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publica ion in Jou nal o An imic obial Chemo he apy ollowing pee e iew. The
e sion o eco d Ma ina R. Pulido, Me i xell Ga cía-Quin anilla, Reyes Ma ín-
Peña, José Miguel Cisne os, Michael J. McConnell, P og ess on he
de elopmen o apid me hods o an imic obial suscep ibili y es ing, Jou nal o
An imic obial Chemo he apy, Volume 68, Issue 12, Decembe 2013, Pages
2710–2717, is a ailable online a :
h ps://academic.oup.com/jac/a icle/68/12/2710/695468,
h ps://doi.o g/10.1093/jac/dk 253”.
REVIEW 1
2
P og ess on he de elopmen o apid me hods o an imic obial suscep ibili y 3
es ing 4
5
Ma ina R. PULIDO, Me i xell GARCÍA-QUINTANILLA, Reyes MARTÍN-PEÑA, 6
José Miguel CISNEROS and Michael J. MCCONNELL* 7
8
Uni o In ec ious Diseases, Mic obiology, and P e en i e Medicine and Biomedical 9
Ins i u e o Se ille (IBiS), Uni e si y Hospi al Vi gen del Rocío/CSIC/Uni e si y o 10
Se illa, 41013, Se illa, Spain. 11
12
13
Au ho o whom co espondence should be add essed: 14
Michael J. McConnell 15
Uni o In ec ious Disease, Mic obiology, and P e en i e Medicine 16
Hospi al Uni e si a io Vi gen del Rocío/Ins i u o de Biomedicina de Se illa 17
A enida Manuel Siu o s/n, 41013 Se illa, Spain 18
e-mail: [email protected] 19
Phone: +34 955923104 20
Fax: +34 955013292 21
22
MRP and MGQ con ibu ed equally o his wo k 23
Running Ti le: Rapid suscep ibili y es ing 24
Key Wo ds: suscep ibili y es ing, an ibio ic esis ance, bac e ia 25
26
Synopsis 27
An imic obial suscep ibili y es ing is essen ial o guiding he ea men o 28
many ypes o bac e ial in ec ions, especially in he cu en con ex o ising a es o 29
an ibio ic esis ance. The mos commonly employed me hods ely on he de ec ion o 30
pheno ypic esis ance by measu ing bac e ial g ow h in he p esence o he an ibio ic 31
being es ed. Al hough hese me hods a e highly sensi i e o he de ec ion o 32
esis ance, hey equi e ha he bac e ial pa hogen is isola ed om he clinical sample 33
be o e es ing and mus employ incuba ion imes ha a e su icien o di e en ia ing 34
esis an om suscep ible isola es. Knowledge ega ding he molecula de e minan s o 35
an ibio ic esis ance has acili a ed he de elopmen o no el app oaches wi h po en ial 36
o apidly de ec ing esis ance in bac e ial pa hogens. PCR-based echniques, mass 37
spec ome y, mic oa ays, mic o luidics, cell lysis-based app oaches and whole 38
genome sequencing ha e all demons a ed he abili y o de ec esis ance in a ious 39
bac e ial species. Howe e , i emains o be de e mined i hese me hods can achie e 40
su icien sensi i i y and speci ici y compa ed o s anda d pheno ypic esis ance es ing 41
ha would jus i y hei use in ou ine clinical p ac ice. In he p esen e iew, we discuss 42
ecen p og ess in he de elopmen o me hods o apid an imic obial suscep ibili y 43
es ing, and highligh he limi a ions o each app oach ha s ill emain be add essed. 44
45
46
In oduc ion 47
The adminis a ion o app op ia e an ibio ic he apy o many ypes o bac e ial 48
in ec ions equi es ha a mic obiologic diagnosis, consis ing o iden i ica ion o he 49
causa i e agen o in ec ion and i s esis ance p o ile, is ob ained. In ou ine clinical 50
p ac ice, his p ocess can equi e be ween 24 and 72 hou s, du ing which empi ic 51
he apy is selec ed based on he suspec ed causa i e o ganism and local epidemiology. 52
The use o me hods ha a e able o apidly de ec an ibio ic esis ance in bac e ial 53
isola es he e o e has he po en ial o educe he du a ion o empi ic he apy and 54
acili a e ea ly ini ia ion o a ge ed ea men wi h p o en an ibio ic ac i i y agains he 55
causa i e agen o in ec ion. This is o impo ance in ligh o s udies ha ha e 56
demons a ed ha he apid a ailabili y o an imic obial suscep ibili y es ing esul s can 57
imp o e pa ien ou comes,1-3 and ha a delay in he ini ia ion o app op ia e an ibio ic 58
he apy is associa ed wi h highe pa ien mo ali y in ce ain bac e ial in ec ions.4, 5 In 59
addi ion, apid an imic obial esis ance es ing may con ibu e o educing heal hca e 60
cos s gi en s udies showing ha he ea ly a ailabili y o an ibio ic suscep ibili y 61
in o ma ion can esul in he o de ing o ewe labo a o y es s, a dec ease in he numbe 62
o in asi e p ocedu es pe o med, and educed hospi al s ay.1, 2 An addi ional 63
o eseeable bene i o he apid a ailabili y o an ibio ic suscep ibili y es ing esul s is 64
i s po en ial o con ibu e o an imic obial s ewa dship e o s, which include he 65
adminis a ion o app op ia e an ibio ic he apy once suscep ibili y es ing esul s a e 66
a ailable.6 In his con ex , he ea ly ini ia ion o adequa e he apy wi h he na owes 67
spec um needed o app op ia e ea men o he in ec ing bac e ia could play a ole in 68
educing he eme gence and ansmission o esis an s ains. In he p esen e iew, we 69
gi e an o e iew o he echniques ha ha e been de eloped o apidly de ec ing 70
an ibio ic esis ance in bac e ial pa hogens (excluding Mycobac e ia, a opic which has 71
ecen ly been e iewed7, 8) and p o ide examples o how hese echniques ha e been 72
employed o de ec ing esis ance in a a ie y o bac e ial pa hogens. In addi ion, we 73
discuss he ques ions ha s ill emain o be add essed ega ding hese app oaches. 74
75
Cu en ly-used me hods 76
The mos widely used me hods o cha ac e izing an ibio ic esis ance in clinical 77
isola es de ec pheno ypic esis ance by measu ing bac e ial g ow h in he p esence o 78
he an ibio ic being es ed. These echniques include b o h mic odilu ion, an imic obial 79
g adien me hods (e.g. E es s ips), disk di usion, and a ious comme cially a ailable 80
au oma ed sys ems (e.g. he Mic oScan WalkAway sys em om Siemens, he Phoenix 81
Au oma ed Mic obiology Sys em om BD Diagnos ics and he Vi ek sys ems om 82
bioMe ieux). In addi ion o hei high sensi i i y o de ec ing an ibio ic esis ance, a 83
majo ad an age o hese echniques is ha hey ha e been highly s anda dized, a 84
p ocess ha has been acili a ed by he elabo a ion o in e na ional guidelines o 85
an imic obial suscep ibili y es ing such as hose published by he CLSI and he 86
EUCAST.9, 10 Fo each o hese me hods, a a ie y o comme cial supplie s p o ides 87
eagen s in eady- o-use o ma s ha acili a e hei use in clinical mic obiology 88
labo a o ies wi h high wo k loads. In he cases o b o h mic odilu ion and an imic obial 89
g adien me hods, an MIC alue is ob ained, which p o ides in o ma ion on he 90
concen a ion o an ibio ic necessa y o inhibi ing bac e ial g ow h. Howe e , despi e 91
hei b oad use and high sensi i i y, he e a e some limi a ions associa ed wi h hese 92
app oaches. These me hods gene ally equi e pu i y cul u e be o e suscep ibili y es ing 93
can be pe o med, al hough some p o ocols may allow o es ing di ec ly wi h clinical 94
samples. In addi ion, because hese echniques measu e bac e ial g ow h in o de o 95
de ec esis ance, incuba ion imes ha a e su icien o di e en ia ing suscep ible om 96

esis an s ains mus be employed. In he ollowing sec ions, we commen on he 97
ad an ages and disad an ages o echniques and p o ocols ha a e being de eloped o 98
apidly iden i ying an ibio ic esis ance wi h espec o hese commonly-used me hods. 99
100
PCR-based echniques 101
PCR-based echniques (bo h con en ional and eal- ime) ely on he sequence 102
speci ic ampli ica ion o nucleic acids. Fo his eason, PCR was ini ially used in 103
mic obiologic es ing o he apid iden i ica ion and quan i ica ion o causa i e agen s 104
o in ec ions h ough he ampli ica ion o sequences speci ic o a pa icula pa hogen.11 105
Wi h inc eased knowledge o he gene ic bases o an ibio ic esis ance ha has been 106
acqui ed in ecen yea s, PCR-based app oaches ha e been de eloped o de ec ing he 107
p esence o gene ic de e minan s o esis ance o a a ie y o an ibio ics o a numbe o 108
di e en bac e ial species. One salien example has been he use o PCR o iden i y 109
me hicillin esis an S aphylococcus au eus (MRSA) h ough he de ec ion o he mecA 110
gene, which encodes a modi ied penicillin-binding p o ein wi h educed a ini y o β-111
lac am an ibio ics. Many PCR assays (bo h con en ional and eal- ime) ha e been 112
desc ibed o de ec ing mecA and/o associa ed sequences,12-16 including comme cial 113
sys ems ha in some cases ha e he abili y o de ec MRSA di ec ly om clinical 114
samples in less han wo hou s.17-19 Examples o comme cialized sys ems ha employ 115
eal- ime PCR o de ec ion a e he BD GeneOhm MRSA assay om Bec on-Dickinson 116
and he GeneXpe sys em om Cepheid, he la e o which showed high sensi i i y 117
(>93%) compa ed o ou ine me hods o de ec ing MRSA in a mul icen e s udy.20 A 118
ecen s udy demons a ing ha he clinical implemen a ion o a es o de ec ion o he 119
mecA gene educed he ime o ecei ing op imal an ibio ic he apy by an a e age o 120
25.4 hou s in pa ien s wi h S. au eus bac e emia illus a es he po en ial u ili y o his 121
me hod.21 PCR-based app oaches ha e also been de eloped o de ec ing ancomycin 122
esis ance associa ed wi h he anA and anB genes, and ha e p ima ily been used o 123
de ec ing esis ance in En e ococcus species. The sensi i i y and speci ici y o hese 124
assays a ies depending on he s udy, howe e a numbe o hese s udies ha e epo ed 125
high alse posi i e a es o he de ec ion o he anB gene, esul ing in educed 126
speci ici y.22-25 Assays o de ec ing he p esence o esis ance genes in G am nega i e 127
bac e ia ha e also been de eloped. Mos no ably, his has included assays o 128
iden i ying nume ous ca bapenemase-encoding genes including, bu no limi ed o, 129
KPC, NDM, IMP, VIM, AmpC, TEM, SHV, and he OXA ca bapenemases in 130
Acine obac e baumannii, Pseudomonas ae uginosa, Klebsiella pneumoniae and 131
Esche ichia coli.26-32 132
The majo ad an age o hese PCR-based app oaches is ha hey can be ca ied 133
ou in a ela i ely sho pe iod o ime, in some cases using clinical samples wi hou he 134
need o pu i y cul u e. PCR hus clea ly has he po en ial o signi ican ly educe u n-135
a ound imes and apidly p o ide in o ma ion on an ibio ic esis ance. The majo 136
limi a ion o his app oach howe e , is ha he p esence o esis ance genes may no 137
always co ela e wi h pheno ypic esis ance. This is less wo isome in cases we e he 138
p esence o a geno ype is highly associa ed wi h pheno ypic esis ance, such as he 139
p esence o he mecA, anA and anB genes. Howe e , o he cases a e no so clea cu , 140
such as he p esence o ca bapenemases in G am nega i e bac e ia. The e a e dozens o 141
dis inc ca bapenemases o which pheno ypic esis ance may depend no only on he 142
p esence o he gene, bu on i s le el o exp ession. One example is he OXA-51 gene 143
o A. baumannii which only p oduces pheno ypic esis ance i i is highly exp essed, o 144
example due o he in eg a ion o mobile inse ion sequences in he gene’s p omo e 145
egion ha inc ease exp ession.33, 34 The de elopmen o echniques ha ely on he 146
measu emen o gene ansc ip s (RNA le els) ins ead o he p esence o a gene may 147
p o ide a po en ial solu ion o his p oblem. PCR-based echniques ha de ec he 148
p esence o esis ance genes a e also unable o de ec no el o uncha ac e ized 149
mechanisms o esis ance o which he gene ic de e minan is unknown. This may be 150
especially impo an in he case o ca bapenemases in G am nega i e bac e ia gi en he 151
con inuous eme gence o new a ian s. This is a c i ical poin gi en ha he inabili y o 152
a echnique o de ec esis ance would lead o he inapp op ia e classi ica ion o a 153
esis an isola e as suscep ible, an e o ha could lead o he adminis a ion o 154
ine ec i e he apy. A inal conside a ion is ha hese me hods do no p o ide MIC 155
alues, which can be use ul o guiding clinical decisions ega ding he apy. 156
In addi ion o using PCR o de ec ing he p esence o gene ic de e minan s o 157
esis ance, he abili y o eal- ime PCR o accu a ely quan i y he numbe o copies o a 158
speci ic nucleic acid in a sample has led o he de elopmen o app oaches ha employ 159
his me hod o measu ing bac e ial g ow h. This app oach moni o s he numbe o 160
bac e ial genome copies p esen du ing g ow h o he isola ed bac e ia in he p esence o 161
he an ibio ic being es ed. Since quan i a i e eal- ime PCR can p o ide p ecise 162
in o ma ion ega ding genome copy numbe s, e y sho incuba ion imes can be used 163
o di e en ia ing suscep ible om esis an s ains. One ecen example is he de ec ion 164
o esis ance o imipenem, cip o loxacin and colis in in clinical isola es o A. baumannii 165
using a eal- ime PCR assay a ge ing highly conse ed sequences o he ompA gene.35 166
As shown in Figu e 1, g ow h o a esis an s ain in he p esence o an ibio ics could be 167
de ec ed wi h ela i ely sho incuba ion imes. This app oach has been applied o a 168
numbe o bac e ial species wi h a ious an ibio ics.36, 37 One ad an age o his 169
app oach wi h espec o he PCR-based app oaches desc ibed abo e is ha i does no 170
depend on he mechanism o esis ance and ha he assay is indi ec ly measu ing 171
pheno ypic esis ance by de ec ing g ow h in he p esence o an ibio ic. The majo 172
disad an age is ha , unlike PCR-based app oaches ha de ec he p esence o esis ance 173
de e minan s, his app oach equi es p e ious cul u e and canno be used di ec ly wi h 174
clinical samples. 175
176
Ma ix-assis ed lase deso p ion/ioniza ion ime o ligh mass spec ome y 177
(MALDI-TOF MS) 178
MALDI-TOF MS iden i ies molecules based on hei ime o ligh h ough a 179
acuum ube a e lase i adia ion o a ma ix which is co-c ys allized wi h he sample. 180
The ime o ligh allows o de e mina ion o he mass/cha ge a io (m/z) o he ions 181
p esen , and a spec um o he sample is gene a ed. The spec um is hen compa ed wi h 182
a e e ence da abase in o de o iden i y he analy e. Simila o PCR, MALDI-TOF was 183
o iginally in oduced in o clinical mic obiology labo a o ies o he iden i ica ion o 184
pa hogens, and has only ecen ly been applied o he de ec ion o esis ance. The use o 185
MALDI-TOF o de ec esis ance mos commonly aims o di e en ia e spec a om 186
esis an and suscep ible isola es using whole cells o c ude ex ac s. Compa ison o 187
inge p in s om E. coli ATCC 700926 and he same s ain ca ying he β-lac amase-188
p oducing plasmid pUC19 de e mined jus one disc imina o y peak co esponding o a 189
β-lac amase.38 Howe e , no pa e n was ound ha could eliably iden i y β-lac amase 190
esis ance in clinical isola es o E. coli, K. pneumoniae and P. ae uginosa.39 The 191
de ec ion o ancomycin- esis an en e ococci has been ecen ly demons a ed by 192
iden i ying disc imina o y peaks be ween anB-posi i e En e ococcus aecium isola es 193
om hose lacking anB.40 In he case o MRSA, di e en esul s ha e been ob ained 194
in indi idual s udies. Some s udies ha e epo ed measu eable di e ences in spec a 195
when compa ing o me hicillin-suscep ible S. au eus isola es,41-44 whe eas o he au ho s 196
below 1.5% and he majo e o a e ( he classi ica ion o a suscep ible isola e as 347
esis an ) mus be below 3.0%.77 One o he key limi a ions ha mus be add essed 348
ega ding many o hese me hods in o de o ensu e ha hey mee hese c i e ia is how 349
o en he e is disco dance be ween he p esence o a esis an de e minan and 350
pheno ypic esis ance. This is o pa icula impo ance wi h he use o echniques ha 351
ely solely on he de ec ion o esis ance de e minan s, such as PCR o gene de ec ion 352
o MALDI-TOF. How hese es s will deal wi h no el o uncha ac e ized esis ance 353
mechanisms mus also be conside ed, since he inabili y o a es o iden i y esis ance 354
will lead o e y majo e o s. Addi ional aspec s ha will need o be add essed on a 355
case-by-case basis a e whe he o no he es s being de eloped equi e highly-quali ied 356
pe sonal, he added expense associa ed wi h he es , and he accep ance o es esul s 357
by clinicians. Howe e , gi en he po en ial bene i s in e ms o imp o ing pa ien 358
ou comes by educing he pe iod o ime du ing which empi ic he apy is adminis e ed 359
and in e ms o educing heal hca e cos s, he con inued de elopmen o hese 360
app oaches is wa an ed. 361
362
363

Acknowledgemen s 364
The au ho s hank Pila Pé ez-Rome o o c i ical eading o he manusc ip and José 365
Luis Fe nández o p o iding he images used in Figu e 2. 366
367
Funding 368
This wo k was unded by a g an om he Eu opean Communi y’s 7 h P og amme 369
F amewo k (MagicBulle ; G an Ag eemen Numbe : 278232) and he Minis e io de 370
Economía y Compe i i idad, Ins i u o de Salud Ca los III - co- inanced by Eu opean’s 371
De elopmen Regional Fund "A way o achie e Eu ope" ERDF, Spanish Ne wo k o 372
he Resea ch in In ec ious Diseases (REIPI RD06/0008/0000). MJM is suppo ed by 373
he Subp og ama Miguel Se e om he Minis e io de Economía y Compe i i idad o 374
Spain (CP11/00314). 375
376
T anspa ency Decla a ion 377
All au ho s decla e no po en ial con lic s o in e es . 378
379
Re e ences 380
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