RESEARCH ARTICLE
Applica ion o mini-MLST and whole genome
sequencing in low di e si y hospi al ex ended-
spec um be a-lac amase p oducing Klebsiella
pneumoniae popula ion
Ma ej BezdicekID
1,2
, Ma ke a Nyk yno a
1,3
, K is ina Ple o a
1
, E a B helo a
1,2
,
I a Kocmano a
4
, Ka el Sedla
3
, Zdenek Racil
1,2
, Ji i Maye
1,2
, Ma ina Lenge o a
1,2
*
1Depa men o In e nal Medicine–Hema ology and Oncology, Uni e si y Hospi al B no, B no, Czech
Republic, 2Depa men o In e nal Medicine–Hema ology and Oncology, Masa yk Uni e si y, B no, Czech
Republic, 3Depa men o Biomedical Enginee ing, B no Uni e si y o Technology, B no, Czech Republic,
4Depa men o Clinical Mic obiology, Uni e si y Hospi al B no, B no, Czech Republic
*Lenge o a.Ma ina@ nb no.cz
Abs ac
S udying bac e ial popula ion di e si y is impo an o unde s and heal hca e associa ed
in ec ions’ epidemiology and has a signi ican impac on dealing wi h mul id ug esis an bac-
e ial ou b eaks. We cha ac e ised he ex ended-spec um be a-lac amase p oducing K.
pneumoniae (ESBLp KPN) popula ion in ou hospi al using mini-MLST. Then we used
whole genome sequencing (WGS) o compa e selec ed isola es belonging o he mos p e -
alen mel ing ypes (MelTs) and he coloniza ion/in ec ion pai isola es collec ed om one
pa ien o s udy he ESBLp KPN popula ion’s gene ic di e si y. A o al o 922 ESBLp KPN
isola es collec ed be ween 7/2016 and 5/2018 we e di ided in o 38 MelTs using mini-MLST
wi h only 6 MelTs o ming 82.8% o all isola es. Fo WGS, 14 isola es om he mos p omi-
nen MelTs collec ed in he moni o ed pe iod and 10 isola es belonging o he same MelTs
collec ed in ou hospi al in 2014 we e andomly selec ed. Resis ome, i ulome and ST we e
MelT speci ic and s able o e ime. A maximum o 23 SNV pe co e genome and 58 SNV
pe co e and accesso y genome we e ound. To de e mine he SNV ela edness cu -o al-
ues, 22 isola es ep esen ing coloniza ion/in ec ion pai samples ob ained om 11 di e en
pa ien s we e analysed by WGS wi h a maximum o 22 SNV in he co e genome and 40
SNV in he co e and accesso y genome wi hin pai s. The mini-MLST showed i s po en ial
o eal- ime epidemiology in clinical p ac ice. Howe e , o ou b eak e alua ion in a low
di e si y bac e ial popula ion, mini-MLST should be combined wi h mo e sensi i e me hods
like WGS. Ou indings showed he e we e only minimal di e ences wi hin he co e and
accesso y genome in he low di e si y hospi al popula ion and gene based SNV analysis
does no ha e enough disc imina o y powe o di e en ia e isola e ela edness. Thus, in e -
genic egions and mobile elemen s should be inco po a ed in o he analysis scheme o
inc ease disc imina o y powe .
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0221187 Augus 13, 2019 1 / 14
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OPEN ACCESS
Ci a ion: Bezdicek M, Nyk yno a M, Ple o a K,
B helo a E, Kocmano a I, Sedla K, e al. (2019)
Applica ion o mini-MLST and whole genome
sequencing in low di e si y hospi al ex ended-
spec um be a-lac amase p oducing Klebsiella
pneumoniae popula ion. PLoS ONE 14(8):
e0221187. h ps://doi.o g/10.1371/jou nal.
pone.0221187
Edi o : D. Ashley Robinson, Uni e si y o
Mississippi Medical Cen e , UNITED STATES
Recei ed: May 27, 2019
Accep ed: July 31, 2019
Published: Augus 13, 2019
Pee Re iew His o y: PLOS ecognizes he
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p ocess; he e o e, we enable he publica ion o
all o he con en o pee e iew and au ho
esponses alongside inal, published a icles. The
edi o ial his o y o his a icle is a ailable he e:
h ps://doi.o g/10.1371/jou nal.pone.0221187
Copy igh : ©2019 Bezdicek e al. This is an open
access a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : Da a accession o
ou p ojec was upda ed in he manusc ip . All aw
In oduc ion
Klebsiella pneumoniae (KPN) equen ly causes communi y and hospi al acqui ed in ec ions
including pneumonia, u ina y ac in ec ions and pyogenic li e abscesses [1,2]. The main
KPN ansmission ese oi s a e he gas oin es inal ac and he hands o hospi al pe sonnel
and pa ien s. Nosocomial KPN isola es o en display highly esis an pheno ypes wi h an
ex ended-spec um be a-lac amases p oducing (ESBLp) KPN p e alence be ween 2% and 55%
[3–5].
Typing me hods o disc imina e di e en bac e ial isola es om he same species a e essen-
ial epidemiological ools. In popula ions wi h a high p e alence o ESBL, he knowledge o
bac e ial popula ion s uc u e and dynamics is especially impo an in ou b eak de ec ion and
in e en ion. To moni o he bac e ial popula ion, cheap, apid and obus me hods a e
needed. In ou p e ious s udy, we p o ed ha mini-MLST, a me hod de i ed om mul i locus
sequence yping (MLST) in which cos ly and ime-consuming sequencing is eplaced wi h
high esolu ion mel ing analysis, is sui able o long e m p ospec i e KPN popula ion sc een-
ing. Cu en ly, besides KPN [6], mini-MLST has been es ablished o S aphyloccoccus au eus
[7], En e ococcus aecium [8] and S ep ococcus pyogenes [9]. Howe e , i s lowe disc imina o y
powe makes mini-MLST insu icien o iden i y ou b eak s ains and i needs o be combined
wi h mo e sensi i e me hods.
Recen ly, whole genome sequencing (WGS) has e olu ionized ou abili y o di e en ia e
be ween bac e ial s ains a he en i e genome’s DNA sequence le el. Fo bac e ial yping,
WGS has wo majo app oaches–co e genome mul ilocus sequence yping (cgMLST) and sin-
gle nucleo ide a ian analysis (SNV) [10]. CgMLST is based on an allele numbe ing sys em o
a p e-de e mined se o genes. An ad an age o cgMLST’s app oach is i s in e -labo a o y po -
abili y and he exis ence o public da abases e.g. h ps://www.cgmls .o g/ncs,h p://
en e obase.wa wick.ac.uk/ o h ps://pubmls .o g/. SNV analysis is based on mapping aw
sequence eads agains a e e ence genome and de ec ing nucleo ides ha a y wi hin he da a-
se . This app oach p o ides an e en highe esolu ion powe han cgMLST, bu is a mo e
compu a ionally in ensi e han cgMLST analysis and in e p e ing he esul s is mo e complex
[11,12].
As gene a ing WGS da a become mo e accessible, apid and cheap, bo lenecks emain in
p ope p e-sequencing sample selec ion and pos -sequencing da a analysis [11]. The main bo -
lenecks include he need o c i ically e alua e he aw sequencing da a, some knowledge and
skills in p og amming and imp o emen s in da a analysis o ansla e he eno mous amoun
o ob ained da a in o unde s andable esul s o heal h p o essionals [13]. Knowledge o he
local bac e ial popula ion’s gene ics cha ac e isa ion is also c ucial o he esul s o be co -
ec ly in e p e ed, as he e a e no gene al h esholds o ela edness [10].
The main objec i es o his s udy we e i) o cha ac e ise he ESBLp KPN popula ion in ou
hospi al using mini-MLST p ospec i e yping ii) o e alua e co e and accesso y genome single
nucleo ide a ian analysis con ibu ion in possible ou b eak de ec ion wi hin he low di e si y
ESBLp KPN hospi al popula ion.
Ma e ial and me hods
Clinical isola es
The s udy was conduc ed a he Uni e si y Hospi al B no (B no, Czech Republic), a e ia y
ca e hospi al wi h mo e han 2,000 beds and 5,000 employees. The e a e mo e han 1,000,000
people ea ed in ou -pa ien clinics and o e 70,000 pa ien s hospi alized e e y yea . Du ing
he sys ema ic s ain collec ion be ween 7/2016 and 5/2018, we collec ed all ESBLp KPN
Molecula yping o low di e si y ESBL-p oducing Klebsiella pneumoniae popula ion
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0221187 Augus 13, 2019 2 / 14
sequencing da a a e a ailable unde he accession
numbe PRJNA515630 in he BioP ojec da abase
(di ec link was also added o he e ised
manusc ip h ps://www.ncbi.nlm.nih.go /
biop ojec /PRJNA515630.).
Funding: This s udy was suppo ed by he Minis y
o Heal h, Czech Republic - concep ual
de elopmen o esea ch o ganiza ion (FNB ,
65269705)(MB, ML, IK, ZR, JM, KP, EB), Masa yk
Uni e si y G an Agency (MUNI/A/1105/2018)(MB)
and Czech Science Founda ion (GACR 17-01821S)
(MB, ML, MN, KS, IK). The unde s had no ole in
s udy design, da a collec ion and analysis, decision
o publish, o p epa a ion o he manusc ip .
Compe ing in e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
isola es om high isk depa men s (Depa men o In e nal Medicine–Hema ology and
Oncology, Depa men o In e nal Medicine, Ge ia ics and P ac ical Medicine, Depa men
o Anes hesiology and In ensi e Ca e Medicine) and all isola es om neona es, new-bo ns and
child en. In o al, we collec ed 922 ESBLp KPN isola es (Table 1). Fo he pu poses o his
s udy, 24 ESBLp KPN isola es om ou p e ious s udy collec ed be ween 1/2014 and 10/2014
we e also included [14]. All isola es we e collec ed du ing ou ine p ac ice and we e made
comple ely anonymous.
DNA isola ion
Fo mini-MLST, bac e ial genomic DNA (gDNA) was isola ed using Chelex 100 Resin (Bio-
Rad, USA). The Bac e ial cul u e was homogenised in 100 μo 5% w/ Chelex 100 Resin wi h a
o ex. The suspension was incuba ed o 10 min a 100˚C and hen cen i uged o 2 min a
Table 1. Epidemiological da a; n = 922.
Gende no. o isola es (%)
Male 510 (55.3)
Female 412 (44.7)
Pa ien age mean ( ange)
56 yea s (0 days-99 yea s)
Hospi alisa ion/ou pa ien no. o isola es (%)
Hospi alisa ion 863 (93.6)
Ou pa ien 59 (6.4)
mean ( ange)
Hospi alisa ion leng h 27 days (0–724 days)
Hospi alisa ion leng h o collec ion 11 days (0–369 days)
Sou ce no. o isola es (%)
U ine 269 (29.2)
Rec um 226 (24.5)
Blood cul u e 96 (10.4)
O al ca i y swab 84 (9.1)
Th oa swab 49 (5.3)
Spu um 43 (4.7)
Pe ianal swab 39 (4.2)
Wound swab 30 (3.3)
U ina y ca he e 24 (2.6)
Skin swab 13 (1.4)
Handp in 11 (1.2)
Venous ca he e 10 (1.1)
Nose swab 7 (0.8)
S ool 6 (0.7)
B onchoal eola la age luid 4 (0.4)
Vagina 3 (0.3)
Asci es 2 (0.2)
Suc ion ca he e 2 (0.2)
Ea swab 1 (0.1)
Eye swab 1 (0.1)
S oma swab 1 (0.1)
U e h a swab 1 (0.1)
h ps://doi.o g/10.1371/jou nal.pone.0221187. 001
Molecula yping o low di e si y ESBL-p oducing Klebsiella pneumoniae popula ion
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15,500 c . A supe na an con aining gDNA was ans e ed in o a clean mic o ube. Fo WGS,
gDNA was pu i ied using DNeasy Blood & Tissue Ki (Qiagen GmbH). The DNA concen a-
ion was measu ed using NanoD op (The mo Scien i ic, USA).
Mini-MLST
Mini-MLST was pe o med wi h p ime s desc ibed by Ande sson, Tong [6] on a Ro o Gene
6000 pla o m (Co be Resea ch, Aus alia). The 20 μL eac ion olume con ained 10 μL 2×
SensiFAST HRM mix (Bioline Reagen s, UK), 0.4 μM o each p ime , 1 μL o ex ac ed geno-
mic DNA (30 ng) and deionized wa e o a inal olume o 20 μL. The mo cycling pa ame e s
we e: 95˚C o 3 min, 40 cycles o 95˚C o 5 s, 65˚C o 10 s and 72˚C o 20 s, hen one cycle
o 95˚C o 2 min and 50˚C o 20 s, ollowed by HRM amping om 70 o 95˚C, inc easing
by 0.2˚C a each s ep. The esul s we e in e p e ed using he cu en e sion o ou con e sion
key, which is a ailable o ee download a h p://www.cmbg .cz/mini-mls / 6353.
Whole genome sequencing and analysis
Pu i ied gDNA was sh edded using S220 Focused-ul asonica o (Co a is, USA). WGS lib a -
ies we e p epa ed wi h KAPA Hype P ep Ki s (Roche, Swi ze land) and a quali y check was
pe o med using 2100 Bioanalyze (Agilen Technologies, USA). The Illumina MiSeq pla o m
was used o WGS and 250-bp pai ed-end sequencing was pe o med. The aw sequencing
da a we e deposi ed in he NCBI BioP ojec da abase unde he p ojec ID PRJNA515630
(h ps://www.ncbi.nlm.nih.go /biop ojec /PRJNA515630). The ob ained eads we e quali y
checked using Fas QC (Bab aham Bioin o ma ics, UK) and assembled using Bu ows-
Wheele Aligne [15]. Ridom SeqSphe e+ (Ridom, DE) seed genome Klebsiella pneumoniae
subsp. pneumoniae NTUH-K2044 (NC_012731.1) was used as he e e ence genome. To
emo e unmapped eads, eads wi h poo quali y and duplica es, SAM ools we e used [16].
A e e e ence mapping, all posi ions wi h less han 10×co e age and all ambiguous posi ions
(less common base ep esen ed a leas 10% o bases in he a ge posi ion) we e emo ed om
u he analysis. UGENE so wa e was used o ob ain consensus sequences [17]. Resis ome
and i ulome analysis was ca ied ou using public online da abases (h p://www.
genomicepidemiology.o g/,h p://bigsdb.pas eu . /). The cgMLST was pe o med using
Ridom SeqSphe e+ (Ridom, DE) wi h an inco po a ed KPN cgMLST scheme. This included
2,358 a ge genes whose alleles we e used o gene a e a cgMLST dendog am. Fo he co e
genome SNV minimum spanning ee 2,042,000 aligned nucleo ide si es we e analysed. In
o al, 4,179,387 aligned nucleo ide si es we e accoun ed o by he co e and accesso y genome
SNV minimum spanning ee analysis. The WGS da a we e used o de e mine he MLST ype
o all sequenced s ains.
Resul s
Mini-MLST
Fo hospi al popula ion cha ac e isa ion, we used mini-MLST o ype all collec ed isola es.
O e all, 38 di e en MelTs we e iden i ied among 922 ESBLp KPN isola es collec ed be ween
7/2016 and 5/2018. MelT145 (30.8%, n = 284), MelT26 (25.9%, n = 239), MelT132 (8.0%,
n = 74), MelT139 (7.7%, n = 71), MelT269 (5.4%, n = 50), MelT281 (5.0%, n = 46), MelT266
(2.6%, n = 24) and MelT20 (2.3%, n = 21) we e he mos p edominan (Fig 1). The emaining
30 MelTs we e p esen in less han 2% o all isola es. Fig 1 shows he p opo ions o ce ain
MelTs in ou hospi al popula ion a e qui e s able (MelT145, MelT26), while an inc ease
(MelT132) o dec ease (MelT139) can be obse ed in o he s. Mini-MLST’s disc imina o y
Molecula yping o low di e si y ESBL-p oducing Klebsiella pneumoniae popula ion
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Fig 1. Compa ison o MelTs dis ibu ion in moni o ed pe iod (7/2016-5/2018).
h ps://doi.o g/10.1371/jou nal.pone.0221187.g001
Molecula yping o low di e si y ESBL-p oducing Klebsiella pneumoniae popula ion
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powe was D = 0.8189. The mini-MLST esul s we e used o basic popula ion so ing and
WGS sample selec ion.
Whole genome sequence analysis
Whole genome sequence sample selec ion. We andomly selec ed 14 isola es om he
ou mos p edominan MelTs om 7/2016-5/2018 (3 MelT145 isola es, 5 MelT26 isola es, 3
MelT132 isola es and 3 MelT139 isola es). Because hese MelTs we e also p esen in ou hospi-
al du ing ou p e ious s udy [14], we added 10 isola es isola ed in ou hospi al in 2014 (3
MelT145 isola es, 1 MelT26 isola e, 3 MelT132 isola es and 3 MelT139 isola es).
To se SNV ela edness cu -o alues, 22 isola es ep esen ing coloniza ion ( ec al swab)/
in ec ion (blood cul u e) pai samples ob ained om 11 di e en pa ien s we e analysed using
WGS. Pai sample isola es belonged o 6 di e en MelTs (2 MelT26 isola es, 2 MelT130 iso-
la es, 6 MelT132 isola es, 6 MelT145 isola es, 4 MelT266 isola es and 2 MelT281 isola es). Fo
all 46 sequenced isola es, we pe o med in silico MLST, esis ome and i ulome analysis,
cgMLST and SNV analysis o he co e genome and accesso y genome.
The MLST, esis ome and i ulome analysis. Fi s , in silico STs we e de e mined and
esis ome and i ulome we e compa ed o all 46 samples (Fig 2). MLST analysis (based on
se en house-keeping genes poB,gapA,mhd,pgi,phoE,in B and onB) showed ST uni o mi y
wi hin indi idual MelT g oups, excep isola e S13 om MelT26 which, unlike he o he iso-
la es in his MelT g oup, was ST29. This isola e was he only MelT26 de ec ed in 2014. All
o he sequenced MelT26 isola es om 2016 and 2017 we e ST1271. ST1271 and ST29 only di -
e in he MLST scheme’s phoE allele (allele phoE 4 in ST1271 agains allele phoE 6 in ST26)
(S1 Table).
Compa ing esis ome and i ulome co ela ed wi h he MLST analysis esul s and con-
i med a high le el o simila i y inside he MelTs (see de ailed esul s in S1 Table). Isola es clus-
e ed oge he , as in he in silico MLST analysis (including sepa a ion o isola e S13 om o he
isola es) wi h one excep ion in MelT132, which was di ided in o wo lineages, A and B,
acco ding o he di e en esis ome and i ulome composi ion. Lineage A included S19, S20,
S21 and S44; lineage B included S22, S24, S25, S26, S35 and S36.
cgMLST and SNV analysis. CgMLST analysis p o ides mo e disc imina o y powe han
adi ional MLST. In his s udy, we used a cgMLST scheme consis ing o 2,358 a ge s p o-
cessed by Ridom SeqSphe e+ so wa e (Ridom, DE). A cgMLST dend og am based on 2,251
a ge s p esen in all analysed s ains was cons uc ed (Fig 2). The o he 107 a ge s om he
o iginal 2,358 a ge s we e absen in some o all o he s ains and we e excluded om all u -
he analyses. The cgMLST di ided 46 isola es in o 7 clea ly dis inguishable clus e s co e-
sponding o hei MelTs g oups, wi h wo excep ions. The S13 isola e di e ed om he o he
MelT26 isola es in 584 a ge s, while he a e age numbe o di e en alleles wi hin he o he
MelTs anged om 0 o 19 di e ences be ween wo samples o he same MelT. The second
excep ion was MelT132, which was di ided in o wo subpopula ions wi h 127 di e en alleles
be ween hem. Bo h excep ions co ela ed wi h he p e ious in silico MLST and esis ome and
i ulome analysis. The a e age numbe o di e en alleles be ween indi idual MelTs was 1827
( ange om 1809 o 1844 di e ences).
SNV analysis was ca ied ou o bo h a co e genome (n = 2,251 genes p esen in all ana-
lysed s ains) and co e wi h accesso y genome (n = 4,084 genes p esen in all analysed s ains).
In he co e genome, 29,535 SNV posi ions we e iden i ied in o al. In gene al, he co e genome
SNV numbe inside each MelT a ies om 0 o 23 SNV (MelT145 0–23 SNV, n = 12; MelT139
0–21 SNV, n = 8; MelT26 0–20 SNV, n = 8; MelT266 0–14 SNV, n = 4; MelT132 clus e A
0–22 SNV, n = 5; MelT132 clus e B 0–4 SNV, n = 6; MelT281 0 SNV, n = 2 and MelT130 0
Molecula yping o low di e si y ESBL-p oducing Klebsiella pneumoniae popula ion
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Molecula yping o low di e si y ESBL-p oducing Klebsiella pneumoniae popula ion
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SNV, n = 2) (Fig 3A). The S13 isola e di e ed by 2,311 SNV om o he MelT26 isola es, while
be ween indi idual MelTs, he numbe o SNV anged om 9,595 o 9,821. The MelT132 iso-
la es we e di ided in o wo lineages, A and B, simila o he cgMLST analysis. Bo h MelT132
lineages di e ed wi h 137 SNV be ween hem. The comple e dis ance ma ix o he co e
genome SNV analysis is showed in S2 Table.
The co e and accesso y genome SNV analysis was done wi h 4,084 gene a ge s p esen in
all 48 isola es (Fig 3B). 61,343 SNV posi ions we e iden i ied wi h he SNV coun wi hin each
MelT anging om 0 o 58 (MelT145 0–58 SNV, n = 12; MelT139 0–53 SNV, n = 8; MelT26
3–32 SNV, n = 8; MelT266 2–50 SNV, n = 4; MelT132 lineage A 0–36 SNV, n = 5; MelT132
lineage B 1–6 SNV, n = 6; MelT281 1 SNV, n = 2 and MelT130 1 SNV, n = 2). The a e age
SNV numbe be ween indi idual MelTs was 20,196 SNV ( ange om 19,936 o 20,358 SNV).
The dis ance ma ix o he co e and accesso y genome’s SNV analysis is showed in S3 Table.
The majo opology di e ence be ween he co e (A) and he co e and accesso y (B) genome
panels was he posi ion exchange be ween he MelT281 and he MelT132 clus e s. Addi ion-
ally, using he co e and accesso y genome panel (B), he MelT26 and he MelT132 clus e s
we e linked wi h he S13 isola e ha was only linked o MelT26 clus e when using he co e
genome panel (A).
In ec ion and colonisa ion pai isola es analysis. We analysed 11 pai s (each pai
included a ec al swab and a blood cul u e) o ESBLp KPN isola es ob ained om 11 pa ien s
o se he SNV cu -o , which de e mined he ela edness o isola es (Fig 2). F om 22 ESBLp
KPN isola es, 6 dis inc STs we e iden i ied: ST405 (n = 6), ST433 (n = 6), ST323 (n = 4),
ST1271 (n = 2), ST458 (n = 2) and ST23 (n = 2). The isola es om each pa ien sha e he same
ST wi hin he pai . The co e genome’s SNV numbe wi hin each pai a ies in ange om 0 o
22 SNV, while oge he wi h he accesso y genome’s SNV numbe , a ies in ange om 0 o 40
SNV. Based on pai ed sample analysis, we es ablished ela edness cu -o alues o ou ESBLp
KPN popula ion as he highes SNV numbe wi hin pai isola es, 22 SNV o he co e genome
and 40 SNV o he co e wi h he accesso y genome. Bo h he highes SNV alues we e o pai
S39/S40 ha had ime lapses be ween samples o 47 days. The nex highes SNV alues we e
only 3 SNVs o he co e genome and 5 SNVs o he co e and accesso y genome, despi e ime
lapses o up o 90 days."
Discussion
We a e cu en ly using he ollowing p o ocol in ou ine p ac ice. ESBLp KPN collec ed om
high- isk depa men s a e p ospec i ely es ed wi h mini-MLST o de e mine he MelT. When
he s ains MelT di e , ansmission is unlikely. When we obse e an inc eased incidence o
one MelT, we in es iga e he po en ial epidemiological linkages and hen we decide i he e is a
possible ou b eak and need o WGS analysis. Meanwhile, ea ly epidemiological measu es can
be implemen ed o p e en u he sp ead o in ec ion.
KPN mini-MLST is a cheap, apid and obus me hod o epidemiological s ain yping
in oduced by Ande sson, Tong [6], wi h ad an ages in i s obus ness, ep oducibili y and
po abili y be ween labo a o ies as i is based on a well-es ablished MLST me hod. In s udies
wi h a la ge numbe o samples, mini-MLST also can be used o sample so ing and p e-
selec ing o mo e de ailed analysis. We e alua ed his me hod o p ospec i e ESBLp KPN
Fig 2. WGS esul s o 46 selec ed ESBLp KPN isola es. The cgMLST dendog am was made using Ridom Seqsphe e+ so wa e and is based on sequence simila i y in he
2,251 co e genome a ge s sha ed by all 46 isola es. Mini-MLST was done expe imen ally and MLST was done in silico using WGS da a. Mini-MLST, MLST, cgMLST and
SNV analysis o pai samples (blood cul u e/ ec al swab pai s a e highligh ed in he same colou ). The ime lapse indica es ime be ween collec ing he colonizing and
in ec ing isola es. �Samples ob ained om one pa ien , bu wi h no ec al swab.
h ps://doi.o g/10.1371/jou nal.pone.0221187.g002
Molecula yping o low di e si y ESBL-p oducing Klebsiella pneumoniae popula ion
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0221187 Augus 13, 2019 8 / 14
Molecula yping o low di e si y ESBL-p oducing Klebsiella pneumoniae popula ion
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0221187 Augus 13, 2019 9 / 14