneumoniae producen hormonas vegetales.

Conclusiones:

Se encontró una distribución en mosaico de los genes de asociación con plantas y de virulencia en K. variicola y K. quasipneumoniae. Principalmente en K. variicola se encontraron algunos genes involucrados en la colonización de plantas. Se propone el término plantanosis para las infecciones humanas de origen vegetal.

Palabras clave:
infecciones bacterianas; infecciones de bacterias Gram-negativas; infecciones Enterobacteriaceae

Introduction

The taxonomy of the genus Klebsiella has been periodically revised. Currently, this genus includes K. pneumoniae subsp. pneumoniae, also known as KpI, and four novel species: Klebsiella quasipneumoniae, also known as KpII - with two subspecies: quasipneumoniae (KpII-A) and similipneumoniae (KpII-B); Klebsiella variicola (KpIII) and Klebsiella michiganensis.¹1. Brisse S, Passet V, Grimont PA. Description of Klebsiella quasipneumoniae sp. nov., isolated from human infections, with two subspecies, Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a junior heterotypic synonym of Klebsiella variicola. Int J Syst Evol Microbiol 2014;64(9):3146-3152. https://doi.org/10.1099/ijs.0.062737-0
https://doi.org/10.1099/ijs.0.062737-0... K. variicola and K. quasipneumoniae are sister species of K. pneumoniae. K. variicola has been isolated from plant tissues,²2. Martinez-Romero E, Silva-Sanchez J, Barrios H, Rodriguez-Medina N, Martinez-Barnetche J, Tellez-Sosa J, et al. Draft genome sequences of Klebsiella variicola plant isolates. Genome Announc 2015;3(5):e01015-15. https://doi.org/10.1128/genomeA.01015-15
https://doi.org/10.1128/genomeA.01015-15... ^,33. Rosenblueth M, Martinez L, Silva J, Martinez-Romero E. Klebsiella variicola, a novel species with clinical and plant-associated isolates. System Appl Microbiol 2004;27(1):27-35. https://doi.org/10.1078/0723-2020-00261
https://doi.org/10.1078/0723-2020-00261... fungal gardens of leaf-cutter ant colonies,⁴4. Pinto-Tomas AA, Anderson MA, Suen G, Stevenson DM, Chu FS, Cleland WW, et al. Symbiotic nitrogen fixation in the fungus gardens of leaf-cutter ants. Science 2009;326(5956):1120-1123. cotton disease vectors (specifically of the insect, Nezara viridula),⁵5. Medrano EG, Forray MM, Bell AA. Complete genome sequence of a Klebsiella pneumoniae strain isolated from a known cotton insect boll vector. Genome Announc 2014;2(4):e00850-14. https://doi.org/10.1128/genomeA.00850-14
https://doi.org/10.1128/genomeA.00850-14... human⁶6. Garza-Ramos U, Martinez-Romero E, Silva-Sanchez J. SHV-type extended-spectrum beta-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico. Salud Publica Mex 2007;49(6):415-421. https://doi.org/10.1590/S0036-36342007000600008
https://doi.org/10.1590/S0036-3634200700... ^,77. Garza-Ramos U, Moreno-Dominguez S, Hernandez-Castro R, Silva-Sanchez J, Barrios H, Reyna-Flores F, et al. Identification and characterization of imipenem-resistant Klebsiella pneumoniae and susceptible Klebsiella variicola isolates obtained from the same patient. Microb Drug Resist 2016;22(3):179-184. https://doi.org/10.1089/mdr.2015.0181
https://doi.org/10.1089/mdr.2015.0181... ^,88. Garza-Ramos U, Silva-Sanchez J, Martinez-Romero E, Tinoco P, Pina-Gonzales M, Barrios H, et al. Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola. BMC Microbiol 2015;15:64. https://doi.org/10.1186/s12866-015-0396-6
https://doi.org/10.1186/s12866-015-0396-... ^,99. Maatallah M, Vading M, Kabir MH, Bakhrouf A, Kalin M, Naucler P, et al. Klebsiella variicola is a frequent cause of bloodstream infection in the stockholm area, and associated with higher mortality compared to K. pneumoniae. PloS One 2014;9(11):e113539. https://doi.org/10.1371/journal.pone.0113539
https://doi.org/10.1371/journal.pone.011... ^,1010. Seki M, Gotoh K, Nakamura S, Akeda Y, Yoshii T, Miyaguchi S, et al. Fatal sepsis caused by an unusual Klebsiella species that was misidentified by an automated identification system. J Med Microbiol 2013;62(5):801-803. https://doi.org/10.1099/jmm.0.051334-0
https://doi.org/10.1099/jmm.0.051334-0... and animal infections¹¹11. Brisse S, Duijkeren E. Identification and antimicrobial susceptibility of 100 Klebsiella animal clinical isolates. Vet Microbiol 2005;105(3-4):307-312. https://www.sciencedirect.com/science/journal/03781135/30
https://www.sciencedirect.com/science/jo... including bovine mastitis.¹²12. Podder MP, Rogers L, Daley PK, Keefe GP, Whitney HG, Tahlan K. Klebsiella species associated with bovine mastitis in Newfoundland. PloS One 2014;9(9):e106518. https://doi.org/10.1371/journal.pone.0106518
https://doi.org/10.1371/journal.pone.010... Unlike K. variicola, K. quasipneumoniae has been described exclusively in hospital settings.¹³13. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci USA 2015;112(27):E3574-E3581. https://doi.org/10.1073/pnas.1501049112
https://doi.org/10.1073/pnas.1501049112... A recent genomic comparative study with a large number of Klebsiella genomes showed that the nif operon was detected in all the genomes of K. variicola, in half of those of K. quasipneumoniae, but only in one strain of K. pneumoniae.¹³13. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci USA 2015;112(27):E3574-E3581. https://doi.org/10.1073/pnas.1501049112
https://doi.org/10.1073/pnas.1501049112... Additionally, K. pneumoniae and K. variicola shared some virulence determinants that cause infections in humans.¹⁴14. Andrade BG, de Veiga Ramos N, Marin MF, Fonseca EL, Vicente AC. The genome of a clinical Klebsiella variicola strain reveals virulence-associated traits and a pl9-like plasmid. FEMS Microbiol Lett 2014;360(1):13-16. https://doi.org/10.1111/1574-6968.12583
https://doi.org/10.1111/1574-6968.12583... ^,1515. Garza-Ramos U, Silva-Sanchez J, Barrios H, Rodriguez-Medina N, Martinez-Barnetche J, Andrade V. Draft genome sequence of the first hypermucoviscous Klebsiella variicola clinical isolate. Genome Announc 2015;3(2):e01352-14. https://doi.org/10.1128/genomeA.01352-14
https://doi.org/10.1128/genomeA.01352-14...

Some Klebsiella isolates have been misclassified¹⁶6. Garza-Ramos U, Martinez-Romero E, Silva-Sanchez J. SHV-type extended-spectrum beta-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico. Salud Publica Mex 2007;49(6):415-421. https://doi.org/10.1590/S0036-36342007000600008
https://doi.org/10.1590/S0036-3634200700... ^,177. Garza-Ramos U, Moreno-Dominguez S, Hernandez-Castro R, Silva-Sanchez J, Barrios H, Reyna-Flores F, et al. Identification and characterization of imipenem-resistant Klebsiella pneumoniae and susceptible Klebsiella variicola isolates obtained from the same patient. Microb Drug Resist 2016;22(3):179-184. https://doi.org/10.1089/mdr.2015.0181
https://doi.org/10.1089/mdr.2015.0181... and consequently the taxonomic descriptions of their genome sequences in NCBI repositories are inaccurate. Here we used correctly identified K. variicola and K. quasipneumoniae genomes for the comparison of virulence and plant colonization determinants of this genus of enterobacteria.

Materials and methods

K. variicola and K. quasipneumoniae genomes included in the study

A total of 41 Klebsiella genomes were included in the study, 31 of which corresponded to K. variicola, eight to K. quasipneumoniae and two to K. pneumoniae (table I). Of these genomes, 19 of K. variicola and six K. quasipneumoniae were originally misidentified.

In silico analysis of virulence, plant-associated determinants and efflux pumps, regulators, heavy metal resistance and β-lactamases

A set of 138 proteins was selected from previous reports¹⁸18. Fouts DE, Tyler HL, DeBoy RT, Daugherty S, Ren Q, Badger JH, et al. Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice. PLoS Genetics 2008;4(7):e1000141. https://doi.org/10.1371/journal.pgen.1000141
https://doi.org/10.1371/journal.pgen.100... ^,1919. Lin L, Wei C, Chen M, Wang H, Li Y, Li Y, et al. Complete genome sequence of endophytic nitrogen-fixing Klebsiella variicola strain DX120E. Stand Genomic Sci 2015;10:22. https://doi.org/10.1186/s40793-015-0004-2
https://doi.org/10.1186/s40793-015-0004-... and the Pasteur Institute webpage (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html) for their putative roles in host interactions. Among these proteins, 84 were included for their involvement in virulence. Two efflux pump clusters, 11 efflux pump regulators, four heavy metal clusters, three β-lactamase family proteins (LEN-, SHV- and OKP-type), as well as 35 plant-association proteins that included nitrogen fixation enzymes (encoded by the nif operon with 20 genes), were also included. All genes encoding these proteins/enzymes were further searched in 41 Klebsiella spp. genomes by BLAST ²⁰20. Garcia-Vallve S, Palau J, Romeu A. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Mol Biol Evol 1999;16(9):1125-1134. https://doi.org/10.1093/oxfordjournals.molbev.a026203
https://doi.org/10.1093/oxfordjournals.m... (Genome BLASTp option with default values). A cluster analysis using the average linkage based on the Jacquard similarity coefficient and the respective dendrogram (constructed with UPGMA) were constructed using the DendroUPGMA program.²⁰20. Garcia-Vallve S, Palau J, Romeu A. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Mol Biol Evol 1999;16(9):1125-1134. https://doi.org/10.1093/oxfordjournals.molbev.a026203
https://doi.org/10.1093/oxfordjournals.m... In the case of the LEN-, SHV- and OKP-type families of β-lactamases, a phylogenic reconstruction was performed using the maximum-likelihood approach based on the JTT matrix-based model and 100 bootstrap replications (Mega v6.06).²¹21. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Bol Evol 2011;28(10):2731-2739. https://doi.org/10.1093/molbev/msr121
https://doi.org/10.1093/molbev/msr121... In addition, phylogenetic analyses were carried out on the amino acid sequences of FimV fimbrial proteins (KVR801v1_60088) and NifH nitrogenase (KVR801v1_120019) proteins.

PCR screening of fimV gene

Specific oligonucleotides for the fimV gene (fimV-F 5’-TTTGCGGATACTGACCAGGG-3’ and fimV-R 5’-GGTTACCACGGTCAGCGTAA-3’) were designed. Twenty-one K. variicola isolates from plants and humans were analyzed by PCR as previously described.⁸8. Garza-Ramos U, Silva-Sanchez J, Martinez-Romero E, Tinoco P, Pina-Gonzales M, Barrios H, et al. Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola. BMC Microbiol 2015;15:64. https://doi.org/10.1186/s12866-015-0396-6
https://doi.org/10.1186/s12866-015-0396-...

In vitro assays of plant growth promotion

The mechanisms involved in plant growth promotion, such as phytohormone production (auxin [indole-3-acetic acid] and gibberellins), phosphate solubilization, siderophore production and lytic enzyme activities were evaluated in six K. variicola isolated from plants, and in 15 K. variicola and eight K. pneumoniae clinical isolates. The production of auxin was analyzed according to Khalid and collagues²²22. Khalid A, Tahir S, Arshad M, Ahmad Z. Relative efficiency of rhizobacteria for auxin biosynthesis in rhizosphere and non-rhizosphere soils. Aust J Soil Res 2004;42(8):921-926. https://doi.org/10.1071/SR04019
https://doi.org/10.1071/SR04019... with Azotobacter vinelandii and Salmonella enteritidis as positive and negative controls, respectively. Phosphate solubilization was evaluated using the methodology described by Mehta and colleagues²³23. Mehta S, Nautiyal CS. An efficient method for qualitative screening of phosphate-solubilizing bacteria. Curr Microbiol 2001;43(1):51-56. https://doi.org/10.1007/s002840010259
https://doi.org/10.1007/s002840010259... using Azotobacter vinelandii and E. coli DH5-α as bacterial controls. Siderophore production was evaluated using chrome azurol S according to Schwyn and Neilands²⁴24. Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Anal Biochem 1987;160(1):47-56. https://doi.org/10.1016/0003-2697(87)90612-9
https://doi.org/10.1016/0003-2697(87)906... with Pseudomonas fluorescens and Staphylococcus aureus as controls. Activities of lytic enzymes, such as lipases, proteases, esterases and amylases, were evaluated using the methodology described by Malleswari and Bagyanarayana²⁵25. Malleswari D, Bagyanarayana G. Plant growth-promoting activities and molecular characterization of rhizobacterial strains isolated from medicinal and aromatic plants. IOSR-JPBS 2013;6(6):30-37. https://doi.org/10.9790/3008-0663037
https://doi.org/10.9790/3008-0663037... using Bacillus subtilis and E. coli DH5-α as controls.

Ethical committee approval

This study is part of the K. variicola project that was revised and approved by the ethical commission from the Instituto Nacional de Salud Pública on the 21th of June 2011. The present study was carried out at the Instituto Nacional de Salud Pública and at the Centro de Ciencias Genómicas of UNAM, Cuernavaca, Morelos.

Results and discussion

Virulence-associated determinants in K. variicola and K. quasipneumoniae genomes

In silico screening of 84 virulence-determinant proteins in reported genomes of K. variicola, K. quasipneumoniae and K. pneumoniae showed a mosaic distribution in different isolates (table I). Most (>98%, except for FimV) of the virulence determinants that were selected here were originally described in K. pneumoniae NTUH-K2044. From the 84 virulence determinants included in the in silico analysis, 20 were found in at least one K. variicola genome and 11 in at least one K. quasipneumoniae genome. The proteins coded by the urease (UreA) and fimbriae gene cluster MrkABCDFHIJ were present in all the isolates of K. variicola and K. quasipneumoniae. The gene coding glucuronic acid transferase (WabG) was also present in all K. variicola and K. pneumoniae, but was absent in a single K. quasipneumoniae genome.

Siderophores, such as salmochelin (IroN), aerobactin (IucA and receptor IutA), KfuABC cluster, enterobactin (EntB) and yersiniabactin cluster YbtAESTX, were unequally distributed among Klebsiella. The most prevalent siderophores were enterobactin and KfuABC, in 93.5% (29/31) of K. variicola and in 100% (8/8) of the K. quasipneumoniae isolates. The aerobactin (IucA) was not identified, however the receptor of aerobactin (IutA) was present in 87% (27/31) and salmochelin (IroN) in 39% (12/31) of K. variicola isolates; however, in the K. quasipneumoniae genomes, both aerobactin receptor and salmochelin siderophore were present in 100% of the isolates. The siderophore yersiniabactin YbtAESTX cluster was identified in only one K. variicola isolate (MGH20) and in none of the K. quasipneumoniae genomes.

The genes encoding enzymes from the glycerate pathway (Gcl, GlxK), GlxR, and Hyi) were identified in K. variicola isolates (41.9 to 45.1%). The allantoinase cluster AllABCDRS genes and the YlbE-F and YbbW were present in few K. variicola genomes at 3.2, 6.4 and 3.2%, respectively. The GlxK-R and Hyi encoding genes were present only in K. quasipneumoniae genomes, in 75% of them. The genes for the two-component system KvgAS proteins were identified in 12.9% (4/31) of the K. variicola genomes and were absent from the K. quasipneumoniae genomes. The mucoviscosity-associated protein Wzy-K1 was identified in one K. variicola isolate (3.2%-1/31).

The Jacquard index analysis of the virulence-associated determinants that are shared between the K. variicola, K. quasipneumoniae and K. pneumoniae genomes is shown in figure 1. Three main clusters were obtained, two of which were closely related and could be distinguished only by a single siderophore difference. Cluster 1 grouped sixteen K. variicola and two K. quasipneumoniae subsp. similipneumoniae genomes that shared the siderophores IroN (11/18 genomes); receptor IutA, KfuABC and EntB (15/18 genomes); WabG, Uge, the fimbriae cluster MrkABCDFHIJ, FimV (15/18 genomes) and urease UreA. Cluster 2 includes ten K. variicola genomes and mostly contains the virulence-determinants proteins of cluster 1, except for IroN. Genes associated with all of the K. variicola genomes from cluster 2 were those encoding Glc, GlxKR and Hyi proteins that are involved in the glycerate pathway. This pathway together with allantoinase cluster genes are involved in allantoin metabolism as a nitrogen source.²⁶26. Cusa E, Obradors N, Baldoma L, Badia J, Aguilar J. Genetic analysis of a chromosomal region containing genes required for assimilation of allantoin nitrogen and linked glyoxylate metabolism in Escherichia coli. J Bacteriol 1999;181(24):7479-7484. Cluster 3 consists of seven K. quasipneumoniae and one K. variicola genomes. All of the genomes of cluster 3 contain the Uge, WabG, IroN (except K. variicola KTE92 genome), IutA, KfuABC, EntB, UreA and MrkABCDFHIJ proteins. FimV protein was present in three (42.8%-3/7) of the K. quasipneumoniae genomes, corresponding to Klebsiella quasipneumoniae subsp. similipneumoniae (KpII-B).

K. pneumoniae NTUH-K2044 and K. pneumoniae MGH78578 each independently clustered together with K. pneumoniae BIDMC90 and K. variicola DSM15298, respectively (figure 1). The K. pneumoniae BIDMC90 genome shared with K. pneumoniae NTUH-K2044 fifteen virulence determinants. Finally, K. pneumoniae MGH78578 and K. variicola DSM15298 were different from the other clusters due to the absence of siderophore KfuABC, which was present in all of the K. variicola, K. quasipneumoniae and K. pneumoniae NTUH-K2044, and to the presence of the two-component system KvgAS, which was only present in the DX120E, MGH76 and UCICRE10 genomes (figure 1).

In silico analysis and prevalence of Fimbrial FimV protein

Previously a total of 114 unique proteins were identified in K. variicola genomes.⁸8. Garza-Ramos U, Silva-Sanchez J, Martinez-Romero E, Tinoco P, Pina-Gonzales M, Barrios H, et al. Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola. BMC Microbiol 2015;15:64. https://doi.org/10.1186/s12866-015-0396-6
https://doi.org/10.1186/s12866-015-0396-... Excluding transposons and tra-genes that are involved in horizontal transfer and selecting those that are involved in metabolism and cellular structure, 13 structural proteins were found to be unique for K. variicola, from which a fimbrial protein was chosen for further analysis (fimbria have been shown to mediate host interaction). The fimbrial FimV protein was identified in 90.3% (28/31) of the K. variicola (except for KTE92, B1 and BIDMC90) and in 37.5% (3/8) of K. quasipneumoniae but not in K. pneumoniae NTUH-K2044 and MGH78578 genomes. An additional BLASTp search in GenBank using a non-redundant protein sequence (nr-BLASTp protein-protein BLAST) identified the FimV protein in K. variicola genomes described in GenBank (except for KTE92, B1 and BIDMC90) and in the misclassified K. pneumoniae genomes that actually corresponded to K. variicola species (table I). The phylogenetic analysis of FimV proteins generally showed high amino acid conservation in K. variicola (>98%) (data not shown); however, in a few K. variicola strains, for example the wrongly named “K. pneumoniae 342”, a lower amino acid identity was observed (92 to 94%). Conversely, K. quasipneumoniae subsp. similipneumoniae, 07A044, 700603 and MGH44 FimV amino acid sequence had 91% similarity. FimV protein sequence was not found in bona fide K. pneumoniae; nevertheless it was identified in related Enterobacteriaceae such as Citrobacter koseri, Citrobacter freundii, Enterobacter, Salmonella enterica and Escherichia albertii, all with an amino acid similarity less than 53%. The FimV protein is also widely distributed in Escherichia coli and Shigella sonnei, with an amino acid identity of 33% to that from K. variicola.

The PCR screening of the fimV gene was carried out in a collection of twenty-one K. variicola isolates (see Materials and methods). The fimV gene was identified in 86.9% (20/23) of the K. variicola isolates. This prevalence is similar to that from in silico analysis (90.3%). This indicates that the fimV gene is not universally encoded in K. variicola genomes.

Plant-associated proteins in the K. variicola, K. quasipneumoniae and K. pneumoniae genomes

Plant-associated proteins were identified in K. variicola and K. quasipneumoniae isolates. Twenty-seven plant-associated determinants were found in at least one K. variicola or K. quasipneumoniae isolate. In general, 21 plant-associated determinants were contained in all of the K. variicola, K. quasipneumoniae and K. pneumoniae genomes that were included in the study. The NifJ-NifQ nitrogen fixation cluster was identified in all of the K. variicola genomes, in 62.5% (5/8) of K. quasipneumoniae genomes and was absent from the K. pneumoniae NTUH-K2044 and MGH78578 genomes. The NifH protein has high amino acid identity among all K. variicola genomes and in a phylogenetic analysis it is grouped in a cluster different from the corresponding sequence from the K. quasipneumoniae or other species. Holt and colleagues¹³13. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci USA 2015;112(27):E3574-E3581. https://doi.org/10.1073/pnas.1501049112
https://doi.org/10.1073/pnas.1501049112... identified only one K. pneumoniae genome that contained the nifJ-nifQ operon. In this work, we identified another K. pneumoniae genome (from KPNIN29) that contains the nif operon in addition to that described by Holt and colleagues.¹³13. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci USA 2015;112(27):E3574-E3581. https://doi.org/10.1073/pnas.1501049112
https://doi.org/10.1073/pnas.1501049112... Hazen and colleagues²⁷27. Hazen TH, Zhao L, Sahl JW, Robinson G, Harris AD, Rasko DA, et al. Characterization of Klebsiella sp. strain 10982, a colonizer of humans that contains novel antibiotic resistance alleles and exhibits genetic similarities to plant and clinical Klebsiella isolates. Antimicrob Agents Chemother 2014;58(4):1879-1888. https://doi.org/10.1128/AAC.01605-13
https://doi.org/10.1128/AAC.01605-13... identified the absence of nifJ-nifQ gene cluster in K. pneumoniae NTUH-K2044, MGH-78578, 1162281, JH1, MS 92-3, 1191100241, ATCC13884 and KCTC-2242 genomes. Here we identified a conserved genetic context and gene synteny of nifJ-nifQ operon both in K. variicola and K. quasipneumoniae genomes (data not shown). Our results confirm that nitrogen fixation seems to be a characteristic trait of K. variicola.

Other differences identified in this work between the K. variicola, K. quasipneumoniae and K. pneumoniae genomes are genes encoding cellulases (CelK and BglH), catalases (KPK_4954 and KPK_2333) and hemagglutinins (HecA). While BglX cellulose is present in all K. variicola, K. quasipneumoniae and K. pneumoniae isolates, CelK and BglH were absent from K. pneumoniae NTUH-K2044 and MGH78578 and from 87.5% of the K. quasipneumoniae isolates.

The BglH enzyme, which has specificity towards 1,4-b glucosidic bonds and that most likely acts by hydrolyzing short cello-oligosaccharides, was present in 61.2% (19/31) of K. variicola isolates. The CelK gene encoding an enzyme for the decomposition of highly ordered forms of insoluble cellulose was present in K. variicola strains obtained from three different plants 342, T29A and 6A2 representing 10% (3/31) of the isolates. KPK_2233 (catalase) gene was present in 22.5% of the K. variicola genomes (7/31). KPK_4954 gene encoding a cyclic beta 1-2 glycan synthase possibly playing a role in osmotic adaptation was present in 16.1% of the K. variicola isolates (5/31).

HecA/B hemolysin/hemagglutinin secretion protein is involved in plant attachment in Erwinia chrysanthemis. A hecA gene mutant had reduced attachment, cell aggregate formation, and virulence in its host. HecA protein was present in 64.5% (20/31) of the K. variicola isolates. In K. quasipneumoniae, HecA protein is present only in HKUOPLA²⁸28. Lu MG, Jiang J, Liu L, Ma AP, Leung FC. Complete genome sequence of Klebsiella variicola Strain HKUOPLA, a cellulose-degrading bacterium isolated from giant panda feces. Genome Announc 2015;3(5):e01200-15. https://doi.org/10.1128/genomeA.01200-15
https://doi.org/10.1128/genomeA.01200-15... and HKUOPLC²⁹29. Lu MG, Jiang J, Liu L, Ma AP, Leung FC. Complete genome sequence of Klebsiella pneumoniae strain HKUOPLC, a cellulose-degrading bacterium isolated from giant panda feces. Genome Announc 2015;3(6):e01318-15. https://doi.org/10.1128/genomeA.01318-15
https://doi.org/10.1128/genomeA.01318-15... isolates obtained from giant panda feces.

The Jacquard index analysis showed two main clusters (A and B) that were grouped by the absence or presence of HecA, CelK or BglH protein (figure 2). Cluster A included the K. variicola and K. quasipneumoniae genomes that did not contain the HecA and CelK proteins. The K. quasipneumoniae 18A069 genome is the unique genome of this species that does not contain the YqeF (acetyl-CoA acetyltransferase), induce plant colonization and Ada (a regulatory protein of adaptive response) and DinF (DNA-damage-inducible protein F) putative stress response proteins.¹⁸18. Fouts DE, Tyler HL, DeBoy RT, Daugherty S, Ren Q, Badger JH, et al. Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice. PLoS Genetics 2008;4(7):e1000141. https://doi.org/10.1371/journal.pgen.1000141
https://doi.org/10.1371/journal.pgen.100...

Efflux pump, regulators, heavy metal resistance and chromosomal β-lactamase proteins in K. variicola, K. quasipneumoniae and K. pneumonia

The OqxABR efflux pump gene clusters are present in all K. variicola and K. quasipneumoniae genomes examined. The AcrABR protein efflux was identified in 58.0% (18/31) and 100% (8/8), of K. variicola and K. quasipneumoniae. All of the protein regulators were identified (MarAR, SoxSR, RamAR, Rob, SdiA, Fis, EnvR, and RarA) in >98% and 100%, of the K. variicola and K. quasipneumoniae isolates. All efflux pumps and efflux pump regulator proteins were identified in the K. pneumoniae NTUH-K2044 and MGH78578 genomes.

The copper (PcoABCDERS), silver (SilCERS) and tellurium (TerABCDEWXYZ) protein clusters for heavy metal resistance were analyzed. The PcoABCDERS and SilCERS protein clusters were identified together in 22.5% (7/31) and 37.5% (3/8) of the K. variicola and K. quasipneumoniae genomes, respectively, while the K. variicola Bz19 genome contained only the PcoABCDERS protein cluster.

Tellurite resistance is conferred by the TerABCDEWXYZ protein cluster and seemingly is strongly associated with the hypervirulent clonal groups.³⁰30. Passet V, Brisse S. Association of tellurite resistance with hypervirulent clonal groups of Klebsiella pneumoniae. J Clin Microbiol 2015;53(4):1380-1382. https://doi.org/10.1128/JCM.03053-14
https://doi.org/10.1128/JCM.03053-14... Tellurite resistance is needed to colonize macrophages.³¹31. Ponnusamy D, Clinkenbeard KD. Role of tellurite resistance operon in filamentous growth of Yersinia pestis in macrophages. PloS One 2015;10(11):e0141984. https://doi.org/10.1371/journal.pone.0141984
https://doi.org/10.1371/journal.pone.014... The TerABCDEWXYZ proteins cluster was identified in 12.9% (4/31) and 12.5% (1/8), of K. variicola and K. quasipneumoniae, all of human origin. K. variicola 8917 isolate was described as hypermucoviscous.¹⁵15. Garza-Ramos U, Silva-Sanchez J, Barrios H, Rodriguez-Medina N, Martinez-Barnetche J, Andrade V. Draft genome sequence of the first hypermucoviscous Klebsiella variicola clinical isolate. Genome Announc 2015;3(2):e01352-14. https://doi.org/10.1128/genomeA.01352-14
https://doi.org/10.1128/genomeA.01352-14...

In the analysis of chromosomal β-lactamase, we found that all the K. pneumoniae genomes that were identified as K. variicola contained the LEN-type β-lactamases allele. The K. pneumoniae genomes that were correctly identified as K. quasipneumoniae contained the OKP-type alleles, while the K. pneumoniae NTUH-K2044 and MGH78578 genomes contained the SHV-type alleles. The chromosome-encoded β-lactamases corresponded to constitutive genes of these bacterial species (figure 3A). The OKP-A1 and OKP-B1 β-lactamases is characteristic of K. quasipneumoniae subsp. quasipneumoniae (KpII-A) and K. quasipneumoniae subsp. similipneumoniae (KpII-B), respectively.

β-lactamase genes were present in most of the K. variicola and K. quasipneumoniae genomes and were previously annotated as “Beta-lactamase or class A beta-lactamase”. In K. pneumoniae B1 and K. variicola, BZ19 the corresponding sequence was annotated as “Beta-lactamase TEM” and had an amino acid identity of 99 and 100%, respectively, with LEN-2 b-lactamase. In the Klebsiella sp. 1.1.55 genome, β-lactamase was annotated as “LEN family Class A β-lactamase” and had a 99% of amino acid identity with LEN-2. Figure 3B shows the phylogenetic analysis of K. quasipneumoniae chromosomal β-lactamase genes. The β-lactamases were annotated as class A beta-lactamase, or SHV-1 or TEM proteins. However, all these proteins corresponded to OKP-A1 and OKP-B1 β-lactamases, respectively, in subspecies of K. quasipneumoniae (KpII-A, KpII-B) (figure 3B). Chromosomal LEN- and OKP-β-lactamase proteins were not properly annotated and could have contributed to the bacterial species misclassification, if these chromosomal β-lactamase proteins are considered for Klebsiella classification.

In vitro assays of plant growth-promoting mechanisms in K. variicola and K. pneumonia

Indole acetic acid (an auxin) has the ability to regulate root growth, differentiation of vascular tissues, elongation, apical dominance, initiation of lateral roots and maturation.³²32. Woodward AW, Bartel B. Auxin: regulation, action, and interaction. Ann Bot 2005;95(5):707-735. https://doi.org/10.1093/aob/mci083
https://doi.org/10.1093/aob/mci083... This molecule also functions as an important signaling molecule in the regulation of plant growth, expansion, cell differentiation and cell division regulation.³³33. Ryu RJ, Patten CL. Aromatic amino acid-dependent expression of indole-3-pyruvate decarboxylase is regulated by TyrR in Enterobacter cloacae UW5. J Bacteriol 2008;190(21):7200-7208. https://doi.org/10.1128/JB.00804-08
https://doi.org/10.1128/JB.00804-08... K. variicola and K. pneumoniae isolates were capable of producing indole acetic acid (table II).

Gibberellins (GA) are involved in seed germination, seedling emergence, stem and leaf growth, flower induction, vegetative regulation of reproductive dormancy buds and fruit growth.³⁴34. Cassan F, Bottini R, Schneider G, Piccoli P. Azospirillum brasilense and Azospirillum lipoferum hydrolyze conjugates of GA20 and metabolize the resultant aglycones to GA1 in seedlings of rice dwarf mutants. Plant Physiol 2001;125(4):2053-2058. https://doi.org/10.1104/pp.125.4.2053
https://doi.org/10.1104/pp.125.4.2053... ^,3535. Yamaguchi S. Gibberellin metabolism and its regulation. Annu Rev Plant Biol 2008;59:225-251. https://doi.org/10.1146/annurev.arplant.59.032607.092804
https://doi.org/10.1146/annurev.arplant.... Almost all of the K. variicola and K. pneumoniae isolates (except for two K. pneumoniae isolates) were capable of producing gibberellins.

In addition to nitrogen, phosphorus is a nutrient that is required by plants.³⁶36. Naik PR, Raman G, Narayanan KB, Sakthivel N. Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil. BMC Microbiol 2008;8:230. https://doi.org/10.1186/1471-2180-8-230
https://doi.org/10.1186/1471-2180-8-230... K. variicola and K. pneumoniae isolates were not able to solubilize phosphate (table II), which is a common characteristic among plant associated bacteria. Klebsiella genus, unlike other plant growth promoting bacteria, did not produce chitinases or proteases that may help fighting pathogenic fungi of plants (table II). Siderophores are synthesized mainly by Gram-negative bacteria, fungi, yeast and some plants (phytosiderophores) and act as specific chelating agents. They are soluble in aqueous solutions at neutral pH and are considered as secondary metabolites.³⁷37. Ahmed E, Holmstrom SJ. Siderophores in environmental research: roles and applications. Microbial Biotechnol 2014;7(3):196-208. https://doi.org/10.1111/1751-7915.12117
https://doi.org/10.1111/1751-7915.12117... Siderophores may be important in the process of colonizing plant and human tissues.³⁸38. D’Onofrio A, Crawford JM, Stewart EJ, Witt K, Gavrish E, Epstein S, et al. Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chemistry & Biology 2010;17(3):254-264. https://doi.org/10.1016/j.chembiol.2010.02.010
https://doi.org/10.1016/j.chembiol.2010.... Different siderophores were identified in silico in the genome of K. variicola, K. quasipneumoniae and K. pneumoniae. In agreement with these findings, the in vitro siderophore assay was positive for all of the K. variicola and K. pneumoniae isolates that were tested.

The name K. variicola means from various places and novel reports confirmed the adequacy of its name. This species has now been identified in diverse plants, such as bitter gourd and chili, as well as in insects such as Nezara viridula. It is also significant that isolates have been obtained from different human samples, such as urine, pus and bile.¹⁷17. Martínez-Romero E, Rodríguez-Medina N, Beltrán-Rojel M, Pérez-Rueda E, Garza-Ramos U. Genome misclassification of Klebsiella variicola and Klebsiella quasipneumoniae isolated from plants, animals and humans. Salud Publica Mex . 2018;60. https://doi.org/10.21149/8149
https://doi.org/10.21149/8149...

K. variicola isolates were additionally obtained from the fecal microbiota of two cohorts of Malawian infants/children, but their genomes could not be included in this work because they are fragmented into a high number of contigs. Of note, the K. variicola strain has been identified in giant panda feces,²⁸28. Lu MG, Jiang J, Liu L, Ma AP, Leung FC. Complete genome sequence of Klebsiella variicola Strain HKUOPLA, a cellulose-degrading bacterium isolated from giant panda feces. Genome Announc 2015;3(5):e01200-15. https://doi.org/10.1128/genomeA.01200-15
https://doi.org/10.1128/genomeA.01200-15... and corresponds to other example of misclassification and corresponds to K. quasipneumoniae.¹⁷17. Martínez-Romero E, Rodríguez-Medina N, Beltrán-Rojel M, Pérez-Rueda E, Garza-Ramos U. Genome misclassification of Klebsiella variicola and Klebsiella quasipneumoniae isolated from plants, animals and humans. Salud Publica Mex . 2018;60. https://doi.org/10.21149/8149
https://doi.org/10.21149/8149... The first OXA-181 (carbapenemase)-producing K. variicola isolate was identified (by rpoB gene analysis) in fresh vegetables that were commercialized between different continents (Asia-Europe).³⁹39. Zurfluh K, Poirel L, Nordmann P, Klumpp J, Stephan R. First detection of Klebsiella variicola producing OXA-181 carbapenemase in fresh vegetable imported from Asia to Switzerland. Antimicrob Resist Infect Control 2015;4:38. https://doi.org/10.1186/s13756-015-0080-5
https://doi.org/10.1186/s13756-015-0080-... This work showed the fist carbapenem-resistance K. variicola strain and may suggest possible emergence paths of resistance in Enterobacteriaceae.

Gene flow may occur among different related Klebsiella species, mediated by plasmid transfer. The plasmid of K. variicola pBz19 showed high identity to pl9 (both associated with IncN incompatibility) from K. pneumoniae isolate.¹⁴14. Andrade BG, de Veiga Ramos N, Marin MF, Fonseca EL, Vicente AC. The genome of a clinical Klebsiella variicola strain reveals virulence-associated traits and a pl9-like plasmid. FEMS Microbiol Lett 2014;360(1):13-16. https://doi.org/10.1111/1574-6968.12583
https://doi.org/10.1111/1574-6968.12583... K. variicola DX120E contains pKV1 and pKV2 plasmids. The pKV1 plasmid is very similar to the pKp5-1 plasmid that was identified in K. pneumoniae KP5-1.¹⁹19. Lin L, Wei C, Chen M, Wang H, Li Y, Li Y, et al. Complete genome sequence of endophytic nitrogen-fixing Klebsiella variicola strain DX120E. Stand Genomic Sci 2015;10:22. https://doi.org/10.1186/s40793-015-0004-2
https://doi.org/10.1186/s40793-015-0004-... Plasmid pKV2 is most similar to plasmid pKOXM1C from K. oxytoca strain M1, suggesting plasmid exchange between these bacterial species. Additional analyses of plasmids are required specially in K. variicola from different niches to further understand their role in host interaction.

Conclusions

This work provides a new molecular framework for distinguishing different organisms of the Klebsiella genus. The in silico analyses showed that the genomes of K. variicola and K. quasipneumoniae shared a set of virulence-associated genes encoding mainly siderophores (aerobactin and enterobactin KfuABC cluster), urea metabolism, lipopolysaccharide biosynthesis enzymes (WabG and Uge), and fimbriae genes encoding the MrkABCDFHIJ gene cluster. The in vitro analysis indicated the presence of different siderophores in distinct isolates and species. Siderophores may have redundant functions given that they all are involved in bacterial iron acquisition. The number of siderophores in a single cell and their type however, may be important to regulate the flow of iron incorporation into the bacterial cell, a process that is linked to invasion of host tissues and thus virulence.

FimV, on the other hand, could be considered a common character in K. variicola strains distinguishing them from those of K. pneumoniae; however, not all K. variicola genomes contained this gene, as shown by both in silico and PCR assays. To explain the mosaic distribution of putative host-interaction genes in Klebsiella genomes, we envisage the possibility that Klebsiella cells may acquire virulence genes by horizontal transfer from closely related bacteria. However, this possibility is tempered by the alternative process of gene loss in bacterial evolution, which could be rather significant in the conformation of Klebsiella genomes as has been shown in other bacteria.⁴⁰40. McNally A, Thomson NR, Reuter S, Wren BW. ‘Add, stir and reduce’: Yersinia spp. as model bacteria for pathogen evolution. Nature Rev MicroBiol 2016;14(3):177-190. https://doi.org/10.1038/nrmicro.2015.29
https://doi.org/10.1038/nrmicro.2015.29... K. variicola and K. quasipneumoniae strains with different genetic repertoires have the capacity to adapt to plant and clinical settings. Previously, we suggested that a different epidemiological dynamics occurred with K. variicola in comparison to K. pneumoniae with K. variicola coming from the environment,⁴¹41. Martinez J, Martinez L, Rosenblueth M, Silva J, Martinez-Romero E. How are gene sequence analyses modifying bacterial taxonomy? The case of Klebsiella. Int Microbiol 2004;7(4):261-268. we further refine here that the environment refers most probably to the plant environment. Plants can be a reservoir for K. variicola isolates that may opportunistically infect humans or animals. K. variicola infection in humans thus seems to be a case of “phytonosis”, a term we suggest for symbiotic bacteria plant-borne, parallel to the term zoonosis bacteria pathogen animal-borne.

Acknowledgments

This work was funded by the Consejo Nacional de Ciencia y Tecnología (Conacyt), SEP-Conacyt - grant number 256988. We thank Angelina Quiterio for her excellent laboratory assistance and Michael Dunn and J. Martínez-Romero for their help in reviewing the manuscript.

References

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