- Email: [email protected]
A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine
A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae in the clinical routine Erica Lourenc¸o Fonseca, Nilceia da Veiga Ramos, Bruno G. Nascimento Andrade, Lena L.C.S. Morais, Michel F. Abanto Marin, Ana Carolina P. Vicente PII: DOI: Reference:
S0732-8893(17)30009-3 doi: 10.1016/j.diagmicrobio.2017.01.005 DMB 14273
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
22 October 2016 9 January 2017 12 January 2017
Please cite this article as: Fonseca Erica Louren¸co, da Veiga Ramos Nilceia, Nascimento Andrade Bruno G., Morais Lena L.C.S., Abanto Marin Michel F., Vicente Ana Carolina P., A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae in the clinical routine, Diagnostic Microbiology and Infectious Disease (2017), doi: 10.1016/j.diagmicrobio.2017.01.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola and
RI
Running title: bla for Klebsiella identification in clinics
PT
Klebsiella quasipneumoniae in the clinical routine
SC
Erica Lourenço Fonsecaa,*, Nilceia da Veiga Ramosa, Bruno G. Nascimento Andradea,
Laboratório de Genética Molecular de Microrganismos. Instituto Oswaldo
MA
a
NU
Lena L. C. S. Moraisb, Michel F. Abanto Marina, Ana Carolina P. Vicentea
Cruz/FIOCRUZ. Avenida Brasil 4365, Manguinhos, Rio de Janeiro, Brasil. Laboratório de Microbiologia Ambiental, Seção de Meio Ambiente, Instituto Evandro
e-mails:
AC CE P
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Chagas, Belém, Pará, Brasil.
D
b
Erica Lourenço Fonseca: [email protected] Nilceia da Veiga Ramos: [email protected] Bruno G. Nascimento Andrade: [email protected] Lena Lilian Canto de Sá Morais: [email protected] Michel F. Abanto Marin: [email protected] Ana Carolina P. Vicente: [email protected]
ACCEPTED MANUSCRIPT Abstract word count: 53
PT
Text body word count: 1004
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* Corresponding author: Erica L. Fonseca. Laboratório de Genética Molecular de Micorganismos, Instituto Oswaldo Cruz/FIOCRUZ, Brasil. Phone number: +55-21-3865-
NU
SC
8168. e-mail: [email protected]
Abstract
MA
Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae are difficult to differentiate phenotypically, leading to misinterpretation of their infection prevalence.
D
We propose a multiplex PCR for blaSHV, LEN and OKP and their flanking gene (deoR). Since
TE
this scheme focuses only on chromosomal genes, it will be feasible for Klebsiella
AC CE P
identification in the clinical routine.
Keywords: class A β-lactamase; Klebsiella species; molecular identification; blaLEN; blaSHV; blaOKP.
ACCEPTED MANUSCRIPT The opportunistic pathogen Klebsiella pneumoniae has been classified in three phylogenetic groups KpI, KpII-A/KpII-B and KpIII (Brisse and Verhoef, 2001), and the
PT
chromosomal class A β-lactamase blaSHV, blaOKP-A/blaOKP-B and blaLEN genes had been
RI
directly associated with each of these groups, respectively (Haeggman et al., 2004). Recently, it has been shown that these groups corresponded to the species K. pneumoniae
SC
(KpI), Klebsiella quasipneumoniae subsp. quasipneumoniae (KpII-A) Klebsiella
NU
quasipneumoniae subsp. similipneumoniae (KpII-B) and Klebsiella variicola (KpIII) (Brisse et al., 2014, Chen et al., 2016, Rosenblueth et al., 2004). However, some of the
MA
current phenotypic tests are unable to distinguish efficiently these Klebsiella species (Alves et al., 2006, Berry et al., 2015), leading to an underestimation of K. variicola and K.
D
quasipneumoniae infections, since they are often misidentified as K. pneumoniae (Brisse et
TE
al., 2004, Chen et al., 2016, Seki et al., 2013). In fact, there is no biochemical test capable
AC CE P
of differentiating concomitantly these three Klebsiella species in the routine of clinical microbiology laboratories (Brisse et al., 2014, Rosenblueth et al., 2004). For example, adonitol fermentation test distinguishes K. pneumoniae (adonitol-positive) from the other two species, but it is not able to differentiate K. variicola from K. quasipneumoniae (adonitol-negative). Moreover, such phenotypic traits are unstable characters, as already observed for K. variicola adonitol-postive and K. pneumoniae adonitol-negative strains (Alves et al., 2006), which leads to confusion and species misidentification. Even mass spectrometry, which is a highly specific molecular technique, is not able to distinguish those Klebsiellla species (Van Veen et al., 2010). Therefore, the routine identification of Klebsiella species remains difficult to be determined, raising the need for implementing a robust species marker that could rapidly and accurately distinguish them. The introduction
ACCEPTED MANUSCRIPT of a new identification approach will improve the control of Klebsiella infections, since these species have distinct pathogenic and epidemiological characteristics.
PT
Severe human infections caused by K. variicola and K. quasipneumoniae have been
RI
recently demonstrated by the assessment of genomic information and phylogenetic
al., 2014, Ozer et al., 2016, Seki et al., 2013).
SC
analyses (Arena et al., 2015, Breurec et al., 2016, Garza-Ramos et al., 2015, Maatallah et
NU
Therefore, due to the impact of K. variicola and K. quasipneumoniae on clinics, and the lack of a suitable identification approach to microbiological routine, we proposed here a
MA
PCR scheme targeting the chromosomal bla genes for rapidly distinguishing Klebsiella species.
D
BLAST Genome analyses revealed that blaSHV, blaOKP and blaLEN are embedded in
TE
the same genetic context in the K. pneumoniae, K. quasipneumoniae and K. variicola
AC CE P
genomes, respectively. In these species, their corresponding bla genes are flanked by the chromosomally-encoded deoR and a gene coding for an ATPase that are part of bacterial stable genome (Figure 1A). Based on this finding, we designed primers targeting each specific bla and the chromosomal gene flanking them (deoR) in the three Klebsiella species (Figure 1A). This strategy avoids the recovery of plasmid-borne blaSHV alleles, and only the chromosomal species-specific bla gene would be amplified. For this purpose, a global multiple alignment with 200 blaSHV, 38 blaOKP (17 blaOKPA
and 21 blaOKP-B) and 37 blaLEN alleles was performed to design primers specific to each of
these three genes. The blaSHV alleles were retrieved from “The Lactamase Engineering Database – LACED” (http://www.laced.uni-stuttgart.de/) (Thai and Pleiss, 2010). The blaLEN and blaOKP alleles were retrieved from GenBank based on the LEN and OKP
ACCEPTED MANUSCRIPT enzymes described in the “LEN, OXY, and OKP ß-lactamases Database” (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). A global multiple alignment was
PT
also performed with the chromosomal region flanking the bla gene (deoR) that is shared
RI
among K. pneumoniae, K. variicola and K. quasipenumoniae genomes retrieved from GenBank.
SC
The Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) was used to design the primers
NU
SHV-F, LEN-F, OKP-F and DEOR-R, which were applied in a multiplex PCR scheme for the identification of K. pneumoniae, K. variicola and K. quasipneumoniae according to the
MA
obtained amplicon size (Table 1, Figure 1A and B, Figure 2). PCR reactions were performed with the GoTaq Flexi DNA Polymerase and dNTP Mix (Promega), according to
D
manufacturer instructions, under the following conditions: an initial denaturation step of 5
TE
min at 95°C, followed by 40 amplification cycles (30s at 94°C, 30s at 55°C and 1 min at
AC CE P
72°C) and a final elongation step of 10 min at 72°C. The PCR products were submitted to electrophoresis on 2.5% agarose gel in 1X TAE buffer at 60 V for two hours. Amplicon size was estimated with a 100 bp molecular weight marker (Promega). The specificity and reproducibility of primers were tested by amplifying the bla genes from 150 K. pneumoniae and Klebsiella sp. isolates previously identified by VITEK2. Non-Klebsiella enterobacteria (Escherichia coli strains), with and without a plasmid-borne blaSHV, were included in the PCR reactions as controls. Primer specificity were also checked in silico by BLASTn against the non-redundant nucleotide collection and the complete and draft Klebsiella genomes.
ACCEPTED MANUSCRIPT The proposed PCR strategy showed to be specific and reproducible. A unique fragment of a specific size was obtained for each of the 150 isolates. Ninety-seven isolates
PT
were positive for a 995 bp-fragment (blaSHV), 32 isolates were positive for a 485 bp-
RI
fragment (blaLEN), and 21 isolates were positive for a 348 bp-fragment (blaOKP), allowing the identification of K. pneumoniae, K. variicola and K. quasipneumoniae, respectively
SC
(Figure 1 and Figure 2). The identity of amplicons with the corresponding bla genes was
NU
confirmed by sequencing. No amplification was obtained for the negative controls. These results demonstrated the reliability of the PCR scheme proposed here, and the occurrence of
MA
K. variicola and K. quasipneumoniae misidentification as K. pneumoniae based on the biochemical tests (Brisse et al., 2004), and call attention to the current underestimation and
D
impact of the former species in human infections.
TE
Several studies have stressed the need for a system to identify Klebsiella in clinical
AC CE P
microbiology laboratories (Berry et al., 2015; Seki et al., 2013). Here we proposed a PCR strategy specifically targeting the chromosomal class A β-lactamase genes that properly distinguish K. pneumoniae, K variicola and K. quasipneumoniae in a fast, reliable and single step fashion, which is particularly appropriated to be applied in the clinical identification routine.
Acknowledgements We thank the team of curators of the Institut Pasteur MLST and whole genome MLST databases for curating the data and making them publicly available at http://bigsdb.web.pasteur.fr/. We also acknowledged the support by CAPES, FAPERJ, CNPq and Oswaldo Cruz Institute grant.
ACCEPTED MANUSCRIPT
Funding
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This work was supported by FAPERJ and PNPD-CAPES fellowships and CNPq
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grant.
NU
SC
Conflict of Interest: none
References
MA
Alves MS, Dias RC, de Castro AC et al. Identification of clinical isolates of indole-positive
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10.1128/JCM.00940-06.
D
and indole-negative Klebsiella spp. J Clin Microbiol 2006; 44: 3640-3646. doi:
AC CE P
Arena F, Henrici De Angelis L, Pieralli F et al. Draft Genome Sequence of the First Hypermucoviscous Klebsiella quasipneumoniae subsp. quasipneumoniae Isolate from a Bloodstream Infection. Genome Announc 2015; 3: e00952-15. doi: 10.1128/genomeA.00952-15.
Berry GJ, Loeffelholz M, Williams-Bouyer N. An Investigation into Laboratory Misidentification of a Bloodstream Klebsiella variicola Infection. J Clin Microbiol 2015; 53: 2793-2794. doi:10.1128/JCM.00841-15.
ACCEPTED MANUSCRIPT Breurec S, Melot B, Hoen B et al. Liver Abscess Caused by Infection with CommunityAcquired Klebsiella quasipneumoniae subsp. quasipneumoniae. Emerg Infect Dis
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2016; 22: 529-531. doi: 10.3201/eid2203.151466.
Brisse S, Passet V, Grimont PA. Description of Klebsiella quasipneumoniae sp. nov.,
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isolated from human infections, with two subspecies, Klebsiella quasipneumoniae
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subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a
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junior heterotypic synonym of Klebsiella variicola. Int J Syst Evol Microbiol 2014; 64:
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3146-3152.
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Brisse S, van Himbergen T, Kusters K et al. Development of a rapid identification method
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for Klebsiella pneumoniae phylogenetic groups and analysis of 420 clinical isolates. Clin Microbiol Infect 2004; 10: 942-945.
Brisse S, Verhoef J. Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping. Int J Syst Evol Microbiol 2001; 51: 915-924.
Chen M, Li Y, Li S et al. Genomic identification of nitrogen-fixing Klebsiella variicola, K. pneumoniae and K. quasipneumoniae. J Basic Microbiol 2016; 56: 78-84. doi: 10.1002/jobm.201500415.
ACCEPTED MANUSCRIPT
Garza-Ramos U, Silva-Sanchez J, Barrios H et al. Draft Genome Sequence of the First
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Hypermucoviscous Klebsiella variicola Clinical Isolate. Genome Announc 2015; 3:
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e01352-14. doi: 10.1128/genomeA.01352-14.
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Haeggman S, Löfdahl S, Paauw A et al. Diversity and evolution of the class A
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chromosomal beta-lactamase gene in Klebsiella pneumoniae. Antimicrob Agents
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Chemother 2004; 48: 2400-2408. doi: 10.1128/AAC.48.7.2400-2408.2004.
Maatallah M, Vading M, Kabir MH et al. Klebsiella variicola is a frequent cause of
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bloodstream infection in the Stockholm area, and associated with higher mortality
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compared to K. pneumoniae. PLoS One 2014; 9: e113539. doi:
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10.1371/journal.pone.0113539.
Ozer EA, Morris AR, Krapp F et al. Draft Genome Sequence of a Multidrug-Resistant Klebsiella quasipneumoniae subsp. similipneumoniae Isolate from a Clinical Source. Genome Announc 2016; 4: e00422-16. doi: 10.1128/genomeA.00422-16.
Rosenblueth M, Martínez L, Silva J et al. Klebsiella variicola, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol 2004; 27: 27-35.
ACCEPTED MANUSCRIPT Seki M, Gotoh K, Nakamura S et al. Fatal sepsis caused by an unusual Klebsiella species that was misidentified by an automated identification system. J Med Microbiol 2013;
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62: 801-803.
Thai QK, Pleiss J. SHV Lactamase Engineering Database: a reconciliation tool for SHV b-
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lactamases in public databases. BMC Genomics 2010; 11: 563.
van Veen SQ, Claas EC, Kuijper EJ. High-throughput identification of bacteria and yeast
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by matrix-assisted laser desorption ionization-time of flight mass spectrometry in
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conventional medical microbiology laboratories. J Clin Microbiol 2010; 48: 900-907.
ACCEPTED MANUSCRIPT Table 1: Primers for Klebsiella species identification Primer
Primer sequence (5’ – 3’)b
Size
(nucleotide position)c
product
SC
AGAAGCATCCTGCTGTGCG
blaSHV alleles
blaLEN alleles
485bp
(nt 551-581)
blaOKP alleles
348bp
(nt 696-714)
3’ end of the deoR (182 nts downstream bla)
The forward primers SHV-F, LEN-F and OKP-F were all used in combination with DeoR-
R reverse primer in the multiplex PCR producing the amplicons sizes indicated in last column. b
The LEN-F and OKP-F primers were designed with degenerated bases due to
polymorphisms in blaLEN-26 and between blaOKP-A and blaOKP-B alleles. c
995bp
(nt 48-69)
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a
GGCCGGYGAGCGGGGCTCA
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DeoR-R
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OKP-F
CACGCTGCGYAAACTACTGACYGCGCAGCA
MA
LEN-F
GCTGGCGGTACACGCCAGCCCG
D
SHV-F
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namea
Target gene
nucleotide position of primer annealing considering the start codon of each bla gene.
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ACCEPTED MANUSCRIPT
Primer LEN-F
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Primer SHV-F
Primer OKP-F
ACCEPTED MANUSCRIPT Figure 1: Schematic representation of bla genomic context in K. pneumoniae, K. variicola and K. quasipneumoniae (A) and alignment of the bla gene regions targeted
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by each primer (B). A- The synteny among the three species is represented by gray blocks
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and colored arrows, whose directions indicated orientation of transcription. The bla and deoR genes are highlighted and the corresponding targeted sites of the PCR primers are
SC
illustrated. B- The blocks of alignment show the specificity of each bla primer relative to its
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corresponding bla gene. The shaded nucleotides represent the bla gene regions targeted by each primer. The nucleotide position where the primers anneal in each bla gene is indicated
MA
by the horizontal numbered lines on the top of alignments. The alignments demonstrate that each primer anneals exclusively in its corresponding bla gene due to the presence of
D
polymorphisms in the other genes. One polymorphism was observed between blaOKP-A and
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blaOKP-B regions targeted by OKP-F, which presents a degenerated base in this position in
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order to contemplate all blaOKP-A and blaOKP-B alleles.
ACCEPTED MANUSCRIPT 1
2
3
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M
995bp
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600bp
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485bp
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100bp
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348bp
Figure 2: Multiplex PCR strategy for Klebsiella species identification. The sizes of each fragment produced with the primer combinations OKP-F/DeoR-R, LEN-F/DeoR-R and SHV-F/DeoR-R are indicated by arrows. M: 100 bp DNA Ladder (Invitrogen); lane 1: blaOKP amplicon; lane 2: blaLEN amplicon; lane 3: blaSHV amplicon.
ACCEPTED MANUSCRIPT HIGHLIGHTS
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- A one-step and reliable diagnostic PCR for Klebsiella species identifications was proposed. - The chromosomal class A β-lactamase bla gene is a robust species marker.
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- The scheme robustness was determined by primers specificity and PCR reproducibility.
S0732-8893(17)30009-3 doi: 10.1016/j.diagmicrobio.2017.01.005 DMB 14273
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
22 October 2016 9 January 2017 12 January 2017
Please cite this article as: Fonseca Erica Louren¸co, da Veiga Ramos Nilceia, Nascimento Andrade Bruno G., Morais Lena L.C.S., Abanto Marin Michel F., Vicente Ana Carolina P., A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae in the clinical routine, Diagnostic Microbiology and Infectious Disease (2017), doi: 10.1016/j.diagmicrobio.2017.01.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola and
RI
Running title: bla for Klebsiella identification in clinics
PT
Klebsiella quasipneumoniae in the clinical routine
SC
Erica Lourenço Fonsecaa,*, Nilceia da Veiga Ramosa, Bruno G. Nascimento Andradea,
Laboratório de Genética Molecular de Microrganismos. Instituto Oswaldo
MA
a
NU
Lena L. C. S. Moraisb, Michel F. Abanto Marina, Ana Carolina P. Vicentea
Cruz/FIOCRUZ. Avenida Brasil 4365, Manguinhos, Rio de Janeiro, Brasil. Laboratório de Microbiologia Ambiental, Seção de Meio Ambiente, Instituto Evandro
e-mails:
AC CE P
TE
Chagas, Belém, Pará, Brasil.
D
b
Erica Lourenço Fonseca: [email protected] Nilceia da Veiga Ramos: [email protected] Bruno G. Nascimento Andrade: [email protected] Lena Lilian Canto de Sá Morais: [email protected] Michel F. Abanto Marin: [email protected] Ana Carolina P. Vicente: [email protected]
ACCEPTED MANUSCRIPT Abstract word count: 53
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Text body word count: 1004
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* Corresponding author: Erica L. Fonseca. Laboratório de Genética Molecular de Micorganismos, Instituto Oswaldo Cruz/FIOCRUZ, Brasil. Phone number: +55-21-3865-
NU
SC
8168. e-mail: [email protected]
Abstract
MA
Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae are difficult to differentiate phenotypically, leading to misinterpretation of their infection prevalence.
D
We propose a multiplex PCR for blaSHV, LEN and OKP and their flanking gene (deoR). Since
TE
this scheme focuses only on chromosomal genes, it will be feasible for Klebsiella
AC CE P
identification in the clinical routine.
Keywords: class A β-lactamase; Klebsiella species; molecular identification; blaLEN; blaSHV; blaOKP.
ACCEPTED MANUSCRIPT The opportunistic pathogen Klebsiella pneumoniae has been classified in three phylogenetic groups KpI, KpII-A/KpII-B and KpIII (Brisse and Verhoef, 2001), and the
PT
chromosomal class A β-lactamase blaSHV, blaOKP-A/blaOKP-B and blaLEN genes had been
RI
directly associated with each of these groups, respectively (Haeggman et al., 2004). Recently, it has been shown that these groups corresponded to the species K. pneumoniae
SC
(KpI), Klebsiella quasipneumoniae subsp. quasipneumoniae (KpII-A) Klebsiella
NU
quasipneumoniae subsp. similipneumoniae (KpII-B) and Klebsiella variicola (KpIII) (Brisse et al., 2014, Chen et al., 2016, Rosenblueth et al., 2004). However, some of the
MA
current phenotypic tests are unable to distinguish efficiently these Klebsiella species (Alves et al., 2006, Berry et al., 2015), leading to an underestimation of K. variicola and K.
D
quasipneumoniae infections, since they are often misidentified as K. pneumoniae (Brisse et
TE
al., 2004, Chen et al., 2016, Seki et al., 2013). In fact, there is no biochemical test capable
AC CE P
of differentiating concomitantly these three Klebsiella species in the routine of clinical microbiology laboratories (Brisse et al., 2014, Rosenblueth et al., 2004). For example, adonitol fermentation test distinguishes K. pneumoniae (adonitol-positive) from the other two species, but it is not able to differentiate K. variicola from K. quasipneumoniae (adonitol-negative). Moreover, such phenotypic traits are unstable characters, as already observed for K. variicola adonitol-postive and K. pneumoniae adonitol-negative strains (Alves et al., 2006), which leads to confusion and species misidentification. Even mass spectrometry, which is a highly specific molecular technique, is not able to distinguish those Klebsiellla species (Van Veen et al., 2010). Therefore, the routine identification of Klebsiella species remains difficult to be determined, raising the need for implementing a robust species marker that could rapidly and accurately distinguish them. The introduction
ACCEPTED MANUSCRIPT of a new identification approach will improve the control of Klebsiella infections, since these species have distinct pathogenic and epidemiological characteristics.
PT
Severe human infections caused by K. variicola and K. quasipneumoniae have been
RI
recently demonstrated by the assessment of genomic information and phylogenetic
al., 2014, Ozer et al., 2016, Seki et al., 2013).
SC
analyses (Arena et al., 2015, Breurec et al., 2016, Garza-Ramos et al., 2015, Maatallah et
NU
Therefore, due to the impact of K. variicola and K. quasipneumoniae on clinics, and the lack of a suitable identification approach to microbiological routine, we proposed here a
MA
PCR scheme targeting the chromosomal bla genes for rapidly distinguishing Klebsiella species.
D
BLAST Genome analyses revealed that blaSHV, blaOKP and blaLEN are embedded in
TE
the same genetic context in the K. pneumoniae, K. quasipneumoniae and K. variicola
AC CE P
genomes, respectively. In these species, their corresponding bla genes are flanked by the chromosomally-encoded deoR and a gene coding for an ATPase that are part of bacterial stable genome (Figure 1A). Based on this finding, we designed primers targeting each specific bla and the chromosomal gene flanking them (deoR) in the three Klebsiella species (Figure 1A). This strategy avoids the recovery of plasmid-borne blaSHV alleles, and only the chromosomal species-specific bla gene would be amplified. For this purpose, a global multiple alignment with 200 blaSHV, 38 blaOKP (17 blaOKPA
and 21 blaOKP-B) and 37 blaLEN alleles was performed to design primers specific to each of
these three genes. The blaSHV alleles were retrieved from “The Lactamase Engineering Database – LACED” (http://www.laced.uni-stuttgart.de/) (Thai and Pleiss, 2010). The blaLEN and blaOKP alleles were retrieved from GenBank based on the LEN and OKP
ACCEPTED MANUSCRIPT enzymes described in the “LEN, OXY, and OKP ß-lactamases Database” (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). A global multiple alignment was
PT
also performed with the chromosomal region flanking the bla gene (deoR) that is shared
RI
among K. pneumoniae, K. variicola and K. quasipenumoniae genomes retrieved from GenBank.
SC
The Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) was used to design the primers
NU
SHV-F, LEN-F, OKP-F and DEOR-R, which were applied in a multiplex PCR scheme for the identification of K. pneumoniae, K. variicola and K. quasipneumoniae according to the
MA
obtained amplicon size (Table 1, Figure 1A and B, Figure 2). PCR reactions were performed with the GoTaq Flexi DNA Polymerase and dNTP Mix (Promega), according to
D
manufacturer instructions, under the following conditions: an initial denaturation step of 5
TE
min at 95°C, followed by 40 amplification cycles (30s at 94°C, 30s at 55°C and 1 min at
AC CE P
72°C) and a final elongation step of 10 min at 72°C. The PCR products were submitted to electrophoresis on 2.5% agarose gel in 1X TAE buffer at 60 V for two hours. Amplicon size was estimated with a 100 bp molecular weight marker (Promega). The specificity and reproducibility of primers were tested by amplifying the bla genes from 150 K. pneumoniae and Klebsiella sp. isolates previously identified by VITEK2. Non-Klebsiella enterobacteria (Escherichia coli strains), with and without a plasmid-borne blaSHV, were included in the PCR reactions as controls. Primer specificity were also checked in silico by BLASTn against the non-redundant nucleotide collection and the complete and draft Klebsiella genomes.
ACCEPTED MANUSCRIPT The proposed PCR strategy showed to be specific and reproducible. A unique fragment of a specific size was obtained for each of the 150 isolates. Ninety-seven isolates
PT
were positive for a 995 bp-fragment (blaSHV), 32 isolates were positive for a 485 bp-
RI
fragment (blaLEN), and 21 isolates were positive for a 348 bp-fragment (blaOKP), allowing the identification of K. pneumoniae, K. variicola and K. quasipneumoniae, respectively
SC
(Figure 1 and Figure 2). The identity of amplicons with the corresponding bla genes was
NU
confirmed by sequencing. No amplification was obtained for the negative controls. These results demonstrated the reliability of the PCR scheme proposed here, and the occurrence of
MA
K. variicola and K. quasipneumoniae misidentification as K. pneumoniae based on the biochemical tests (Brisse et al., 2004), and call attention to the current underestimation and
D
impact of the former species in human infections.
TE
Several studies have stressed the need for a system to identify Klebsiella in clinical
AC CE P
microbiology laboratories (Berry et al., 2015; Seki et al., 2013). Here we proposed a PCR strategy specifically targeting the chromosomal class A β-lactamase genes that properly distinguish K. pneumoniae, K variicola and K. quasipneumoniae in a fast, reliable and single step fashion, which is particularly appropriated to be applied in the clinical identification routine.
Acknowledgements We thank the team of curators of the Institut Pasteur MLST and whole genome MLST databases for curating the data and making them publicly available at http://bigsdb.web.pasteur.fr/. We also acknowledged the support by CAPES, FAPERJ, CNPq and Oswaldo Cruz Institute grant.
ACCEPTED MANUSCRIPT
Funding
PT
This work was supported by FAPERJ and PNPD-CAPES fellowships and CNPq
RI
grant.
NU
SC
Conflict of Interest: none
References
MA
Alves MS, Dias RC, de Castro AC et al. Identification of clinical isolates of indole-positive
TE
10.1128/JCM.00940-06.
D
and indole-negative Klebsiella spp. J Clin Microbiol 2006; 44: 3640-3646. doi:
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Arena F, Henrici De Angelis L, Pieralli F et al. Draft Genome Sequence of the First Hypermucoviscous Klebsiella quasipneumoniae subsp. quasipneumoniae Isolate from a Bloodstream Infection. Genome Announc 2015; 3: e00952-15. doi: 10.1128/genomeA.00952-15.
Berry GJ, Loeffelholz M, Williams-Bouyer N. An Investigation into Laboratory Misidentification of a Bloodstream Klebsiella variicola Infection. J Clin Microbiol 2015; 53: 2793-2794. doi:10.1128/JCM.00841-15.
ACCEPTED MANUSCRIPT Breurec S, Melot B, Hoen B et al. Liver Abscess Caused by Infection with CommunityAcquired Klebsiella quasipneumoniae subsp. quasipneumoniae. Emerg Infect Dis
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2016; 22: 529-531. doi: 10.3201/eid2203.151466.
Brisse S, Passet V, Grimont PA. Description of Klebsiella quasipneumoniae sp. nov.,
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isolated from human infections, with two subspecies, Klebsiella quasipneumoniae
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subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a
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junior heterotypic synonym of Klebsiella variicola. Int J Syst Evol Microbiol 2014; 64:
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3146-3152.
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Brisse S, van Himbergen T, Kusters K et al. Development of a rapid identification method
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for Klebsiella pneumoniae phylogenetic groups and analysis of 420 clinical isolates. Clin Microbiol Infect 2004; 10: 942-945.
Brisse S, Verhoef J. Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping. Int J Syst Evol Microbiol 2001; 51: 915-924.
Chen M, Li Y, Li S et al. Genomic identification of nitrogen-fixing Klebsiella variicola, K. pneumoniae and K. quasipneumoniae. J Basic Microbiol 2016; 56: 78-84. doi: 10.1002/jobm.201500415.
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Garza-Ramos U, Silva-Sanchez J, Barrios H et al. Draft Genome Sequence of the First
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Hypermucoviscous Klebsiella variicola Clinical Isolate. Genome Announc 2015; 3:
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e01352-14. doi: 10.1128/genomeA.01352-14.
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Haeggman S, Löfdahl S, Paauw A et al. Diversity and evolution of the class A
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chromosomal beta-lactamase gene in Klebsiella pneumoniae. Antimicrob Agents
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Chemother 2004; 48: 2400-2408. doi: 10.1128/AAC.48.7.2400-2408.2004.
Maatallah M, Vading M, Kabir MH et al. Klebsiella variicola is a frequent cause of
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bloodstream infection in the Stockholm area, and associated with higher mortality
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compared to K. pneumoniae. PLoS One 2014; 9: e113539. doi:
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10.1371/journal.pone.0113539.
Ozer EA, Morris AR, Krapp F et al. Draft Genome Sequence of a Multidrug-Resistant Klebsiella quasipneumoniae subsp. similipneumoniae Isolate from a Clinical Source. Genome Announc 2016; 4: e00422-16. doi: 10.1128/genomeA.00422-16.
Rosenblueth M, Martínez L, Silva J et al. Klebsiella variicola, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol 2004; 27: 27-35.
ACCEPTED MANUSCRIPT Seki M, Gotoh K, Nakamura S et al. Fatal sepsis caused by an unusual Klebsiella species that was misidentified by an automated identification system. J Med Microbiol 2013;
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62: 801-803.
Thai QK, Pleiss J. SHV Lactamase Engineering Database: a reconciliation tool for SHV b-
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lactamases in public databases. BMC Genomics 2010; 11: 563.
van Veen SQ, Claas EC, Kuijper EJ. High-throughput identification of bacteria and yeast
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by matrix-assisted laser desorption ionization-time of flight mass spectrometry in
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conventional medical microbiology laboratories. J Clin Microbiol 2010; 48: 900-907.
ACCEPTED MANUSCRIPT Table 1: Primers for Klebsiella species identification Primer
Primer sequence (5’ – 3’)b
Size
(nucleotide position)c
product
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AGAAGCATCCTGCTGTGCG
blaSHV alleles
blaLEN alleles
485bp
(nt 551-581)
blaOKP alleles
348bp
(nt 696-714)
3’ end of the deoR (182 nts downstream bla)
The forward primers SHV-F, LEN-F and OKP-F were all used in combination with DeoR-
R reverse primer in the multiplex PCR producing the amplicons sizes indicated in last column. b
The LEN-F and OKP-F primers were designed with degenerated bases due to
polymorphisms in blaLEN-26 and between blaOKP-A and blaOKP-B alleles. c
995bp
(nt 48-69)
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a
GGCCGGYGAGCGGGGCTCA
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DeoR-R
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OKP-F
CACGCTGCGYAAACTACTGACYGCGCAGCA
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LEN-F
GCTGGCGGTACACGCCAGCCCG
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SHV-F
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namea
Target gene
nucleotide position of primer annealing considering the start codon of each bla gene.
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Primer LEN-F
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Primer SHV-F
Primer OKP-F
ACCEPTED MANUSCRIPT Figure 1: Schematic representation of bla genomic context in K. pneumoniae, K. variicola and K. quasipneumoniae (A) and alignment of the bla gene regions targeted
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by each primer (B). A- The synteny among the three species is represented by gray blocks
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and colored arrows, whose directions indicated orientation of transcription. The bla and deoR genes are highlighted and the corresponding targeted sites of the PCR primers are
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illustrated. B- The blocks of alignment show the specificity of each bla primer relative to its
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corresponding bla gene. The shaded nucleotides represent the bla gene regions targeted by each primer. The nucleotide position where the primers anneal in each bla gene is indicated
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by the horizontal numbered lines on the top of alignments. The alignments demonstrate that each primer anneals exclusively in its corresponding bla gene due to the presence of
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polymorphisms in the other genes. One polymorphism was observed between blaOKP-A and
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blaOKP-B regions targeted by OKP-F, which presents a degenerated base in this position in
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order to contemplate all blaOKP-A and blaOKP-B alleles.
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2
3
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995bp
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600bp
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485bp
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100bp
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348bp
Figure 2: Multiplex PCR strategy for Klebsiella species identification. The sizes of each fragment produced with the primer combinations OKP-F/DeoR-R, LEN-F/DeoR-R and SHV-F/DeoR-R are indicated by arrows. M: 100 bp DNA Ladder (Invitrogen); lane 1: blaOKP amplicon; lane 2: blaLEN amplicon; lane 3: blaSHV amplicon.
ACCEPTED MANUSCRIPT HIGHLIGHTS
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- A one-step and reliable diagnostic PCR for Klebsiella species identifications was proposed. - The chromosomal class A β-lactamase bla gene is a robust species marker.
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- The scheme robustness was determined by primers specificity and PCR reproducibility.