1
T ends in Helicobac e pylo i esis ance o cla i h omycin: om
pheno ypic o genomicapp oaches
And eia T.Ma ques1, Jo ge M. B.Ví o 1,2, And eaSan os3, MónicaOleas o3 and Filipa F.Vale1,*
MINI REVIEW
Ma ques e al., Mic obial Genomics 2020;6
DOI 10.1099/mgen.0.000344
Recei ed 09 Augus 2019; Accep ed 10 Feb ua y 2020; Published 02 Ma ch 2020
Au ho a ilia ions: 1Hos –Pa hogen In e ac ions Uni , Resea ch Ins i u e o Medicines (iMed- ULisboa), Facul y o Pha macy, Uni e sidade de Lisboa,
Lisboa, Po ugal; 2Depa men o Biochemis y and Human Biology, Facul y o Pha macy, Uni e sidade de Lisboa, 1649 003 Lisbon, Po ugal; 3Na ional
Re e ence Labo a o y o Gas oin es inal In ec ions, Depa men o In ec ious Diseases, Na ional Ins i u e o Heal h D Rica do Jo ge, Lisbon, Po ugal.
*Co espondence: Filipa F. Vale, ale. ilipa@ gmail. com
Keywo ds: Helicobac e pylo i; cla i h omycin; esis ance; 23S ibosomal RNA subuni ; nex - gene a ion sequencing; poin mu a ions.
Abb e ia ions: CLA, cla i h omycin; EF- Tu, elonga ion ac o he mo uns able; EPI, e lux pump inhibi o ; FISH, luo escence in si u hyb idiza ion;
FRET, luo escence esonance ene gy ans e ; MIC, minimal inhibi o y concen a ion; NGS, nex - gene a ion sequencing; PPI, p o on pump inhibi o ;
RND, esis ance- nodula ion- cell di ision; 23S RNA, 23S ibosomal RNA; WHO, Wo ld Heal h O ganiza ion.
Accession numbe s o an um isola es o H. pylo i CLA- esis an sequenced genomes: SRR9930173–SRR9930179.
000344 © 2020 The Au ho s
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.
Abs ac
Fo a long ime Helicobac e pylo i in ec ions ha e been ea ed using he mac olide an ibio ic, cla i h omycin. Cla i h omycin
esis ance is inc easing wo ldwide and is he mos common cause o H. pylo i ea men ailu e. He e we e iew he mecha-
nisms o an ibio ic esis ance o cla i h omycin, de ailing he indi idual and combina ions o poin mu a ions ound in he 23S
RNA gene associa ed wi h esis ance. Addi ionally, we conside he me hods used o de ec cla i h omycin esis ance, empha-
sizing he use o high- h oughpu nex - gene a ion sequencing me hods, which we e applied o 17 newly sequenced pai s o H.
pylo i s ains isola ed om he an um and co pus o a ecen colonized paedia ic popula ion. This se o isola es was com-
posed o six pai s o esis an s ains whose pheno ype was associa ed wi h wo poin mu a ions ound in he 23S RNA gene:
A2142C and A2143G. O he poin mu a ions we e ound simul aneously in he same gene, bu , acco ding o ou esul s, i is
unlikely ha hey con ibu e o esis ance. Fu he , among suscep ible isola es, genomic a ia ions compa ible wi h mu a ions
p e iously associa ed wi h cla i h omycin esis ance we e de ec ed. Exposu e o cla i h omycin may selec low- equency a i-
an s, esul ing in a p og essi e inc ease in he esis ance a e due o selec ion p essu e.
Da a SuMMaRy
The genome accession numbe s and me ada a a e p esen ed
in he Reposi o ies sec ion and in Table1.
IN RoDuc IoN
Helicobac e pylo i is a long- las ing human (s omach) a -
elle companion causing gas i is, pep ic ulce and gas ic
ca cinoma [1, 2]. Cla i h omycin (CLA) has been he basis
o H. pylo i ea men because o i s low minimal inhibi o y
concen a ion (MIC), good mucosal di usion and ela i ely
small e ec on gas ic acidi y [3]. Fo yea s, his ea men
consis ed o iple he apy ha combined CLA wi h ei he
amoxicillin o me onidazole, and a p o on pump inhibi o
(PPI) [4]. Howe e , he e icacy o iple he apy is in decline,
mos ly due o H. pylo i esis ance o CLA, con ibu ing o
he inc easing bu den o mul id ug- esis an G am- nega i e
in ec ion. This global an ibio ic c isis was ecognized by he
Wo ld Heal h O ganiza ion (WHO), who in 2017 published
a lis o an ibio ic- esis an p io i y pa hogens o esea ch
and de elopmen o new an ibio ics, including CLA- esis an
H. pylo i [5].
CLA is a mac olide de i ed om e y h omycin, whose
bac e ios a ic ac i i y depends on i s abili y o inhibi he
bac e ial p o ein syn hesis [6]. P o ein syn hesis is c i ical o
li e and is pe o med in a e y old nanomachine, he ibo-
some. This nanomachine a ose om he molecula e olu ion
ha p eda ed he i s li ing cells [7, 8]. Ribosomes ha e wo
subuni s, he la ge and he small, made o RNA and p o eins,
p esen ing basic machine y o syn he ize polypep ides: bo h
subuni s join, emb acing an mRNA molecule, a e ecog-
ni ion o a s a e sequence, and bo h ha e a simila si e o
ecei e/dock he RNA, and inally a simila exi ing unnel
o he nascen p o ein chain [9]. Bu , as hey a e e y old,
enough ime has passed o he e olu ion o mechanisms and
compounds o s all his c ucial nanomachine. O ganisms
who had ha abili y in s a a ion imes could s op o he s
om consuming esou ces ha a e undamen al o hei
2
Ma ques e al., Mic obial Genomics 2020;6
own su i al. Acco dingly, he e a e se e al molecules ha
a ge di e en pa s o he ibosome, all o hem p oduced
by bac e ia, such as chlo amphenicol, e acyclines, s ep o-
mycin and e y h omycin [10].
E y h omycin is a na u al an ibio ic p oduced by Saccha-
opolyspo a e y h aea ( o me ly S ep omyces e y h aeus)
whose o iginal s ain p oduced ou ypes o molecules:
e y h omycin A, B, C and D [11]. E y h omycin A is a
mac olide (a mac ocyclic lac one wi h a ing o 12 o mo e
membe s de i ed om a polyke ide) ha inhibi s bac e ial
p o ein syn hesis by in e e ing wi h aminoacyl ansloca-
ion, p e en ing he ans e o he RNA bound a he A
si e o he RNA complex o he P si e o he RNA complex
(Fig.1a) [12]. E y h omycin may be bac e ios a ic o bac e-
icidal, depending on he o ganism and d ug concen a ion.
Howe e , he ac ion o mac olides is no simple. A ecen
e iew poin s o o he impo an mac olide ac ions, such as
being modula o s o pep ide bond o ma ion, no only ibo-
some unnel plugs [10].
This e iew is abou H. pylo i esis ance o a small man- made
al e a ion o e y h omycin: he 6- O- me hyl e he o e y h-
omycin A, named cla i h omycin (CLA) (Fig.1B). CLA
inhibi s bac e ial p o ein syn hesis by e e sibly binding
o he 50S ibosomal subuni [6]. I s mechanism o ac ion
is o bind o he pep idyl ans e ase loop o he V domain
o 23S ibosomal RNA (23S RNA) gene, which esul s in
s uc u al changes and dissocia ion o pep idyl- RNA om
ibosome, in e e ing wi h nascen pep ide chain elonga ion
[13]. Misuse o an ibio ics accele a es esis ance, namely
when a ge ing ibosome, which is e y lexible and a mino
con o ma ional change could be enough o a oid he ac ion
o na u al o semi- syn he ic an ibio ics [14]. Bac e ial popula-
ions ha e a a iabili y o RNA sequences ha assu es he
su i al o some o i s membe s, which we will show using
ou own esul s.
cLa esis ance mechanism
H. pylo i’s an imic obial esis ance is mainly acqui ed by poin
mu a ions, which a e ansmi ed e ically by bina y ission,
esul ing in a p og essi e inc ease in he esis ance a e due
o selec ion p essu e.
Se e al PCR- based s udies ha e demons a ed ha poin
mu a ions in he pep idyl ans e ase loop o he V domain
o 23S RNA gene a e esponsible o he CLA esis -
ance pheno ype in clinical H. pylo i s ains om a ious
geog aphical loca ions [15–20]. These mu a ions a e able
o dis up he pep idyl ans e ase loop con o ma ion
and inhibi he binding be ween CLA and he 23S RNA,
educing i s e iciency and leading o a esis ance pheno ype
[18].
Two copies o he 23S RNA ope on a e p esen in he H.
pylo i genome [19, 21] and, o mos H. pylo i s ains, mu a-
ions a e gene ally ound in bo h copies; ne e heless, a
he e ozygo e pheno ype is su icien o con e in e media e
esis ance o CLA [16, 22]. Resis an s ains can be di ided
in o wo g oups: a high le el o esis ance (MIC >64 mg l−1)
and a low le el o esis ance (0.5≤MIC≤1 mg l−1).
The mos p e alen and well- documen ed mu a ions in H.
pylo i occu in wo speci ic adjacen nucleo ide posi ions,
an adenine- o- guanine ansi ion a ei he posi ion 2142
(A2142G) o 2143 (A2143G), o , less equen ly, an adenine-
o- cy osine ans e sion a posi ion 2142 (A2142C), and
hese mu a ional e en s a e esponsible o mo e han 90 %
o CLA esis ance in de eloped coun ies [23]. In pa icula ,
mu a ion a posi ion 2143 is usually associa ed wi h di e en
le els o esis ance (MICs anging om 2 o 256 mg l
−1
), while
s ains wi h mu a ion a posi ion 2142 equen ly exhibi a
mo e es ic ed esis ance (MIC o 64 mg l
−1
). These wo mu a-
ions (2142 and 2143) we e o iginally desc ibed as posi ions
2058 and 2059, based on Esche ichia coli 23S RNA sequence
[15, 16, 18, 24] and la e changed o 2143 and 2144 acco ding
o he H. pylo i 23S RNA sequence GenBank U27270 [20].
A e Taylo e al. de e mined he DNA sequences o he wo
copies o he 23S RNA gene om H. pylo i s ain UA802
and compa ed he sequences om CLA- esis an s ains, hey
p oposed ha he posi ions associa ed wi h CLA esis ance
we e nucleo ides 2142 and 2143 [19] and mos in es iga o s
choose o use his nomencla u e. Fu he mo e, i has been
epo ed ha o he mu a ions a e able o con e CLA esis -
ance, including mu a ions A2115G, G2141A, A2144T and
T2289C [25–28], whils C2694A and T2717C ha e been
associa ed wi h low esis ance le els [29, 30]. Fig.2 illus a es
poin mu a ions ha con e CLA esis ance in he model o
he 23S RNA domains V and VI. The con e sion T2182C
is one con o e sial mu a ion and has been epo ed as no
being equi ed o CLA esis ance, as well as con e ing low-
le el o high- le el esis ance, wi h an MIC >64 mg l
−1
[30–33].
O he mu a ions ha e been desc ibed in he li e a u e (i.e.
G1939A, C2147G, G2172T, T2215C and C2245T), bu hei
ole in ailu e o CLA- based he apy is s ill no p o en o is
no being consis en ly epo ed [34–38]. Table2 summa izes
a comple e lis wi h desc ibed single and combined poin
mu a ions con e ing CLA esis ance (sea ch ca ied ou in
Impac S a emen
An ibio ic- esis an Helicobac e pylo i s ains a e
inc easing in p e alence, as acknowledged ecen ly
by he Wo ld Heal h O ganiza ion when H. pylo i was
included in a lis o bac e ia ha pose he g ea es h ea
o human heal h. We e iewed he mechanisms o esis -
ance o cla i h omycin, p oducing a comple e compen-
dium o all mu a ions ound in he 23S RNA gene ha
ha e been associa ed wi h esis ance o his mac olide
an ibio ic. Addi ionally, we desc ibed he pheno ypic and
geno ypic me hods used o de ec ion o cla i h omycin
esis ance, placing pa icula emphasis on he use ul-
ness o whole- genome sequencing in de ec ing he
esis ance and p og ession owa ds esis ance ound in
mino i y genomic a ian s.
3
Ma ques e al., Mic obial Genomics 2020;6
Table 1. Associa ion be ween poin mu a ions in he 23S RNA and he
cla i h omycin- esis an pheno ype o 17 pai s o H. pylo i isola es om
he an um and co pus
Genome Pheno ype Posi ion o he
mu a ion
Cla i h omycin MIC
(mg l−1)
10 087A Resis an A2143G 8
10087C Resis an A2143G 8
10 103A Resis an A2142C, G2212A >256
10103C Resis an A2142C, G2212A >256
10 120A Resis an A2143G, C2759T 12
10120C Resis an A2143G, C2759T 12
10 198A Resis an A2143G, C2772T 24
10198C Resis an A2143G, C2772T 24
10 211A Resis an A2143G 12
10211C Resis an A2143G 12
10 212A Resis an A2143G 2
10212C Resis an A2143G 2
10 104A Suscep ible – –
10104C Suscep ible – –
10 127A Suscep ible T2182C –
10127C Suscep ible T2182C –
10 128A Suscep ible – –
10128C Suscep ible – –
10 133A Suscep ible – –
10133C Suscep ible – –
10 144A Suscep ible – –
10144C Suscep ible – –
10 147A Suscep ible* – –
10147C Suscep ible* – –
10 201A Suscep ible – –
10201C Suscep ible – –
10 210A Suscep ible – –
10210C Suscep ible – –
10 215A Suscep ible – –
10215C Suscep ible – –
10 218A Suscep ible – –
10218C Suscep ible – –
10 222A Suscep ible – –
10222C Suscep ible – –
*Wi h esis an clones.
A, an um isola e; C, co pus isola e.
Fig. 1. Two- dimensional chemical s uc u e o e y h omycin A (a) and
CLA (b). CLA is he 6- O- me hyl e he o e y h omycin A. S uc u es om
PubChem (accessed Augus 2019) [67].
July 2019, using he keywo ds CLA esis ance, H. pylo i, 23S
RNA and e lux pumps, and a ‘snowball’ sea ch – pu suing
e e ences o e e ences).
o he esis ance mechanisms
Ano he possible mechanism o CLA esis ance is mul id ug
e lux pump sys ems. E lux o an imic obial compounds
is commonly obse ed in bac e ia, educing in acellula
an imic obial concen a ion [39, 40]. E lux pumps o he
esis ance- nodula ion- cell di ision (RND) amily a e espon-
sible o mac olide esis ance in G am- nega i e bac e ia and
his mechanism has also been p oposed o H. pylo i [41, 42].
Th ee pu a i e ope ons ha e been desc ibed as an RND
e lux sys em in H. pylo i: he ABC (hp0605–hp0607), he DEF
(hp0969–hp0971, ecen ly denomina ed cznABC) and he GHI
(hp1327–hp1329, ecen ly denomina ed czcAB- c dB), wi h he
he ABC ope on being mos simila o mul id ug e lux pumps.
The he A, he D and he G genes encode an ou e - memb ane
p o ein TolC homologue o E. coli, while he B/he C, he E/he F
and he H/he I genes a e homologues o he ac A/ac B genes,
encoding a memb ane usion and RND cy oplasmic pump
p o eins, espec i ely [39, 43, 44]. A ou h gene clus e
including ORF hp1489–1487 was desc ibed, whe e hp1489
is a TolC homologue and hp1488 shows simila i ies wi h he
ac A gene [45]. In H. pylo i a syne gis ic e ec be ween 23S
RNA mu a ions and e lux pumps is likely o be p esen in
esis an s ains; he o me lowe s he CLA a ini y o he
ibosome and he la e exc e es he an ibio ic. In some CLA-
esis an s ains p esen ing 23S RNA mu a ions, i was shown
ha he p esence o e lux pumps inhibi o s (EPIs) was able o
dec ease he MIC o mos o he s udied s ains by ou old.
The EPIs lead o an augmen ed in acellula CLA concen a-
ion, which binds o he ibosome e en in he p esence o
mu a ions. Howe e , in hese cases, he inal MICs we e s ill
in he esis an ange [46]. Examina ion o he gene ic a i-
an s o hese ou e lux pumps e ealed ha CLA esis an
4
Ma ques e al., Mic obial Genomics 2020;6
Fig. 2. Seconda y s uc u e model o he pep idyl ans e ase cen e in he domain V (g een) and domain VI (blue) o he H. pylo i 23S
RNA. The indica ed poin mu a ion posi ions co espond o single mu a ions (no equi ing combina ion wi h o he mu a ions) con e ing
low- le el o high- le el CLA esis ance. Mu a ions 2141 (G2141A), 2144 (A2144T), 2182 (T2182C), 2190 (T2190C), 2195 (C2195T), 2244
(T2244C), 2694 (C2694A) and 2711 (T2711C) a e indica ed by black ci cles and he mos p e alen mu a ions 2142 (A2142G) and 2143
(A2143G) a e indica ed by ed ci cles. Image a ailable a (and modi ied om) h p://www. na.icmb.u exas.edu (accessed July 2019) [68]).
The ci cled nucleo ides indica e he posi ions o mu a ions ha con e CLA esis ance in H. pylo i (de ails and e e ences a e gi en in
Table2).
s ains a e mo e p one o single- nucleo ide a ian s in all ou
clus e s o e lux genes, wi h signi ican di e ences o clus e
he ABC [47]. Al hough he exac unde lying mechanism is
unclea , i may in ol e an ibio ic e lux. To he bes o ou
knowledge, he e lux pumps alone (ei he by mu an a ian s
o by di e en ial exp ession p o ile) ha e no been associa ed
wi h H. pylo i CLA esis ance.
O he no el candida es o CLA esis ance likely p esen a
syne gis ic e ec wi h 23S RNA poin mu a ions. Indeed,
a e he exposu e o a suscep ible s ain o low doses o
CLA, mu a ions in genes in B ( ansla ion ini ia ion ac o
IF-2) and pl22 ( ibosomal p o ein L22) con e ed low- le el
esis ance o CLA (low MIC alue), while mu a ions in hese
genes plus mu a ions in 23S RNA inc eased he MIC alue.
Addi ionally, compa a i e p o eomics analysis highligh ed he
possible in ol emen o ou e - memb ane p o eins in CLA
esis ance. Resis an s ains in compa ison o suscep ible
s ains p esen ed up egula ed U easeB subuni and EF- Tu
(elonga ion ac o he mo uns able) and down egula ed Ho C
(e lux pump) and OMP31 [48]. Finally, he spoT [bi unc-
ional (p)ppGpp syn hase] gene is in ol ed in ole ance o
CLA, up egula ing anspo e genes (HP0939, HP1017,
HP0497 and HP0471) [49]. O e all, he mechanisms besides
mu a ions in 23S RNA appea o po en ia e he e ec o ibo-
somal mu a ions by in e e ing ei he wi h o he ansla ion
machine y o wi h an ibio ic anspo , educing i s in acel-
lula concen a ion.
De ec ion me hods
An imic obial suscep ibili y es ing should be pe o med
whene e possible o guide he apy selec ion. Fo CLA, o
which he in i o esis ance is p edic i e o unsuccess ul
ea men s, his es assumes pa icula ele ance in he
managemen o H. pylo i in ec ion, especially in high p ima y
esis ance egions [50]. Pheno ypic and geno ypic me hods
can be used o es suscep ibili y o CLA. Se e al pheno ypic
5
Ma ques e al., Mic obial Genomics 2020;6
Table 2. Poin mu a ions in he 23S RNA egion and MIC alues associa ed wi h CLA- esis an H. pylo i s ains
Posi ion* Mu a ion MIC (mg l−1)Re e ences
1592† A1592G+T2182C 1 [69]
A1592G+C2195T 2 [69]
A1592G + T1644C + A1821G + G1826A + T1830C+T2182C 1 [69]
1592† A1592T + A1821G + G1826A + T1830C+T2182C 4 [69]
1652† A1652G 2 [69]
1694† G1694A + A1738G + C1953T 4 [69]
1738† A1738G + G1826C+C2195T 1 [69]
1769† C1769T 1 [69]
1821 A1821G+T2182C 1 [31]
A1821G + G1826A + T1830C+T2182C 4 [31]
1826† G1826A+A2143G 4 [69]
1939 G1939A + T1942C+A2142G+C2147G 1 [35]
G1939A+A2306G 0.064 o 0.5 [34]
1944 C1944T+G2212A 0.064 o 0.5 [34]
1953 C1953T+A2143G+T2182C+A2223G+T2244C 1.5 o 256 [70]
2115 A2115G 1.24 o 4 [25, 26]
A2115G+A2141G 4 [22]
A2115G+A2142G 4 [26]
A2115G+A2143G 4 [26]
A2115G+A2144T 4 [26]
2141 G2141A 1.25 o 28 [25–27]
2142 A2142C 64 o >256 [15, 27, 71–73]
A2142C+A2142G na [73]
A2142C+A2143G na [71]
A2142C+G2212A >256 This wo k
A2142C+A2142G+A2143G na [71]
2142 A2142G 0.1 o 256 [16, 22, 26, 27, 35, 38, 72–76]
A2142G+A2143G 4 [26, 71, 73]
A2142G+A2144T na [71]
A2142G+T2182C 1 o 256 [76, 77]
A2142G+C2195T ≤32 [74]
A2142G+A2223G >32 [74]
A2142G+A2143G+A2144T 256 [26]
A2142G+T2182C+C2195T >32 [74]
Con inued

6
Ma ques e al., Mic obial Genomics 2020;6
Posi ion* Mu a ion MIC (mg l−1)Re e ences
2143 A2143G 0.1 o 256 [16, 22, 26, 27, 32, 35, 38, 71–76, 78]
A2143G+A2174G ≤32 [74]
A2143G+T2182C 0.5 o 45 [36, 74, 76–78]
A2143G+A2223G ≤32 [74]
A2143G+G2224A 4 [27]
A2143G+T2244C 3 o 48 [70]
A2143G+C2245T 16 [27]
A2143G+C2759T 12 This wo k
A2143G+C2772T 24 This wo k
A2143G+T2182C+T2190C 52 [78]
A2143G+T2182C+C2195T 53.3 [78]
A2143G+T2182C+A2223G 80 [78]
A2143G+T2182C+T2244C 1.5 o 96 [70]
A2143G+C2195T+A2223G ≤32 [74]
A2143G+A2223G+T2244C 1.5 o 96 [70]
A2143G+T2182C+A2223G+T2244C 1.5 o 256 [70]
A2143G+T2182C+T2244C+A2302G 1.5 o 256 [70]
2143 A2143T+T2182C+G2172T+G2254T na [36]
2144 A2144T 3 o 134 [25–27]
2182 T2182C 1 o >64 [31, 32, 72, 74, 76, 78]
T2182C+T2244C 3 o 48 [70]
T2182C+T2190C+C2694A >0.5 [30]
2190 T2190C na [79]
2195 C2195T na [79]
C2195T+T2182C+A2223G+T2244C 1.5 o 256 [70]
2224 G2224A+T2289C 64 [28]
G2224A+C2245T+T2289C >256 [28]
2244‡ T2244C 1.5 o 5 [70, 80]
2289 T2289C 8 [28]
2694 C2694A 1 [30]
2711§ T2711C 0.5 o 1 [29]
na, no a ailable.
*Consolida ed posi ion acco ding he nomencla u e published by Taylo e al.[19] (1997), which co esponds o he e e ence sequence H. pylo i
23S RNA gene (GenBank: U27270.1) nucleo ide.
†Ma a e al. [69] desc ibe hese posi ions as 1593, 1653, 1695, 1739, 1770 and 1827, espec i ely ( e e ence sequence GenBank: U27270.1).
‡Khademi e al. [80] desc ibe his posi ion as 2243.
§Fon ana e al. [29] desc ibe his posi ion as 2717.
Table 2. Con inued
7
Ma ques e al., Mic obial Genomics 2020;6
Table 3. Geno ypic me hods used o de ec mac olide esis ance in H.
pylo i
Based on 23S
RNA gene
Me hod Re e ence
Wi h
ampli ica ion
RFLP [55]
PCR ollowed by e e se hyb idiza ion [57, 58]
Oligonucleo ide liga ion assay [81]
DNA enzyme immunoassay [82, 83]
P e e en ial homoduplex o ma ion
assay
[84]
Real- ime PCR [56, 85]
3’-misma ched e e se p ime PCR [86]
Mic oelec onic chip a ay [87]
Dual- p iming oligonucleo ide‐based
mul iplex PCR
[88]
Mic oa ay [89]
D ople digi al PCR [90]
Wi hou
ampli ica ion
Pep ide nucleic acid- luo escence in si u
hyb idiza ion
[59, 91]
Nex - gene a ion sequencing [92]
me hods ha e been de eloped, such as he aga dilu ion
me hod, which is conside ed o be he e e ence me hod in
compa ison o o he echniques. The MIC b eakpoin o
CLA, which is based on epidemiological cu - o alues, is
0.25 mg l−1 o suscep ible and 0.5 mg l−1 o esis an s ains
[51]. This me hod is e y ime- consuming and is a ely
pe o med in ou ine labo a o ies. The E- es me hod, based
on g adien di usion, wi h he abili y o p oduce an MIC
esul , is cu en ly he me hod o choice in mos o he clinical
labo a o ies pe o ming an imic obial suscep ibili y es ing
o H. pylo i, since is adap ed o slow- g owing bac e ia. A
good co ela ion has been ound be ween his me hod and
he aga dilu ion me hod [51]. B eakpoin suscep ibili y
es ing is a simpli ied e sion o he aga dilu ion me hod.
I consis s o inocula ing a line o he s ain o be es ed on
an aga pla e con aining an an ibio ic concen a ion equal
o he b eakpoin concen a ion ha de ines esis ance. The
b o h dilu ion me hod is seldom used due o he di icul y o
g owing H. pylo i in b o h media. Howe e , i is possible o
use i and ob ain accep able MIC esul s i he b o h is supple-
men ed wi h se um o de ib ina ed blood [52–54]. Finally, he
simples and mos economical me hod o ou ine suscep i-
bili y es ing is he disk di usion me hod, which is gene ally
no ecommended o slow- g owing bac e ia, al hough i has
been alida ed o de ec mac olide esis ance accu a ely in H.
pylo i, while e y h omycin is he ecommended an ibio ic o
mac olide suscep ibili y es ing.
In H. pylo i, gi en he low numbe o ch omosomal poin
mu a ions con e ing CLA esis ance (Table 2), accu a e
geno ypic me hods ha e de eloped exponen ially. These
es s a e as e han he pheno ypic me hods, and a e easy o
es ablish in ou ine p ac ice. The mos common is based on
ampli ica ion o he 23S RNA by PCR, ollowed by di e en
de ec ion me hods. PCR- RFLP was one o he i s me hods
o be de eloped, making use o h ee di e en es ic ion
enzymes, one o each o he h ee mos common poin mu a-
ions (A2142/3G and A2142C) [55]. This labo ious me hod,
based on a p o ile band esol ed in an aga ose gel, was apidly
eplaced by as e and mo e sensi i e me hods, such as eal-
ime PCR, which allows he de ec ion o H. pylo i as well as
CLA esis ance- associa ed poin mu a ions in a single eac-
ion. This es is usually based on a bip obe and luo escence
esonance ene gy ans e (FRET), allowing easy disc imina-
ion o he h ee mu a ions by mel ing cu e analysis, wi h he
po en ial o being used di ec ly on gas ic biopsies o on s ool
specimens, inc easing he sensi i i y o he me hod o de ec
mixed suscep ible and esis an popula ions in a single sample
[56]. O he PCR- based me hods can a y in hei de ec ion
me hod, o example combining mul iplex con en ional
PCR and amplicon de ec ion ia a e e se hyb idiza ion and
alkaline phospha ase eac ion on a memb ane s ip coa ed
wi h highly speci ic p obes complemen a y o he selec i ely
ampli ied nucleic acid sequences [57, 58]. Using PCR- based
molecula me hods on non- in asi e samples such as s ools
makes suscep ibili y es ing easie and mo e p ac ical o
pe o m, and he e o e such me hods a e widely a ailable.
Geno ypic me hods no in ol ing DNA ampli ica ion ha e
also been de eloped, using luo escence in si u hyb idiza ion
(FISH), which is based on p obes ha hyb idize wi h speci ic
RNA sequences o mic o- o ganisms. The bes pe o mance is
achie ed using pep ide nucleic acid p obes, which a e usually
smalle han ypical DNA p obes, inc easing hei abili y o
pene a e he bac e ial cell wall, and a e mo e esis an o
deg ada ion by nucleases and p o eases [59].
Finally, he de elopmen o high- h oughpu nex - gene a ion
sequencing (NGS) me hods has allowed he use o whole-
genome sequencing as a genome- based yping me hod,
addi ionally enabling an ibio ic esis ance de e minan s o
be in e ed. Table3 p esen s an o e iew o some o he geno-
ypic me hods used o de ec mac olide esis ance in H. pylo i.
NGS o de ec ing esis ance: an example
As p e iously s a ed, i has been epo ed ha o he mu a-
ions a e able o con e CLA esis ance, bu hei ole is s ill
con o e sial. We he e o e used a se o an um and co pus
pai s o H. pylo i clinical isola es in o de o cla i y he ole o
hese less common mu a ions, as well as he powe o NGS o
de ec esis ance. The genomes o 17 pai s o isola es om a
pool o colonies om he an um and co pus o a paedia ic
popula ion we e sequenced, Illumina MiSeq assembled wi h
SPAdes3.13 [60] and analysed. An imic obial suscep ibili y
es ing was pe o med by disk di usion o e y h omycin,
and he E- es was used o de e mine he MIC o CLA o
he esis an s ains. Six pai s o isola es we e CLA- esis an
(12/34 isola es) and a o al o six poin mu a ions we e
de ec ed (Table 1). All pai s o CLA- esis an isola es o
8
Ma ques e al., Mic obial Genomics 2020;6
Table 4. Genomes o Helicobac e pylo i s ains suscep ible o CLA
p esen ing a ian s ma ching desc ibed mu a ions in 23S RNA o CLA
esis ance
Genome Posi ion Change Va ian equency (%) Co e age
10 222A 2142 A -> G 6.3 191
10222C 2142 A -> G 6.9 275
10 147A 2142 * A -> G 2.5 204
2301 A -> G 4.7 235
10147C 2142 A -> G 12.9 240
2301 A -> G 12.0 259
*Al hough ewe han 10 eads we e obse ed wi h mu a ion A
-> G, he esul is p esen ed because o he consis ency be ween
an um and co pus isola es.
an um and co pus o he same pa ien displayed he same
mu a ions. All hese isola es ha e mu a ions a posi ions
2142 and 2143, wi h A2143G (83.3 % o isola es, 10/12) being
p edominan , ollowed by A2142C (16.7 % o isola es, 2/12).
In de ail, h ee pa ien s had he single mu a ion A2143G o
bo h an um and co pus isola es (6/12 isola es), and h ee
pa ien s had double mu a ions, A2142C+G2212A o one
case (2/12 isola es), A2143G+C2759T o he second case
(2/12 isola es) and A2143G+C2772T o he las case (2/12
isola es). The MICs o CLA a ied be ween 2 and 24 mg l−1
o isola es ha bou ing he mu a ion A2143G, while he MIC
was >256 mg l−1 o he wo isola es om he same pa ien wi h
he mu a ion A2142C (Table1). These alues a e in he ange
o hose desc ibed ex ensi ely in he li e a u e o hese mu a-
ions, making i di icul o p edic he ole o he emaining
mu a ions ound simul aneously.
Two addi ional mu a ions ound in combina ion wi h A2143G,
C2759T and C2772T, bo h loca ed a 23S RNA domain VI,
a e epo ed o he i s ime (Fig.2 and Table2). A T2182C
mu a ion was ound in wo suscep ible isola es (an um and
co pus om same pa ien ), which is in ag eemen wi h i s
con o e sial ole in CLA esis ance [30–33].
Based on he esul s om ou se ing, we conclude ha he
esis ance pheno ype is ela ed o he p esence o he well-
desc ibed 2142 and 2143 poin mu a ions, while he p esence
o he o he epo ed mu a ions pe se canno con e a esis -
ance pheno ype, co obo a ing p e ious s udies. Rega ding
he p edic ion o esis ance le els, we canno make a conclu-
si e s a emen abou he ole o hese less common o new
mu a ions, since he MICs desc ibed a e all in he ange o
he p e iously desc ibed alues o isola es ha bou ing he
well- desc ibed 2142 and 2143 poin mu a ions. Ne e heless,
his example o he applica ion o NGS o CLA esis ance
de e mina ion shows he powe o genome sequencing o
co ec ly de ec ing esis ance in clinical s ains. In ac , all
o he cases wi h MIC alues ha we e compa ible wi h
esis ance o CLA p esen ed he mos common mu a ions
in he 23S RNA gene associa ed wi h esis ance (Table1).
The con inuous educ ion o he cos o genome sequencing,
coupled wi h he mul i- asking pe o mance o NGS (e.g. i
is also use ul o molecula yping, popula ion gene ics o
epidemiology), makes his echnique a s ong candida e o
in oduc ion in o clinical labo a o ies o he es ing o an i-
bio ic esis ance.
Genomic a iabili y o he 23S RNa gene:
genome-based p edic ion o e olu ion owa ds cLa
esis ance
H. pylo i genomes a e highly di e se and no in equen ly
s ains in ec ing he same hos a e gene ically di e en i-
a ed [1, 61]. Mo eo e , H. pylo i genomes e ol e apidly
du ing ch onic in ec ion [62, 63] and labo a o y cul u e
[64], e idencing i s ema kable adap i e capaci y o i s
niche. The genomes a ailable o each bac e ial s ain a e
ypically he canonical genomes, i.e. he mos equen esi-
dues o nucleo ides a each posi ion. The a ia ion wi hin
a bac e ial genome is commonly dis ega ded so ha a
consensus wo king sequence is a ailable and also due o
he di icul y o asce aining i i ep esen s a ue a ia ion
o a sequencing e o [64]. Impo an ly, he 23S RNA gene
is p esen in wo copies in he H. pylo i genome, which
due o hei simila i y a e assembled in he same locus.
In ac , a common cause o he unde es ima ion o gene
numbe is he collapse o gene copies in o a single locus
ha due o highly simila i y in sequence a e challenging
o assemble as sepa a e loci [65]. To unde s and he di e -
si y wi hin H. pylo i clinical isola es, we ha e wo ked wi h
he same se o H. pylo i s ains and looked o genomic
a ian s in he 23S RNA gene p esen ing a co e age le el
>100×, and a leas 10 copies o he a ian . Sequence eads
we e mapped o a canonical genome and a BAM ile was
p oduced using SAM ools [66]. Va ian calling o e BAM
iles was pe o med wi h Geneious 8.1.9, selec ing a ian s
a a equency a ian >1 % and maximum a ian P- alue
10E-6. Two ou o 11 pai s o s ains (18.1%) pheno ypically
suscep ible o CLA p esen ed a ian s ma ching mu a ions
associa ed wi h CLA esis ance (Table 4). Exposu e o
CLA may selec low- equency a ian s ha lead o he a-
peu ic ailu e and he eme gence o a esis an s ain. This
appea ed o be he case o he pai 10 147A/10147C, which
is suscep ible o CLA bu p esen ed a ew colonies g owing
a high CLA concen a ion (Tables1 and 4), sugges ing a
geno ype e ol ing owa ds CLA esis ance. Fo one o he
pai s o esis an isola es (10 103A and 10103C), a genomic
a ia ion was also obse able: mos o he eads had he
mu a ion A2142C, bu he mu a ion A2142G was also ound
wi h a equency o 28.5 and 28.2 %, espec i ely (Table1).
Bo h mu a ions a e associa ed wi h CLA esis ance.
High- dep h whole- genome sequencing is hus a powe ul
echnique no only o de e mine he mu a ion associa ed
wi h CLA esis ance, bu also o p edic he e olu ion
in e ed by a ian call analysis, examining he dep h o
eads mapped o each nucleo ide a ia ion posi ion known
o be associa ed wi h esis ance. In addi ion, al hough he
co ela ion be ween CLA esis ance de ec ed by pheno ypic
9
Ma ques e al., Mic obial Genomics 2020;6
and geno ypic me hods is in gene al good, he la e is much
mo e accu a e in de ec ing low numbe s o mu a ed bac e-
ial cells wi hin a pool o o he wise suscep ible bac e ia.
Acco ding o ou da a, he genomic a iabili y showed ha
abou one i h o he suscep ible isola es coming om a
pool o colonies is likely o ha e expe ienced mic oe o-
lu ion e en s, i.e. sha e a common ances o and p esen
dis inc genomic al e a ions wi hin pa ien . In hese cases,
CLA may ac as a selec i e agen o he mino i y and
esis an a ian s. The e o e, he in oduc ion o NGS
o CLA esis ance es ing is addi ionally impo an as in
addi ion o iden i ying s ains ha a e esis an , i may
also con ibu e o he iden i ica ion o a ian s in which
he esis an geno ype is in mino i y, a oiding he selec ion
o his esis an subpopula ion.
conclusion
The esis ance mechanism o CLA is mainly insc ibed on
h ee mu a ions ound on he 23S RNA gene, impai ing
a ge ecogni ion by he an ibio ic. In his s udy, mu a-
ions A2142C and A2143G we e also de ec ed in he 23S
RNA gene V domain o CLA- esis an H. pylo i. Mu a ion
T2182C was ound in suscep ible isola es. Se e al me hods
allow he de ec ion o CLA esis ance. NGS no only allows
us o e i y he p esence o mu a ions in canonical genomes
associa ed wi h esis ance, bu is also use ul o asce ain
e olu ion owa ds esis ance e idenced by genomic a i-
a ion ma ching known mu a ions associa ed wi h CLA
esis ance. The e will be always an ibio ic esis ance o
old and new molecules, because ha is how bac e ia had
su i ed o millions o yea s. The e o e, he s a egy o
o e come CLA esis ance should be o con inue o s udy H.
pylo i biology o ind new a ge s o elimina e he bac e ia
and o design new molecules.
Funding in o ma ion
F. F. V. is he ecipien o a p ojec g an (PTDC/BTM- SAL/28978/2017)
om he Fundação pa a a Ciência e a Tecnologia (FCT), which suppo ed
his wo k. J. V.’s esea ch g oup was inanced by New England Biolabs,
Inc. (USA).
Acknowledgemen s
Whole- genome sequencing was pe o med a Unidade de Tecnologia
e Ino ação (Depa amen o de Gené ica Humana, Ins i u o Nacional de
Saúde Dou o Rica do Jo ge, Lisbon, Po ugal).
Con lic s o in e es
The au ho s decla e ha he e a e no con lic s o in e es .
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