mic oo ganisms
A icle
Genomic Analysis o P ophages om Klebsiella pneumoniae
Clinical Isola es
And eia T. Ma ques 1,* , Luís Tanoei o 1, Aida Dua e 2,3 , Luisa Gonçal es 4, Jo ge M. B. Ví o 1
and Filipa F. Vale 1,*


Ci a ion: Ma ques, A.T.; Tanoei o, L.;
Dua e, A.; Gonçal es, L.; Ví o ,
J.M.B.; Vale, F.F. Genomic Analysis o
P ophages om Klebsiella pneumoniae
Clinical Isola es. Mic oo ganisms 2021,
9, 2252. h ps://doi.o g/10.3390/
mic oo ganisms9112252
Academic Edi o : Igo V. Babkin
Recei ed: 15 Sep embe 2021
Accep ed: 25 Oc obe 2021
Published: 28 Oc obe 2021
Publishe ’s No e: MDPI s ays neu al
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Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
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A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Pa hogen Genome Bioin o ma ics and Compu a ional Biology, Resea ch Ins i u e o
Medicines (iMed-ULisboa), Facul y o Pha macy, Uni e sidade de Lisboa, 1649-003 Lisboa, Po ugal;
[email p o ec ed] (L.T.); j i o @ .ulisboa.p (J.M.B.V.)
2Facul y o Pha macy, Uni e sidade de Lisboa, A . Gama Pin o, 1649-003 Lisboa, Po ugal;
[email p o ec ed]
3Cen o de In es igação In e disciplina Egas Moniz, Ins i u o Uni e si á io Egas Moniz,
2829-511 Mon e da Capa ica, Po ugal
4Clinical Pa hology Uni , Hospi al SAMS, Cidade de Gabela, 1849-017 Lisboa, Po ugal;
[email p o ec ed]
*Co espondence: [email p o ec ed] (A.T.M.); [email p o ec ed] o [email p o ec ed] (F.F.V.)
Abs ac :
Klebsiella pneumoniae is an inc easing h ea o public heal h and ep esen s one o he
mos conce ning pa hogens in ol ed in li e- h ea ening in ec ions. The esis an and i ulence
de e minan s a e coded by mobile gene ic elemen s which can easily sp ead be ween bac e ia
popula ions and co-e ol e wi h i s genomic hos . In his s udy, we p esen he ull genomic sequences,
inse ion si es and phylogene ic analysis o 150 p ophages ound in 40 K. pneumoniae clinical isola es
ob ained om an ou b eak in a Po uguese hospi al. All s ains ha bo ed a leas one p ophage and
we iden i ied 104 in ac p ophages (69.3%). The p ophage size anges om 29.7 o 50.6 kbp, coding
be ween 32 and 78 pu a i e genes. The p ophage GC con en is 51.2%, lowe han he a e age GC
con en o 57.1% in K. pneumoniae. Comple e p ophages we e classi ied in o h ee amilies in he
o de Caudolo i ales:Myo i idae (59.6%), Sipho i idae (38.5%) and Podo i idae (1.9%). In addi ion, an
alignmen and phylogene ic analysis e ealed nine dis inc clus e s. E idence o ecombina ion was
de ec ed wi hin he genome o some p ophages bu , in mos cases, p o eins in ol ed in i al s uc u e,
ansc ip ion, eplica ion and egula ion (lysogenic/lysis) we e main ained. These esul s suppo
he knowledge ha p ophages a e di e se and widely dissemina ed in K. pneumoniae genomes,
con ibu ing o he e olu ion o his species and con e ing addi ional pheno ypes. Mo eo e , we
iden i ied K. pneumoniae p ophages in a se o endolysin genes, which we e ound o code o p o eins
wi h lysozyme ac i i y, clea ing he
β
-1,4 linkages be ween N-ace ylmu amic acid and N-ace yl-D-
glucosamine esidues in he pep idoglycan ne wo k and hus ep esen ing genes wi h he po en ial
o lysin phage he apy.
Keywo ds:
K. pneumoniae genomes; p ophages; bac e iophage; bioin o ma ics; genomic analysis;
compa a i e genomics; phylogeny; sequence anno a ion and compa ison; phage endolysins
1. In oduc ion
Klebsiella pneumoniae is an oppo unis ic and commensal g am-nega i e human pa hogen
p e alen in he hospi al en i onmen . This bac e ium is mainly ound in gas oin es i-
nal and espi a o y ac s and on he skin o heal hy indi iduals, bu in ecen yea s i
has become one o he wo ld’s leading causes o communi y and hospi al-acqui ed in ec-
ions, such as u ina y ac in ec ions (UTIs), pneumonia, sep icaemia, and wound/so
issue in ec ions, wi h an inc easing mo ali y a e, pa icula ly in immunocomp omised
indi iduals, neona es, and he elde ly [1–5].
Due o i s widesp ead dis ibu ion and gene ic plas ici y, K. pneumoniae is one o
he mos impo an mul id ug- esis an (MDR) pa hogens and has been classi ied as an
Mic oo ganisms 2021,9, 2252. h ps://doi.o g/10.3390/mic oo ganisms9112252 h ps://www.mdpi.com/jou nal/mic oo ganisms
Mic oo ganisms 2021,9, 2252 2 o 21
ESKAPE o ganism (En e ococcus aecium,S aphylococcus au eus,K. pneumoniae,Acine obac e
baumannii,Pseudomonas ae uginosa and En e obac e species) [
6
], in which an ibio ic- esis an
s ains a e becoming mo e di icul o ea . K. pneumoniae s ains a e ecu en ly esis an o
an ibio ics a ailable in he apy o ea se ious human diseases, such as luo oquinolones,
aminoglycosides, and be a-lac ams. Among be a-lac ams, penicillins, cephalospo ins and
ca bapenems, he e is inc easing e idence o in ec ions caused by s ains ha ha e become
esis an o imipenem, e apenem and me openem an ibio ics [
5
,
7
–
11
]. Gi en he educ ion
in he e ec i eness o an imic obial he apeu ics o ea K. pneumoniae-associa ed in ec ions,
al e na i e s a egies mus be de eloped in esponse.
Bac e iophages (phages) a e i uses ha in ec bac e ia. Vi uses we e ini ially sug-
ges ed as he i s an imic obial agen s by William Two and Felix d’He elle [
12
,
13
] and
we e a he apy o bac e ial in ec ions [
14
]. Howe e , a e he disco e y o an imic o-
bial compounds, phage he apy was disca ded (excep in Eas e n Eu ope and he o me
So ie Union [
15
]), and om he e on mos s udies add essed p ophage esea ch as a
ool o imp o e ou unde s anding o molecula biology, ho izon al gene ans e and
bac e ial e olu ion.
Mo e ecen ly, gi en he inc ease in he numbe o MDR in ec ions caused by g am-
nega i e bac e ia such as K. pneumoniae, he use o phages o speci ic phage gene p oduc s
has inc eased again as a po en ial al e na i e o cu en an imic obial he apies [
16
]. MDR
isola es o K. pneumoniae we e ound wi h a a iable numbe o p ophages in hei ch o-
mosomes [
17
], and some p ophages ca ied an ibio ic esis ance genes (ARGs) [
18
,
19
],
p omp ing he in e es in bac e iophage esea ch.
Phages o K. pneumoniae ha e been isola ed om a a ie y o sou ces wo ldwide, in-
cluding was ewa e , sewage, seawa e , and human in es inal samples. Such phages belong
o ou o he i e amilies o he o de Caudo i ales, desc ibed as non-en eloped, ailed
phages, wi h icosahed al heads con aining double-s anded DNA: Myo i idae a e cha ac-
e ized by long, s aigh , con ac ile ails; Sipho i idae by long, lexible, non-con ac ile ails;
Podo i idae by sho , non-con ac ile ails; and Acke mann i idae by con ac ile ails wi h up
o ou spikes p esen on each o six ail spike en i ies [20].
Phages a e a g oup o i uses ha in ec bac e ia and make use o bac e ial eplica ion
machine y o become eplica ed, gene a ing p ogeny and eleasing i in he en i onmen
mos ly by p omo ing he cell–hos lysis in he ly ic cycle [
21
,
22
]. In he lysogenic cycle,
phages can in eg a e in he hos genome and emain do man o an unspeci ied amoun
o ime as p ophages, which will be eplica ed as pa o he bac e ial genome wi hou
killing he hos . Unde ce ain condi ions (e.g., in he p esence o en i onmen al s esso s),
p ophages can be excised and induced, assuming a ly ic cycle, and begin ac i ely eplica ing
and p oducing iable phage pa icles [
23
]. In addi ion, du ing he lysogenic cycle, gene ic
changes may happen in p ophage sequences, which lead o cumula i e deg ada ion o
he bac e iophage genome o ans e ence o genes in o he hos ha can con e oxin
p oduc ion and an ibio ic esis ance ai s o he bac e ium genome, hus making he
in ec ion mo e i ulen and di icul o ea [
24
]. Likewise, p ophages can con ibu e
o impo an biological p ope ies o hei bac e ial hos s, such as i ness, i ulence, and
e olu ion [
21
]. E en de ec i e p ophages may p o ide mul iple bene i s o he hos o
su i ing ad e se en i onmen al condi ions [25].
Endolysins (lysins) a e pep idoglycan-hyd olyzing enzymes encoded by phage genes.
In he cell dis up ion s age o he phage ly ic cycle, lysins a e in ol ed in he b eakage
o pep idoglycan o elease phage p ogeny [
26
]. In he case o g am-nega i e bac e ia,
pep idoglycan has a highly conse a i e s uc u e wi h signi ican simila i ies sha ed
among di e en species; he e o e, endolysins a e usually ac i e agains a wide hos
ange [27,28]. Mo eo e , endolysins ea u e easonable selec i i y in a ge ing pa hogenic
species, p ese ing commensal mic o lo a, and he adminis a ion o hese phage-de i ed
enzymes can be easily applied by di e en s a egies, including pa en e al, opical o
o al o mula ions [
27
]. Fo hese easons, he use o endolysins is mo e a ac i e as an
al e na i e an imic obial agen o clinical ea men han he phage i sel ; he e o e, lysins
Mic oo ganisms 2021,9, 2252 3 o 21
ha e been p oposed as al e na i e an imic obial agen s o ea in ec ions in he pos -
an ibio ic e a [
29
–
31
]. Recen esea ch has p oduced some p omising esul s ega ding he
use o endolysins agains K. pneumoniae. A ecombinan endolysin om he K. pneumoniae
phage KP27 was p oduced, and i s pep idoglycan-deg ading ac i i y was demons a ed
agains g am-nega i e bac e ia by he co-incuba ion o bac e ia and endolysin [
32
]. In
a s udy by Walmagh e al. (2013), i e endolysins we e cha ac e ized, including wo
endolysins om K. pneumoniae phages K11 and KP32, and hei mu aly ic ac i i y on he
pep idoglycan o se e al g am-nega i e bac e ial species was demons a ed [
33
]. In ano he
s udy, wo endolysins (ElyA1 and ElyA2) combined wi h colis in we e es ed agains A.
baumannii,P. ae uginosa and K. pneumoniae, and one o hem displayed ac i i y agains
13 ou o 17 s ains o K. pneumoniae [34].
In his wo k, we aimed o e alua e he p ophage p esence in clinical isola es o K.
pneumoniae om an ou b eak in a Po uguese e ia y-ca e hospi al. Also, we aimed o
unde s and how p ophages can con ibu e o he apid e olu ion o his bac e ial pa hogen.
Mo eo e , we ha e iden i ied and cha ac e ized pu a i e endolysin genes encoded by hese
p ophage genomes ha can po en ially be used o phage lysin he apy.
2. Ma e ials and Me hods
2.1. K. pneumoniae Isola es Genomes
A o al o 40 mul iclonal K. pneumoniae isola es om 23 pa ien s hospi alized in in-
ensi e ca e uni a SAMS Hospi al, a Po uguese e ia y-ca e hospi al, we e ecen ly
sequenced by whole genome sequencing (WGS) [
35
] and he genomes we e sc eened o
p ophage p esence.
2.2. P ophage Iden i ica ion
PHASTER (PHAge Sea ch Tool Enhanced Release) [
36
] and P ophage Hun e Tool [
37
]
we e used, allowing o he iden i ica ion and anno a ion o pu a i e p ophages wi hin
con igs o each K. pneumoniae genome (las accessed Janua y 2021). Concu en ly, bac e-
ial genomes we e also anno a ed using he open-access ool RAST: Rapid Anno a ion
using Subsys em Technology [
38
–
40
], and he iden i ied p ophage genes we e ex ensi ely
analysed in e ms o sequence and s uc u e o e alua e i s homology wi h bac e iophage-
de i ed egions. The anno a ion o p ophage coding sequences ound by he h ee di e en
me hods was compa ed (da a no shown).
All p ophage sequences we e manually so ed and cu a ed, and he inse ion si es
we e de e mined as shown below. A comple e p ophage sequence is no o en p esen in
one con ig, and o o e come his limi a ion, whene e possible, p ophages we e sca olded
using BLAST wi h a que y o he p ophages om K. pneumoniae subsp. pneumoniae HS11286
(GenBank Accession: CP003200.1, genome egion om 1288358 o 1338717) and K. pneu-
moniae s ain FDAARGOS_775 (GenBank Accession: NZ_CP040993.1, genome egion om
3328442 o 3378114) o check o homologies in he con igs in a simila way, as desc ibed else-
whe e [
41
]. Bo h genomes we e a ailable on NCBI da abase
(h ps://www.ncbi.nlm.nih.go /
,
las accessed 15 Janua y 2021) as e e ence genomes o K. pneumoniae and, since bo h ha e
in eg a ed p ophages, we also e e ed o hem o de e mine he co ec inse ion si es.
The inse ion si es o he p ophages we e iden i ied whene e he p ophage 5
0
and 3
0
ends
we e con iguously lanked by bac e ial genes in a con ig. The las bac e ial gene be o e he
p ophage sequence and he i s bac e ial gene a e he p ophage sequence we e iden i ied.
Each anno a ed egion was analysed in e ms o nucleo ide sequence wi h BLASTn [
42
]
and phage-limi ed BLASTn (limi ed o bac e iophage- ela ed ax ids: 38018, 10699, 10662,
10744, 10841, 2100421, 28883, 12333, 79205 and 102294) in he NCBI da abase using de aul
pa ame e s. P o ein-coding sequences we e also analysed using egula BLASTp and
phage-limi ed BLASTp (limi ed o bac e iophage- ela ed ax ids: 38018, 10699, 10662, 10744,
10841, 2100421, 28883, 12333, 79205 and 102294). S uc u al homology analyses we e
pe o med using Phy e2 [43].
Mic oo ganisms 2021,9, 2252 4 o 21
2.3. P ophage Classi ica ion
The iden i ied in ac p ophages we e classi ied in silico in o hei espec i e phage
amilies based on he p ophage s uc u al head-neck- ail p o eins using VIRFAM [
44
]. The
p ophage li es yles we e classi ied using PHACTS [
45
] and we e addi ionally assessed by
manually inspec ing he genomes o genes ela ed o li es yle (e.g., in eg ases).
2.4. P ophage Pan-Genome
The co e- and pan-genome o K. pneumoniae in ac p ophages we e de e mined using
Roa y ( e sion 3.13) [
46
], using as se ings o co e genome he genes p esen in a leas
50% o he p ophage in ac genomes, a minimum BLASTp pe cen age iden i y o 40, 50,
60, 70, 80 o 90%, and
−
s op ion. These se ings we e used o de e mine he mos sui able
pa ame e s o de e mining he p ophage pan-genome, as p e iously desc ibed [47].
2.5. P ophage Phylogene ic Analysis
In ac p ophage sequences we e que ied agains all K. pneumoniae phages sequences
a ailable on he PATRIC websi e (h ps://www.pa icb c.o g, las accessed Janua y 2021) [
48
],
which had 256 sequences in Janua y 2021, and agains public da abases using phage-
limi ed BLASTn [
42
] o iden i y simila phages. Hi s wi h a que y co e o a leas 50%
we e conside ed simila phages and hose wi h que y co e s below 50% we e conside ed
close phages.
The p ophage genomes we e aligned using MAFFT e sion 7 [
49
] de aul op ions.
Maximum likelihood phylogene ic ees om he alignmen s we e p oduced using Fas T ee
2.1.11 [
50
]. The p oduced ees we e isualized and anno a ed using In e ac i e T ee O
Li e (iTOL) 6 [51].
2.6. P ophage-Associa ed Vi ulence Fac o s and An ibio ic Resis ance Genes
All p ophage genomic sequences we e sc eened o an ibio ic esis ance genes using
he ResFinde 4.1 da abase (h ps://cge.cbs.d u.dk/se ices/ResFinde -4.1/, las accessed
July 2021) and i ulence genes using Vi ulenceFinde 2.0 (h ps://cge.cbs.d u.dk/se ices/
Vi ulenceFinde /, las accessed July 2021). Simila ly, he Resis ance Gene Iden i ie (RGI)
op ion o The Comp ehensi e An ibio ic Resis ance Da abase (h ps://ca d.mcmas e .
ca/home, las accessed July 2021) was used wi h de aul alues o iden i y esis ance
genes, hei p oduc s, and associa ed pheno ypes ha bo ed by in eg a ed p ophages wi hin
K. pneumoniae s ains.
2.7. Endolysins Iden i ica ion, Gene On ology Analysis and Func ional Anno a ion
Since de ec i e p ophages can also ha bo lysins, we conside ed all p ophages iden-
i ied (in ac and de ec i e) o endolysins iden i ica ion. Toge he wi h ou p ophage
sequences, we also analysed a se o 17 anno a ed phages iden i ied du ing p ophage
phylogene ic analysis, which sha e homology wi h ou p ophages. A o al o 167 p ophage
sequences (150 sequences o iginally iden i ied + 17 phage anno a ed sequences) we e
submi ed o bioin o ma ic analysis o he iden i ica ion o pu a i e phage endolysins
in e ms o sequence homology using BLAST [
42
] and s uc u al homology using he
open-access ools Phy e2 [
43
] and SWISS-MODEL [
52
]. Gene On ology (GO) iden i ie s
and ela ed GO e ms we e assigned o he iden i ied endolysins using he QuickGo web
se e (h p://www.ebi.ac.uk/QuickGO/, las accessed July 2021).
2.8. Endolysin Phylogene ic Analysis
Endolysin genomic and p o eomic sequences we e aligned using MAFFT e sion
7 [
49
] wi h de aul pa ame e s. The genome phylogene ic ee was cons uc ed using he
Jukes–Can o subs i u ion model and he p o eome phylogene ic ee was cons uc ed
using he Le Gascuel subs i u ion model in PHYML 3.3.20180621 (Geneious P ime e sion
2021.1.1). The iden i y ma ix gene a ed du ing he cons uc ion o he phylogene ic ees
Mic oo ganisms 2021,9, 2252 5 o 21
was used o in e nucleo ides and p o eins endolysins iden i y. T ees we e isualized and
anno a ed using In e ac i e T ee O Li e (iTOL) 6 [51].
3. Resul s
3.1. Iden i ica ion and P e alence o P ophages in K. pneumoniae S ains
In he p esen s udy, he genome sequences o 40 K. pneumoniae clinical isola es om
23 pa ien s
we e analysed wi h a web se e ool o iden i ica ion and anno a ion o
p ophage sequences wi hin bac e ial genomes, PHASTER, wi h de aul a gumen s [36].
A o al o 150 p ophage-like elemen s we e de ec ed (Supplemen a y Table S1), o
which 104 we e classi ied as in ac , 39 as incomple e and 7 as ques ionable (Figu e 1a,
Supplemen a y Table S2). One s ain was ound o ha bo only one p ophage, while he
emaining 39 s ains (97.5%) ha e a leas wo p ophages, which indica es ha p ophages
a e abundan in he K. pneumoniae genome. The o al numbe o p ophages pe s ain
anged om 1 o 10, wi h an a e age o 3.7 p ophages pe s ain, and mos s ains ha bo ed
ei he wo (n= 13) o ou (n= 11) p ophages (Figu e 1b).
Figu e 1.
(
A
) Box plo showing he o al, in ac , incomple e, ques ionable, and de ec i e (incomple e + ques ionable)
p ophages o Klebsiella pneumoniae. (
B
) Ba s g aph showing he dis ibu ion o o al p ophages (in ac , incomple e, and
ques ionable) in K. pneumoniae s ains.
A signi ican ly highe p e alence o incomple e and ques ionable p ophages was
expec ed since in ac p ophages a e usually unde s ong selec ion o gene ic deg ada ion
by bac e ia o apid dele ion om bac e ial genomes [
53
,
54
]. Ins ead, we ound ha 97.5%
o he K. pneumoniae s ains con ain in ac p ophages. S ains isola ed om pa ien s 1, 6, 21,
23, 24, 25 and 26 con ained only in ac p ophages, whe eas s ains isola ed om pa ien s
2, 3, 5, 9, 11, 14, 16, 17, 18 and 19 con ained in ac and incomple e p ophages, and s ains
isola ed om pa ien s 4, 7, 8, 10, 13 and 15 con ained in ac , incomple e, and ques ionable
p ophages (Supplemen a y Table S2). Fu he mo e, pa ien s we e colonized wi h one K.
pneumoniae s ain, excep pa ien s 1, 3, 15, 17, 19, 23 and 26, which we e colonized wi h 6, 2,
4, 5, 3, 2 and 2 s ains, espec i ely. I is impo an o no e ha he genomes we e di ided
in o con igs, which implies ha PHASTER may ha e unde es ima ed he co ec numbe
o in ac p ophages (some we e spli in o di e en con igs and iden i ied as incomple e o
ques ionable p ophages).
3.2. Genome Cha ac e is ics o K. pneumoniae P ophages
The sho es ( emnan ) p ophage sequence is 8.9 kbp, and he bigges is 60.8 kbp, wi h
he coding sequences (CDS) numbe anging om 12 o 75. The a e age GC% con en in all
150 p ophages is 52.2% (min 45.1%, max 60.2%), while he a e age bac e ial GC% con en is

Mic oo ganisms 2021,9, 2252 6 o 21
57.1% (min 54.9%, max 57.4%) (Supplemen a y Table S1), which sugges s ho izon al gene
ans e o he p ophage egion.
Genomic analysis o he ups eam and downs eam egions o he p ophage inse ion
si es e ealed ha p ophages a e in eg a ed be ween di e en coding egions: p o eins
in ol ed in me abolic pa hways (18.1%), RNA genes (16.1%), anspo e s (11.1%), e-
combinan p o eins (5.4%), p o ein syn hesis (5.4%), ans e ases (4.7%), ansc ip ional
egula o s (4.0%), memb ane p o eins (2.0%), ibosome biogenesis (1.3%) and sequences
showing homology wi h o he bac e ial genes (8.7%). In he case o some incomple e
p ophages, i was no possible o de e mine he inse ion si e (23.2%) (Supplemen a y
Table S1
). Mo eo e , simila p ophages om di e en K. pneumoniae s ains had a con-
se ed inse ion si e be ween he same wo con iguous genes o K. pneumoniae e e ence
genomes (GenBank Accession: CP003200.1 and NZ_CP040993.1).
Fo he pu pose o ou analysis, incomple e and ques ionable p ophage sequences
di ided in o di e en con igs we e sca olded as desc ibed in ma e ials and me hods.
A e his analysis, p ophages s ill conside ed ques ionable, and incomple e we e g ouped
as de ec i e p ophages. P ophages smalle han 28 kbp we e conside ed no in ac be-
cause hey lacked a p ophage genomic s uc u e and we e di icul o dis inguish om
o he in eg a i e elemen s. Only p ophages wi h iden i ied in eg ase and/o a leas one
gene in ol ed in biological p ocesses (e.g., e minase, endolysin, capsid, ail ibe s) we e
conside ed in ac .
Acco ding o his c i e ion, p ophages we e ound o be in ac in 104 o he
150 p ophage
sequences (63.3%) (Table 1). In ac p ophages ha e an a e age o 50 p edic ed genes (min
32, max 78), 37.4 kbp (min 29.7 kbp, max 50.6 kbp), and 51.2% GC (min 48.3%, max 55.0%).
Table 1.
In ac p ophage genomes cha ac e iza ion. Fea u es such as pa ien , GC%, leng h, CDS, clus e , amily (in
silico-de e mined) and mos ela ed-phage iden i ied a e shown o each p ophage. GC, guanine–cy osine.
P ophages
Pa ien s S ains P ophages GC% Leng h
(kbp) CDS Clus e Family Rela ed Phages Accession
Numbe
Que y
Co e E Value Pe .
Iden
Pa ien 1
Kp4845
PKp4845-1 50,4 33,4 44 C1
Myo i idae
Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4845-2 50,4 33,4 44 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Kp4846
PKp4846-1 50,4 33,4 44 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4846-2 50,4 33,4 44 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Kp4847
PKp4847-1 50,4 33,4 44 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4847-2 50,4 33,4 44 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Kp4848
PKp4848-1 50,4 33,4 44 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4848-2 50,4 33,4 44 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Kp4850
PKp4850-1 50,4 33,4 44 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4850-2 51,2 32,3 43 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Kp4851
PKp4851-1 48,3 46,7 55 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4851-2 50,1 35,6 47 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Pa ien 2 Kp4852
PKp4852-1 50,1 35,6 47 C6
Myo i idae
Klebsiella phage
ST101-KPC2phi6.3 MK416017.1 35% 0.0 95.09%
PKp4852-2 50,1 35,6 47 C3 Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 56% 0.0 96.97%
PKp4852-3 50,1 35,6 47 N/D Pseudomonas phage
VW-6B MF975721.1 31% 0.0 77.91%
PKp4852-4 50,1 35,6 47 C9 Klebsiella phage
ST846-OXA48phi9.1 MK416021.1 77% 0.0 98.39%
Mic oo ganisms 2021,9, 2252 7 o 21
Table 1. Con .
P ophages
Pa ien s S ains P ophages GC% Leng h
(kbp) CDS Clus e Family Rela ed Phages Accession
Numbe
Que y
Co e E Value Pe .
Iden
Pa ien 3
Kp4853
PKp4853-1 50,1 35,6 47 C5
Myo i idae
Klebsiella phage 2
LV-2017 KY271396.1 54% 0.0 94.42%
PKp4853-2 50,1 35,6 47 C6
PKp4853-4 50,1 35,6 47 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
Kp4854
PKp4854-1 50,1 35,6 47 C5 Klebsiella phage 2
LV-2017 KY271396.1 54% 0.0 94.42%
PKp4854-2 50,1 35,6 47 C6
PKp4854-4 50,1 35,6 47 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
Pa ien 4 Kp4855
PKp4855-2 50,1 35,6 47 C5
Myo i idae
Klebsiella phage
ST16-OXA48phi5.3 MK416014.1 28% 0.0 96.13%
PKp4855-4 51,1 31,9 43 N/D
Klebsiella phage
ST974-
OXA48phi18.2
MK448237.1 68% 0.0 96.79%
PKp4855-5 51,1 31,9 43 C2 Klebsiella phage 4
LV-2017 KY271398.1 82% 0.0 96.43%
Pa ien 5 Kp4856 PKp4856-1 51,1 35,1 44 C5 Myo i idae Klebsiella phage 2
LV-2017 KY271396.1 46% 0.0 97.25%
Pa ien 6 Kp4857 PKp4857-1 51,1 35,1 44 C2 Myo i idae Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Pa ien 7 Kp4858
PKp4858-1 51,1 35,1 44 C2 Myo i idae
Klebsiella phage
ST512-
KPC3phi13.6/Klebsiella
phage ST437-
OXA245phi4.1
MK433577.1/
MK416011.1 86% 0.0 97.25%
PKp4858-3 51,1 35,1 44 C3 Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 64% 0.0 95.28%
Pa ien 8 Kp4859
PKp4859-1 51,1 35,1 44 C2 Myo i idae
Klebsiella phage
ST512-
KPC3phi13.6/Klebsiella
phage ST437-
OXA245phi4.1
MK433577.1/
MK416011.1 86% 0.0 97.25%
PKp4859-3 51 34,1 40 C3 Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 64% 0.0 95.28%
Pa ien 9 Kp4860
PKp4860-1 52,2 29,7 41 C9
Myo i idae
Klebsiella phage
ST405-OXA48phi1.1 MK388859.1 88% 0.0 85.05%
PKp4860-2 52,2 29,7 41 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
PKp4860-3 51,3 33,5 43 C9 Klebsiella phage
ST405-OXA48phi1.1 MK388859.1 80% 0.0 86.57%
PKp4860-4 50,2 36,7 43 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
PKp4860-6 50,2 36,7 43 C5 Klebsiella phage 2
LV-2017 KY271396.1 36% 0.0 93.97%
PKp4860-7 50,2 36,7 43 C2 Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
Pa ien 10 Kp4861
PKp4861-1 50,2 36,7 43 C1
Myo i idae
Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 74% 0.0 96.13%
PKp4861-2 50,2 36,7 43 C3 Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 55% 0.0 97.6%
PKp4861-3 50,2 36,7 43 N/D
PKp4861-4 50,2 36,7 43 C6 Klebsiella phage 1
LV-2017 KY271401.1 31% 0.0 96.90%
Pa ien 11 Kp4862 PKp4862-1 50,2 36,7 43 C5 Myo i idae Klebsiella phage 2
LV-2017 KY271396.1 46% 0.0 97.25%
Pa ien 13 Kp4864 PKp4864-1 50,2 36,7 43 C5 Myo i idae Klebsiella phage 2
LV-2017 KY271396.1 35% 0.0 85.93%
Pa ien 14 Kp4865
PKp4865-1 50,2 36,7 43 C9
Myo i idae
Klebsiella phage
ST512-KPC3phi13.3 MK422448.1 73% 0.0 97.23%
PKp4865-2 50,2 36,7 43 C2 Klebsiella phage 4
LV-2017 KY271398.1 53% 0.0 98.48%
PKp4865-3 49,9 37 41 C6 Klebsiella phage
KPP5665-2 MF695815.1 25% 0.0 93.71%
PKp4865-4 54,4 35 48 C9 Klebsiella phage
48ST307 KY271402.1 28% 0.0 94.55%
Mic oo ganisms 2021,9, 2252 8 o 21
Table 1. Con .
P ophages
Pa ien s S ains P ophages GC% Leng h
(kbp) CDS Clus e Family Rela ed Phages Accession
Numbe
Que y
Co e E Value Pe .
Iden
Pa ien 15
Kp4866 PKp4866-3 54,4 35 48 N/D
Myo i idae
PKp4866-6 54,4 35 48 N/D
Kp4867
PKp4867-1 54,4 35 48 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4867-2 54,4 35 48 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Kp4868
PKp4868-1 54,4 35 48 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4868-2 54,4 35 48 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Kp4870
PKp4870-1 54,4 35 48 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4870-2 54,4 35 48 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Pa ien 16 Kp4871
PKp4871-1 50,6 39,1 63 C7
Sipho i idae
Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
PKp4871-2 50,6 39,1 63 C2 Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4871-3 50,6 39,1 63 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
Pa ien 17
Kp4872
PKp4872-1 50,6 39,1 63 C7
Sipho i idae
Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
PKp4872-2 50,6 39,1 63 C2 Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4872-3 52 40,9 57 C4 Podo i idae Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
Kp4873
PKp4873-1 50,2 45,9 51 C2
Sipho i idae
Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4873-2 53,2 34,9 32 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
PKp4873-4 55 33,1 45 C7 Myo i idae Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
Kp4874
PKp4874-1 50,4 39,4 49 C2
Sipho i idae
Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4874-2 50,5 39,5 49 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
PKp4874-4 50,5 39,5 49 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
Kp4875
PKp4875-1 50,5 39,5 49 C2 Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4875-2 50,5 39,5 49 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
PKp4875-4 50,5 39,5 49 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
Kp4876
PKp4876-1 50,5 39,5 49 C2 Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4876-2 50,5 39,5 49 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
PKp4876-4 50,5 39,5 49 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
Pa ien 18 Kp4877
PKp4877-1 50,5 39,5 49 C2
Sipho i idae
Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4877-2 50,5 39,5 49 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
PKp4877-4 50,5 39,5 49 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
Pa ien 19
Kp4878
PKp4878-1 50,4 39,7 49 C7
Sipho i idae
Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
PKp4878-2 50,4 40,1 50 C2 Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4878-3 50,4 40,1 50 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
Kp4879
PKp4879-1 50,4 40,1 50 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
PKp4879-2 50,4 40,1 50 C2 Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4879-3 50,4 40,1 50 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
Mic oo ganisms 2021,9, 2252 9 o 21
Table 1. Con .
P ophages
Pa ien s S ains P ophages GC% Leng h
(kbp) CDS Clus e Family Rela ed Phages Accession
Numbe
Que y
Co e E Value Pe .
Iden
Kp4880
PKp4880-1 49,7 39,3 55 C2 Klebsiella phage 4
LV-2017 KY271398.1 100% 0.0 100%
PKp4880-2 50,4 41,5 61 C7 Klebsiella phage 1
LV-2017 KY271401.1 74% 0.0 99.95%
PKp4880-3 50,6 38,6 56 C4 Klebsiella phage 3
LV-2017 KY271397.1 100% 0.0 100%
Pa ien 21 Kp4882
PKp4882-1 51,4 39,8 67 C8
Sipho i idae
Klebsiella phage
ST101-KPC2phi6.3 MK416017.1 23% 0.0 97.40%
PKp4882-2 50,9 42,2 58 C3 Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 70% 0.0 100.00%
PKp4882-3 50,9 42,2 58 C6 Klebsiella phage
ST147-VIM1phi7.2 MK448232.1 85% 0.0 99.99%
Pa ien 23
Kp4884
PKp4884-1 49,8 45,5 60 C8
Sipho i idae
Klebsiella phage
ST101-KPC2phi6.3 MK416017.1 23% 0.0 97.40%
PKp4884-2 51,2 50,6 77 C3 Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 70% 0.0 100.00%
PKp4884-3 52,1 48,6 75 C6 Klebsiella phage
ST147-VIM1phi7.2 MK448232.1 85% 0.0 99.99%
Kp4885
PKp4885-1 52,1 48,6 75 C8 Klebsiella phage
ST101-KPC2phi6.3 MK416017.1 23% 0.0 97.40%
PKp4885-2 51,5 46,8 78 C3 Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 70% 0.0 100.00%
PKp4885-3 53 46,9 68 C6 Klebsiella phage
ST147-VIM1phi7.2 MK448232.1 85% 0.0 99.99%
Pa ien 24 Kp4886
PKp4886-1 53 47,1 68 C8
Sipho i idae
Klebsiella phage
ST101-KPC2phi6.3 MK416017.1 23% 0.0 97.40%
PKp4886-2 52,7 48,4 73 C3 Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 70% 0.0 100.00%
PKp4886-3 54,9 30,5 45 C6 Myo i idae Klebsiella phage
ST147-VIM1phi7.2 MK448232.1 85% 0.0 99.99%
Pa ien 25 Kp4887
PKp4887-1 54,9 30,2 45 C8 Myo i idae Klebsiella phage
ST101-KPC2phi6.3 MK416017.1 23% 0.0 97.40%
PKp4887-2 54 35,2 43 C3 Sipho i idae Klebsiella phage
ST147-VIM1phi7.1 MK416018.1 70% 0.0 100.00%
PKp4887-3 50,9 40,2 53 C6 Podo i idae Klebsiella phage
ST147-VIM1phi7.2 MK448232.1 85% 0.0 99.99%
Pa ien 26
Kp4849
PKp4849-1 50,4 33,4 44 C1
Myo i idae
Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4849-2 50,4 33,4 44 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
Kp4869
PKp4869-1 54,4 35 48 C1 Klebsiella phage
ST16-OXA48phi5.4 MK416015.1 75% 0.0 96.19%
PKp4869-2 54,4 35 48 C2 Klebsiella phage 4
LV-2017 KY271398.1 70% 0.0 97.67%
3.3. Classi ica ion o K. pneumoniae P ophages
In ac p ophages iden i ied we e in silico assigned o a amily axon using he VIRFAM
websi e [44]. Classi ica ion was based on genes conside ed o be he mos indica i e o i s
amily: majo capsid p o ein, la ge e minase subuni , ail ape measu e p o ein and ail
shea h p o ein. All p ophages could be assigned o a amily. The majo i y,
62 (59.6%) was
assigned o he Myo i idae amily, 40 (38.5%) o he Sipho i idae amily, and 2 (1.9%) o
he Podo i idae amily (Figu e 2). This is in acco dance wi h he es ima ed dis ibu ion
desc ibed in he li e a u e [
55
,
56
]. Based on in o ma ion on he Expansy websi e (h p:
// i alzone.expasy.o g/, las accessed July 2021), Myo i idae a e ypically he la ges phages
wi h a high a iabili y in hei genome sizes, anging om 33 o 244 kbp and coding o 40 o
415 p o eins. In his s udy, all p ophages belonging o he Myo i idae amily ha e genomes
wi h an a e age o 34.8 kbp (min 29.7 kbp, max 46.7 kbp) and coding o 45 p o eins (min
40, max 55). The Sipho i idae amily ound in K. pneumoniae has a genome size o a ound
41.3 kbp (min 35.2, max 50.6) and coding o abou 56 p o eins (min 32, max 78), while he
desc ibed genome size o he Sipho i idae amily is abou 50 kbp and encodes o abou
70 genes. He e, Podo i idae ha e he mo e consis en genome size o abou 40.5 kbp (min
Mic oo ganisms 2021,9, 2252 16 o 21
o ques ionable). Incomple e and ques ionable p ophages o en lack essen ial phage
unc ions [
73
] and he e o e ou u he analysis was ocused on in ac p ophages. Only
a ew s udies ha e cha ac e ized he p e alence o p ophages in K. pneumoniae species,
al hough hey a e gene ic elemen s ha signi ican ly con ibu e o genome a iabili y,
e olu ion, and i ulence o hei bac e ial hos s [32,55,56,72,73,75].
In ou analysis, he size o K. pneumoniae p ophage genomes a ied om 8.9 o
60.8 kbp
, wi h an a e age o 37.4 kbp, which ag ees wi h he li e a u e o wha has al eady
been desc ibed o K. pneumoniae and en e obac e ia [
73
,
75
,
76
]. Using an in silico app oach,
among he 104 in ac p ophages, we ound Myo i idae o be he mos ep esen ed amily
(59.6%), ollowed by Sipho i idae (38.5%) and Podo i idae (1.9%). The same dis ibu ion was
obse ed in o he s udies [
55
,
56
]. Myo i idae, which a e ypically he la ges phage amily,
had size genomes below a e age, while Sipho i idae had sizes sligh ly abo e he a e age.
PHASTER analysis o d a genomes (especially i hese we e dis ibu ed h ough di e en
con igs) could e oneously delimi p ophages, o which all p ophage sequences analysed
we e manually cu a ed. Al hough we ha e manually cu a ed he p ophage inse ion si es
and sca olded p ophages ha we e spli in se e al con igs, his may esul in unexpec ed
o a iable genome sizes. These di e ences may also esul om he acquisi ion o bac e ial
genes adjacen o he p ophage du ing epea ed excision and in eg a ion cycles o loss and
gene ic deg ada ion ha caused he educ ion o i s genome size [76].
P ophages o K. pneumoniae we e ound o be g ea ly simila . Ou compa ison o
104 in ac p ophages
e ealed ha some p ophages sha ed mo e han 50% genome iden i y,
indica ing s ong e olu iona y ela ionships. Compa ing ou p ophage genomes agains
public da abases, we ound 17 Klebsiella phages which sha e simila i y wi h he 104 in ac
p ophages in e ms o que y co e age and iden i y (in some cases highe han 60%) and
his helped us o iden i y nine clus e s composed by iden ical p ophages. Mo eo e , ela ed
clus e s end o g oup p ophages o he same amily. Howe e , a same ime, he p ophages
we e also g ea ly di e si ied. A compa ison o hese nine clus e s e ealed less han 30% o
genome iden i y and we ound a Pseudomonas phage VW-6B ha sha es an iden i y highe
han 31% wi h one o he p ophages iden i ied he e. This may indica e ha he common
ances o o hese wo species was in ec ed by a phage ha co-e ol ed wi h he hos bac e ia
du ing specia ion, o by phage ans e be ween species.
K. pneumoniae p ophages ha e an open pan-genome, meaning ha o each new
p ophage genome added, new genes con ibu e o he pan-genome. Thus, he inclusion o
mo e p ophages is expec ed o aise he numbe o he pan-genome size o 892 genes so a
de e mined, which is also co obo a ed by he high pe cen age o single on genes (43.6%,
389/892). On he o he hand, he educed numbe o co e genes poin s o high sequence
di e si y, only p ese ing essen ial s uc u al genes.
Some p ophages ca y genes ha can al e he ea u es o he hos , anging om in-
c eased hos i ness o inc eased i ulence, and many s udies ha e epo ed he connec ion
o he pa hogen i ulence o he acquisi ion o p ophages [
21
,
77
]. In ac , e en de ec i e
p ophages a e conside ed as po en ial mobile elemen s ca ying i ulence ac o s [
25
].
Thus, al hough open-access esea ch ools did no ind i ulence ac o s, a de ailed analy-
sis showed o he wise, e ealing se e al po en ial i ulence ac o s ha can be ela ed o
bac e ia i ness and in luence he abili y o he bac e ium o colonize i s hos and su i e in
ad e se en i onmen s.
P ophages and hei bac e ial hos s ha e common e olu iona y in e es s since he p o-
li e a ion o he hos also esul s in inc eased p ophage popula ion. Thus, some p ophages
p o ide he bac e ium bene icial ai s, such as inc eased i ness, and con e new i u-
lence ac o s and/o an ibio ic esis ance genes exploi ed o bac e ial pa hogenesis [
25
,
76
].
Acco dingly, we iden i ied se e al pu a i e i ulence ac o s, such as T aR/DksA amily
ansc ip ional egula o , memb ane-associa ed lipop o ein, molecula chape one DnaJ
and o he p o eins wi h unc ions in pe sis ence unde s ess condi ions, in e ac ion wi h
hos cells and egula ion o i ulence gene exp ession. T aR amily egula o s may also
play a ole in p ophage p opaga ion by in e e ing wi h he hos mechanisms o egula ion,

Mic oo ganisms 2021,9, 2252 17 o 21
inc easing he bac e ial conjuga ion, and imp o ing he ansmission o he p ophage by
la e al gene ans e be ween bac e ia [
59
]. Taken oge he , K. pneumoniae may bene i om
ca ying a p ophage due o he pu a i e bene icial genes ca ied by hem. Thus, K. pneumo-
niae p ophages may con e an e olu iona y i ness bene i o he hos due o he p esence o
i ulence ac o s, which should be u he s udy. On he o he hand, ou s udy con i med
he p e ious desc ip ion by Pe digão e al. (2020), whe e pa e ns o esis ance-con e ing
genes ela ed o an imic obial esis ance we e associa ed wi h ch omosomal mu a ions and
plasmid mobiliza ion [
35
]. The p ophages sequences desc ibed he e did no con ain he
genes esponsible by an ibio ic esis ance in hese K. pneumoniae isola es.
P ophages ha e coe ol ed wi h bac e ia o mo e han a billion yea s and ha e de-
eloped e icien s a egies o lyse and hus kill hei bac e ial hos a he end o he ly ic
cycle o p ogeny elease [
16
]. Endolysins a e p o eins used in his ly ic p ocess and
ha e been used in many scien i ic wo ks o he de elopmen o an ibac e ial he apeu-
ics
[71,78–80]
. Depending on clea age si es, hey can be ca ego ized in o ou di e en
g oups: (a) N-ace ylmu amidases (lysozymes), (b) N-ace yl-
β
-D-glucosaminidases (gly-
cosidases) (c) N-ace ylmu amoyl-L-alanine amidases and (d) endopep idases [
71
,
81
]. In
his s udy, we exploi ed he K. pneumoniae sequenced genomes o iden i y endolysins in
hei p ophages and cha ac e ized hem. We iden i ied 132 endolysins (115 endolysins
om ou p ophage genomes and 17 lysins ha bo ed by he mos ela ed phages and he en-
dolysins we e assigned o h ee g oups: lysozymes/mu amidases, glycosidases/chi inases
and endopep idases. The g oup o lysozymes included endolysins ela ed o endolysin
LyzP1 and R21, which a e wo lysozymes o phages P1 and 21, espec i ely, and we e he
i s endolysins showing a signal-ancho - elease (SAR) domain [
82
]. In con as o phage
lysozymes like T4, which accumula e a ully olded and enzyma ically ac i e endolysin in
he cy oplasm, SAR endolysins a e he i s endolysins sec e ed as enzyma ically inac i e
o m ancho ed o he memb ane by he N- e minal SAR domain o a oid p ema u e lysis
o he in ec ed hos [
83
]. The g oup o chi inases a e membe s o glycoside hyd olase
(GH) amily 18 and 19 and a e also a b oad, lysozyme-like supe amily clea ing chi in,
which is he second mos abundan biopolyme on he plane and is a linea , insoluble
homopolyme composed o
β
-1,4 linked subuni s o N-ace yl glucosamine polyme s, a
s uc u e uncommon in bac e ial cell walls. Mos o he bac e ial chi inases isola ed and
sequenced so a a e included in GH amily 18, ha e a molecula weigh ange o 20–60 kDa
and a e smalle han plan chi inases (40–85 kDa) [
70
], which ag ees wi h he molecula
weigh p edic ed he e. The hi d g oup included endopep idases, bu his g oup was only
iden i ied in he ela ed Klebsiella phages, 48ST307 and ST846-OXA48phi9.1.
Ou s udy shows ha he di e si y o K. pneumoniae p ophages is as and con inues
o expand. In addi ion, his genome analysis se es as a basis o he cha ac e iza ion and
e olu iona y ela ionship o p ophages ha bo ed by K. pneumoniae and iden i ica ion o
ele an p o eins, such as endolysins, which may ha e biomedical applica ions o new and
p o ein-based an imic obials. Fu u e wo k will ocus on cloning and cha ac e izing K. pneu-
moniae endolysins and s udying hei lysis ac i i y and po en ial as bac e icidal p oduc s.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/10
.3390/mic oo ganisms9112252/s1. Figu e S1: MAFFT alignmen o whole 104 genomic p ophage
sequences om K. pneumoniae. Anno a ed p ophages which sha e homology wi h ou sequences we e
included. Da ke zones indica e highe iden i y. Clus e s o p ophages wi h iden i ies highe han
50% a e indica ed and numbe ed. Myo i idae, g een; Sipho i idae, blue; and mixed-clus e , yellow;
Figu e S2: Genomic ea u e o he in ac p ophage Kp4852-1 in ela ion o i s Type VI Sec e ion Sys em
componen (blue) along he downs eam egions o he p ophage inse ion si e; Figu e S3: MAFFT
alignmen o 115 p ophage endolysins (A) nucleo ides and (B) amino acids sequences ex ac ed
om K. pneumoniae p ophage sequences and 17 phage endolysins sequences ex ac ed om Klebsiella
which sha e homology wi h ou sequences. Da ke zones indica e highe iden i y: black, 90%;
da k-g ey, 70%, g ey, 50%, and ligh -g ey, 30%; Figu e S4: Ances y cha o (A) lysozyme ac i i y
(GO:0003796), (B) chi inase ac i i y (GO:0004568) and (C) endopep idase ac i i y (GO:0004175).
Table S1.
Genomic de ails o he 150 iden i ied p ophages; Table S2: P ophages classi ica ion (in ac ,
Mic oo ganisms 2021,9, 2252 18 o 21
incomple e, ques ionable). GC, guanine–cy osine. De ec i e, incomple e + ques ionable; Table S3: K.
pneumoniae p ophages pan-genome de e mined wi h Roa y o a p o ein iden i y o 40%. P ophage
genes p esen in mo e han hal o he genomes a e in bold; Table S4: Vi ulence and i ness ac o s
iden i ied o 150 p ophages o K. pneumoniae s ains; Table S5: Endolysins cha ac e iza ion.
Au ho Con ibu ions:
Concep ualiza ion, F.F.V. and A.T.M.; me hodology, F.F.V. and A.T.M.; o mal
analysis, A.T.M., F.F.V. and L.T.; in es iga ion, F.F.V., A.T.M. and J.M.B.V.; esou ces, F.F.V., A.T.M.,
A.D. and L.G.; da a cu a ion, F.F.V. and A.T.M.; w i ing—o iginal d a p epa a ion, A.T.M. and F.F.V.;
w i ing— e iew and edi ing, A.T.M., F.F.V., J.M.B.V., A.D., L.G. and L.T.; isualiza ion, A.T.M., F.F.V.,
J.M.B.V., A.D. and L.T.; supe ision, F.F.V.; p ojec adminis a ion, F.F.V.; unding acquisi ion, F.F.V.
All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
F.F.V. is unded by Fundação pa a a Ciência e a Tecnologia (FCT) h ough an Assis-
an Resea che g an CEECIND/03023/2017, and a p ojec g an (PTDC/BTM-SAL/28978/2017)
ha suppo ed his wo k. The wo k is pa ially suppo ed by Na ional unds om FCT, p ojec s
UIDB/04138/2020 and UIDP/04138/2020.
Da a A ailabili y S a emen :
Da a a e con ained wi hin he a icle o supplemen a y da a. In ac
p ophage sequences we e deposi in GenBank unde he accession OK490394-OK490474.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Re e ences
1.
Podschun, R.; Ullmann, U. Klebsiella spp. as nosocomial pa hogens: Epidemiology, axonomy, yping me hods, and pa hogenici y
ac o s. Clin. Mic obiol. Re . 1998,11, 589–603. [C ossRe ] [PubMed]
2.
Wy es, K.L.; Hol , K.E. Klebsiella pneumoniae popula ion genomics and an imic obial- esis an clones. T ends Mic obiol.
2016
,24,
944–956. [C ossRe ]
3.
Shon, A.S.; Bajwa, R.P.S.; Russo, T.A. Hype i ulen (hype muco iscous) Klebsiella pneumoniae.Vi ulence
2013
,4, 107–118.
[C ossRe ]
4.
He idge, W.P.; Shibu, P.; O’Shea, J.; B ook, T.C.; Hoyles, L. Bac e iophages o Klebsiella spp., hei di e si y and po en ial
he apeu ic uses. J. Med. Mic obiol. 2020,69, 176–194. [C ossRe ] [PubMed]
5.
Coo adia, Y.M.; Johnson, A.P.; Bhana, R.H.; Hu chinson, G.R.; Geo ge, R.C.; Ha e jee, I.E. Mul i esis an Klebsiella pneumoniae in a
neona al nu se y: The impo ance o main enance o in ec ion con ol policies and p ocedu es in he p e en ion o ou b eaks. J.
Hosp. In ec . 1992,22, 197–205. [C ossRe ]
6.
Pendle on, J.N.; Go man, S.P.; Gilmo e, B.F. Clinical ele ance o he ESKAPE pa hogens. Expe Re . An i. In ec . The .
2013
,11,
297–308. [C ossRe ]
7.
Moolchandani, K. An imic obial esis ance su eillance among in ensi e ca e uni s o a e ia y ca e hospi al in sou h India. J.
Clin. Diagn. Res. 2017,11, DC01–DC07. [C ossRe ] [PubMed]
8.
Baue n eind, A.; Ebe lein, E.; Holley, M.; Schweigha , S.; Rosen hal, E. Sp ead o Klebsiella pneumoniae p oducing SHV-5
be a-lac amase among hospi alized pa ien s. In ec ion 1993,21, 18–22. [C ossRe ] [PubMed]
9.
De Champs, C.; Rouby, D.; Guelon, D.; Si o , J.; Si o , D.; Bey ou , D.; Gou gand, J.M. A case-con ol s udy o an ou b eak
o in ec ions caused by Klebsiella pneumoniae s ains p oducing CTX-1 (TEM-3) be a-lac amase. J. Hosp. In ec .
1991
,18, 5–13.
[C ossRe ]
10.
G undmann, H.; Glasne , C.; Albige , B.; Aanensen, D.M.; Tomlinson, C.T.; And ase i´c, A.T.; Can ón, R.; Ca meli, Y.; F ied ich,
A.W.; Giske, C.G.; e al. Occu ence o ca bapenemase-p oducing Klebsiella pneumoniae and Esche ichia coli in he Eu opean su ey
o ca bapenemase-p oducing En e obac e iaceae (EuSCAPE): A p ospec i e, mul ina ional s udy. Lance In ec . Dis.
2017
,17,
153–163. [C ossRe ]
11.
B ad o d, P.A.; U ban, C.; Ma iano, N.; P ojan, S.J.; Rahal, J.J.; Bush, K. Imipenem esis ance in Klebsiella pneumoniae is associa ed
wi h he combina ion o ACT-1, a plasmid-media ed AmpC
β
-lac amase, and he loss o an ou e memb ane p o ein. An imic ob.
Agen s Chemo he . 1997,41, 563–569. [C ossRe ] [PubMed]
12. Two , F.W. An in es iga ion on he na u e o ul a-mic oscopic i uses. Lance 1915,186, 1241–1243. [C ossRe ]
13.
D’Hé elle, F. On an in isible mic obe an agonis ic o dysen e y bacilli. No e by M. F. d’He elle, p esen ed by M. Roux. Comp es
Rendus Academie des Sciences 1917; 165:373–5. Bac e iophage 2011,1, 3–5. [C ossRe ]
14. Sulak elidze, A.; Ala idze, Z.; Mo is, J.G. Bac e iophage The apy. An imic ob. Agen s Chemo he . 2001,45, 649–659. [C ossRe ]
15. Deba is a, J. Phage he apy: Whe e eas mee s wes . Expe Re . An i. In ec . The . 2004,2, 815–819. [C ossRe ]
16.
Lin, D.M.; Koskella, B.; Lin, H.C. Phage he apy: An al e na i e o an ibio ics in he age o mul i-d ug esis ance. Wo ld J.
Gas oin es . Pha macol. The . 2017,8, 162. [C ossRe ] [PubMed]
17.
DeLeo, F.R.; Chen, L.; Po cella, S.F.; Ma ens, C.A.; Kobayashi, S.D.; Po e , A.R.; Cha da, K.D.; Jacobs, M.R.; Ma hema, B.; Olsen,
R.J.; e al. Molecula dissec ion o he e olu ion o ca bapenem- esis an mul ilocus sequence ype 258 Klebsiella pneumoniae.P oc.
Na l. Acad. Sci. USA 2014,111, 4988–4993. [C ossRe ]
Mic oo ganisms 2021,9, 2252 19 o 21
18.
Chen, L.; Cha da, K.D.; DeLeo, F.R.; B yan , K.A.; Jacobs, M.R.; Bonomo, R.A.; K eiswi h, B.N. Genome sequence o a Klebsiella
pneumoniae sequence ype 258 isola e wi h p ophage-encoded K. pneumoniae Ca bapenemase. Genome Announc.
2015
,3, e00659-15.
[C ossRe ]
19.
Kwon, T.; Jung, Y.-H.; Lee, S.; Yun, M.; Kim, W.; Kim, D.-W. Compa a i e genomic analysis o Klebsiella pneumoniae subsp.
pneumoniae KP617 and Pi NDM01, NUHL24835, and ATCC BAA-2146 e eals unique e olu iona y his o y o his s ain. Gu
Pa hog. 2016,8, 34. [C ossRe ]
20.
Fokine, A.; Rossmann, M.G. Molecula a chi ec u e o ailed double-s anded DNA phages. Bac e iophage
2014
,4, e28281.
[C ossRe ]
21.
Fo ie , L.-C.; Sekulo ic, O. Impo ance o p ophages o e olu ion and i ulence o bac e ial pa hogens. Vi ulence
2013
,4, 354–365.
[C ossRe ]
22.
Dennehy, J.J.; Abedon, S.T. Bac e iophage ecology. In Bac e iophages; Sp inge In e na ional Publishing: Cham, Swi ze land, 2020;
pp. 1–42. [C ossRe ]
23.
Feine , R.; A go , T.; Rabino ich, L.; Sigal, N.; Bo o ok, I.; He sko i s, A.A. A new pe spec i e on lysogeny: P ophages as ac i e
egula o y swi ches o bac e ia. Na . Re . Mic obiol. 2015,13, 641–650. [C ossRe ] [PubMed]
24.
Olszak, T.; La ka, A.; Roszniowski, B.; Val ano, M.A.; D ulis-Kawa, Z. Phage li e cycles behind bac e ial biodi e si y. Cu . Med.
Chem. 2017,24, 3987–4001. [C ossRe ] [PubMed]
25.
Wang, X.; Kim, Y.; Ma, Q.; Hong, S.H.; Pokusae a, K.; S u ino, J.M.; Wood, T.K. C yp ic p ophages help bac e ia cope wi h
ad e se en i onmen s. Na . Commun. 2010,1, 147. [C ossRe ] [PubMed]
26. Young, R. Bac e iophage lysis: Mechanism and egula ion. Mic obiol. Re . 1992,56, 430–481. [C ossRe ] [PubMed]
27.
Maciejewska, B.; Olszak, T.; D ulis-Kawa, Z. Applica ions o bac e iophages e sus phage enzymes o comba and cu e bac e ial
in ec ions: An ambi ious and also a ealis ic applica ion? Appl. Mic obiol. Bio echnol. 2018,102, 2563–2581. [C ossRe ] [PubMed]
28.
Bo ysowski, J.; Webe -D ˛ab owska, B.; Gó ski, A. Bac e iophage endolysins as a no el class o an ibac e ial agen s. Exp. Biol. Med.
2006,231, 366–377. [C ossRe ] [PubMed]
29.
Rome o-Calle, D.; Guima ães Bene ides, R.; Góes-Ne o, A.; Billing on, C. Bac e iophages as al e na i es o an ibio ics in clinical
ca e. An ibio ics 2019,8, 138. [C ossRe ]
30.
De Miguel, T.; Rama, J.L.R.; Siei o, C.; Sánchez, S.; Villa, T.G. Bac e iophages and lysins as possible al e na i es o ea
an ibio ic- esis an u ina y ac in ec ions. An ibio ics 2020,9, 466. [C ossRe ] [PubMed]
31.
Rios, A.C.; Mou inho, C.G.; Pin o, F.C.; Del Fiol, F.S.; Jozala, A.; Chaud, M.V.; Vila, M.M.D.C.; Teixei a, J.A.; Balcão, V.M.
Al e na i es o o e coming bac e ial esis ances: S a e-o - he-a . Mic obiol. Res. 2016,191, 51–80. [C ossRe ]
32.
Maciejewska, B.; Roszniowski, B.; Espailla , A.; K˛esik-Szeloch, A.; Majkowska-Sk obek, G.; K opinski, A.M.; B ie s, Y.; Ca a, F.;
La igne, R.; D ulis-Kawa, Z. Klebsiella phages ep esen ing a no el clade o i uses wi h an unknown DNA modi ica ion and
bio echnologically in e es ing enzymes. Appl. Mic obiol. Bio echnol. 2017,101, 673–684. [C ossRe ]
33.
Walmagh, M.; Boczkowska, B.; G ymonp ez, B.; B ie s, Y.; D ulis-Kawa, Z.; La igne, R. Cha ac e iza ion o i e no el endolysins
om G am-nega i e in ec ing bac e iophages. Appl. Mic obiol. Bio echnol. 2013,97, 4369–4375. [C ossRe ]
34.
Blasco, L.; Amb oa, A.; T as oy, R.; Ble io , I.; Moscoso, M.; Fe nández-Ga cia, L.; Pe ez-Nadales, E.; Fe nández-Cuenca, F.;
To e-Cisne os, J.; O eo-Iglesias, J.; e al.
In i o
and
in i o
e icacy o combina ions o colis in and di e en endolysins agains
clinical s ains o mul i-d ug esis an pa hogens. Sci. Rep. 2020,10, 7163. [C ossRe ] [PubMed]
35.
Pe digão, J.; Modes o, A.; Pe ei a, A.L.; Ne o, O.; Ma os, V.; Godinho, A.; Phelan, J.; Cha les on, J.; Spada , A.; de Sessions, P.F.;
e al. Whole-genome sequencing esol es a polyclonal ou b eak by ex ended-spec um be a-lac am and ca bapenem- esis an
Klebsiella pneumoniae in a Po uguese e ia y-ca e hospi al. Mic ob. Genom. 2019,7, mgen000349. [C ossRe ]
36.
A nd , D.; G an , J.R.; Ma cu, A.; Sajed, T.; Pon, A.; Liang, Y.; Wisha , D.S. PHASTER: A be e , as e e sion o he PHAST
phage sea ch ool. Nucleic Acids Res. 2016,44, W16–W21. [C ossRe ] [PubMed]
37.
Song, W.; Sun, H.X.; Zhang, C.; Cheng, L.; Peng, Y.; Deng, Z.; Wang, D.; Wang, Y.; Hu, M.; Liu, W.; e al. P ophage hun e : An
in eg a i e hun ing ool o ac i e p ophages. Nucleic Acids Res. 2019,47, W74–W80. [C ossRe ] [PubMed]
38.
B e in, T.; Da is, J.J.; Disz, T.; Edwa ds, R.A.; Ge des, S.; Olsen, G.J.; Olson, R.; O e beek, R.; Pa ello, B.; Pusch, G.D.; e al.
RAST k: A modula and ex ensible implemen a ion o he RAST algo i hm o building cus om anno a ion pipelines and
anno a ing ba ches o genomes. Sci. Rep. 2015,5, 8365. [C ossRe ]
39.
O e beek, R.; Olson, R.; Pusch, G.D.; Olsen, G.J.; Da is, J.J.; Disz, T.; Edwa ds, R.A.; Ge des, S.; Pa ello, B.; Shukla, M.; e al. The
SEED and he Rapid Anno a ion o mic obial genomes using Subsys ems Technology (RAST). Nucleic Acids Res.
2014
,42, 206–214.
[C ossRe ] [PubMed]
40.
Aziz, R.K.; Ba els, D.; Bes , A.; DeJongh, M.; Disz, T.; Edwa ds, R.A.; Fo msma, K.; Ge des, S.; Glass, E.M.; Kubal, M.; e al. The
RAST Se e : Rapid anno a ions using subsys ems echnology. BMC Genom. 2008,9, 75. [C ossRe ] [PubMed]
41.
Vale, F.; Lehou s, P. Rela ing phage genomes o Helicobac e pylo i popula ion s uc u e: Gene al s eps using whole-genome
sequencing da a. In . J. Mol. Sci. 2018,19, 1831. [C ossRe ]
42.
Bo a yn, G.M.; Camacho, C.; Coope , P.S.; Coulou is, G.; Fong, A.; Ma, N.; Madden, T.L.; Ma en, W.T.; McGinnis, S.D.; Me ezhuk,
Y.; e al. BLAST: A mo e e icien epo wi h usabili y imp o emen s. Nucleic Acids Res. 2013,41, 29–33. [C ossRe ] [PubMed]
43.
Kelley, L.A.; Mezulis, S.; Ya es, C.M.; Wass, M.N.; S e nbe g, M.J.E. The Phy e2 web po al o p o ein modelling, p edic ion and
analysis. Na . P o oc. 2015,10, 845–858. [C ossRe ] [PubMed]
Mic oo ganisms 2021,9, 2252 20 o 21
44.
Lopes, A.; Ta a es, P.; Pe i , M.-A.; Gué ois, R.; Zinn-Jus in, S. Au oma ed classi ica ion o ailed bac e iophages acco ding o hei
neck o ganiza ion. BMC Genom. 2014,15, 1027. [C ossRe ] [PubMed]
45.
McNai , K.; Bailey, B.A.; Edwa ds, R.A. PHACTS, a compu a ional app oach o classi ying he li es yle o phages. Bioin o ma ics
2012,28, 614–618. [C ossRe ] [PubMed]
46.
Page, A.J.; Cummins, C.A.; Hun , M.; Wong, V.K.; Reu e , S.; Holden, M.T.G.; Fookes, M.; Falush, D.; Keane, J.A.; Pa khill, J.
Roa y: Rapid la ge-scale p oka yo e pan genome analysis. Bioin o ma ics 2015,31, 3691–3693. [C ossRe ] [PubMed]
47.
Yaha a, K.; Lehou s, P.; Vale, F.F. Analysis o gene ic ecombina ion and he pan-genome o a highly ecombinogenic bac e iophage
species. Mic ob. Genom. 2019,5, e000282. [C ossRe ] [PubMed]
48.
Da is, J.J.; Wa am, A.R.; Aziz, R.K.; B e in, T.; Bu le , R.; Bu le , R.M.; Chlenski, P.; Con ad, N.; Dicke man, A.; Die ich,
E.M.; e al. The PATRIC bioin o ma ics esou ce cen e : Expanding da a and analysis capabili ies. Nucleic Acids Res.
2020
,48,
D606–D612. [C ossRe ]
49.
Ka oh, K.; S andley, D.M. MAFFT mul iple sequence alignmen so wa e e sion 7: Imp o emen s in pe o mance and usabili y.
Mol. Biol. E ol. 2013,30, 772–780. [C ossRe ] [PubMed]
50.
P ice, M.N.; Dehal, P.S.; A kin, A.P. Fas T ee 2—App oxima ely maximum-likelihood ees o la ge alignmen s. PLoS ONE
2010
,
5, e9490. [C ossRe ] [PubMed]
51.
Le unic, I.; Bo k, P. In e ac i e ee o li e (iTOL) 5: An online ool o phylogene ic ee display and anno a ion. Nucleic Acids
Res. 2021,49, W293–W296. [C ossRe ]
52.
Wa e house, A.; Be oni, M.; Biene , S.; S ude , G.; Tau iello, G.; Gumienny, R.; Hee , F.T.; de Bee , T.A.P.; Remp e , C.; Bo doli,
L.; e al. SWISS-MODEL: Homology modelling o p o ein s uc u es and complexes. Nucleic Acids Res.
2018
,46, W296–W303.
[C ossRe ] [PubMed]
53.
Law ence, J.G.; Hend ix, R.W.; Casjens, S. Whe e a e he pseudogenes in bac e ial genomes? T ends Mic obiol.
2001
,9, 535–540.
[C ossRe ]
54. Kuo, C.-H.; Ochman, H. The ex inc ion dynamics o bac e ial pseudogenes. PLoS Gene . 2010,6, e1001050. [C ossRe ]
55.
Baliga, P.; Sheka , M.; Kallappa, G.S. Genome-wide iden i ica ion and analysis o ch omosomally in eg a ed pu a i e p ophages
associa ed wi h clinical Klebsiella pneumoniae S ains. Cu . Mic obiol. 2021,78, 2015–2024. [C ossRe ] [PubMed]
56.
Ble io , I.; T as oy, R.; Blasco, L.; Fe nández-Cuenca, F.; Amb oa, A.; Fe nández-Ga cía, L.; Pacios, O.; Pe ez-Nadales, E.; To e-
Cisne os, J.; O eo-Iglesias, J.; e al. Genomic analysis o 40 p ophages loca ed in he genomes o 16 ca bapenemase-p oducing
clinical s ains o Klebsiella pneumoniae.Mic ob. Genom. 2020,6, e000369. [C ossRe ] [PubMed]
57.
Cos a, A.R.; Mon ei o, R.; Aze edo, J. Genomic analysis o Acine obac e baumannii p ophages e eals ema kable di e si y and
sugges s p o ound impac on bac e ial i ulence and i ness. Sci. Rep. 2018,8, 15346. [C ossRe ] [PubMed]
58.
Gi a d, M.E.; Gopalk ishnan, S.; G ace, E.D.; Halliday, J.A.; Gou se, R.L.; He man, C. DksA and ppGpp egula e he
σ
S s ess
esponse by ac i a ing p omo e s o he small RNA Ds A and he an i-adap e p o ein I aP. J. Bac e iol.
2018
,200, e00463-17.
[C ossRe ]
59.
Blankschien, M.D.; Po ykus, K.; G ace, E.; Choudha y, A.; Vinella, D.; Cashel, M.; He man, C. T aR, a homolog o a RNAP
seconda y channel in e ac o , modula es ansc ip ion. PLoS Gene . 2009,5, e1000345. [C ossRe ] [PubMed]
60.
Ko acs-Simon, A.; Ti ball, R.W.; Michell, S.L. Lipop o eins o bac e ial pa hogens. In ec . Immun.
2011
,79, 548–561. [C ossRe ]
[PubMed]
61.
Cui, J.; Ma, C.; Ye, G.; Shi, Y.; Xu, W.; Zhong, L.; Wang, J.; Yin, Y.; Zhang, X.; Wang, H. DnaJ (hsp40) o S ep ococcus pneumoniae
is in ol ed in bac e ial i ulence and elici s a s ong na u al immune eac ion ia PI3K/JNK. Mol. Immunol.
2017
,83, 137–146.
[C ossRe ] [PubMed]
62.
Zeng, B.; Wang, C.; Zhang, P.; Guo, Z.; Chen, L.; Duan, K. Hea shock p o ein DnaJ in Pseudomonas ae uginosa a ec s bio ilm
o ma ion ia pyocyanin p oduc ion. Mic oo ganisms 2020,8, 395. [C ossRe ] [PubMed]
63.
Kim, J.-S.; Liu, L.; Vázquez-To es, A. The DnaK/DnaJ chape one sys em enables RNA polyme ase-DksA complex o ma ion in
Salmonella expe iencing oxida i e s ess. MBio 2021,12, e03443-20. [C ossRe ]
64.
Gancz, A.; Kond a ye a, K.; Cohen-Eli, D.; Na on-Venezia, S. Genomics and i ulence o Klebsiella pneumoniae Kpnu95 ST1412
ha bo ing a no el Inc plasmid encoding Blac x-M-15 and Qn s1 causing communi y u ina y ac in ec ion. Mic oo ganisms
2021
,
9, 1022. [C ossRe ] [PubMed]
65.
Cano a, M.J.; Molle, V. Bac e ial se ine/ h eonine p o ein kinases in hos -pa hogen in e ac ions. J. Biol. Chem.
2014
,289,
9473–9479. [C ossRe ] [PubMed]
66.
Ren, J.; Sang, Y.; Lu, J.; Yao, Y.-F. P o ein ace yla ion and i s ole in bac e ial i ulence. T ends Mic obiol.
2017
,25, 768–779.
[C ossRe ]
67.
Sh i as a a, S.; Mande, S.S. Iden i ica ion and unc ional cha ac e iza ion o gene componen s o ype VI sec e ion sys em in
bac e ial genomes. PLoS ONE 2008,3, e2955. [C ossRe ] [PubMed]
68.
Shyn um, D.; Ven e , S.; Moleleki, L.; To h, I.; Cou inho, T. Compa a i e genomics o ype VI sec e ion sys ems in s ains o
Pan oea anana is om di e en en i onmen s. BMC Genom. 2014,15, 163. [C ossRe ]
69.
Wilson, C.A.; K eychman, J.; Ge s ein, M. Assessing anno a ion ans e o genomics: Quan i ying he ela ions be ween p o ein
sequence, s uc u e and unc ion h ough adi ional and p obabilis ic sco es. J. Mol. Biol.
2000
,297, 233–249. [C ossRe ] [PubMed]
70.
Ja ed, S.; Ahmad, M.; Ahmad, M.; Abdin, M.; Hamid, R.; Khan, M.; Musa a , J. Chi inases: An upda e. J. Pha m. Bioallied Sci.
2013,5, 21. [C ossRe ] [PubMed]
Mic oo ganisms 2021,9, 2252 21 o 21
71.
Schmelche , M.; Dono an, D.M.; Loessne , M.J. Bac e iophage endolysins as no el an imic obials. Fu u e Mic obiol.
2012
,7,
1147–1171. [C ossRe ]
72.
Shen, J.; Zhou, J.; Xu, Y.; Xiu, Z. P ophages con ibu e o genome plas ici y o Klebsiella pneumoniae and may in ol e he
ch omosomal in eg a ion o ARGs in CG258. Genomics 2020,112, 998–1010. [C ossRe ]
73.
De Sousa, J.A.M.; Bu e , A.; Haudique , M.; Rocha, E.P.C.; Rendueles, O. Modula p ophage in e ac ions d i en by capsule
se o ype selec o capsule loss unde phage p eda ion. ISME J. 2020,14, 2980–2996. [C ossRe ] [PubMed]
74.
Kopo sa, K.; Mbelle, N.M.; Osei Sekye e, J. Epigenomics, genomics, esis ome, mobilome, i ulome and e olu iona y phy-
logenomics o ca bapenem- esis an Klebsiella pneumoniae clinical s ains. Mic ob. Genom.
2020
,6, mgen000474. [C ossRe ]
[PubMed]
75.
Wang, F.; Wang, D.; Hou, W.; Jin, Q.; Feng, J.; Zhou, D. E olu iona y di e si y o p ophage DNA in Klebsiella pneumoniae
ch omosomes. F on . Mic obiol. 2019,10, 2840. [C ossRe ] [PubMed]
76.
Bobay, L.-M.; Touchon, M.; Rocha, E.P.C. Pe asi e domes ica ion o de ec i e p ophages by bac e ia. P oc. Na l. Acad. Sci. USA
2014,111, 12127–12132. [C ossRe ] [PubMed]
77.
Hayashi, T. Comple e genome sequence o en e ohemo hagic Eschelichia coli O157:H7 and genomic compa ison wi h a labo a o y
s ain K-12. DNA Res. 2001,8, 11–22. [C ossRe ] [PubMed]
78.
Ma suzaki, S.; Rashel, M.; Uchiyama, J.; Saku ai, S.; Ujiha a, T.; Ku oda, M.; Imai, S.; Ikeuchi, M.; Tani, T.; Fujieda, M.; e al.
Bac e iophage he apy: A e i alized he apy agains bac e ial in ec ious diseases. J. In ec . Chemo he .
2005
,11, 211–219.
[C ossRe ]
79. Ghose, C.; Eule , C.W. G am-nega i e bac e ial lysins. An ibio ics 2020,9, 74. [C ossRe ]
80. Fische i, V.A. Bac e iophage lysins as e ec i e an ibac e ials. Cu . Opin. Mic obiol. 2008,11, 393–400. [C ossRe ]
81.
Fen on, M.; McAuli e, O.; O’Mahony, J.; Co ey, A. Recombinan bac e iophage lysins as an ibac e ials. Bioeng. Bugs
2010
,1, 9–16.
[C ossRe ] [PubMed]
82.
Xu, M.; S uck, D.K.; Dea on, J.; Wang, I.-N.; Young, R. A signal-a es - elease sequence media es expo and con ol o he phage
P1 endolysin. P oc. Na l. Acad. Sci. USA 2004,101, 6415–6420. [C ossRe ] [PubMed]
83.
Sun, Q.; Ku y, G.F.; A ockiasamy, A.; Xu, M.; Young, R.; Sacche ini, J.C. Regula ion o a mu aly ic enzyme by dynamic memb ane
opology. Na . S uc . Mol. Biol. 2009,16, 1192–1194. [C ossRe ] [PubMed]