1 NEXT GENERATION 1 ANTIMICROBIAL SUSCEPTIBILITY ...
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NEXT GENERATION 1
ANTIMICROBIAL SUSCEPTIBILITY TESTING 2
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Alex van Belkum1 and Wm. Michael Dunne Jr2 4
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bioMérieux SA1, Unit Microbiology, R&D Microbiology, La Balme Les Grottes, France; 6
bioMérieux Inc.2, Unit Microbiology, R&D Microbiology, Durham NC, USA 7
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Corresponding author: Alex van Belkum, bioMérieux SA, Unit Microbiology, R&D 12
Microbiology, 3, Route de Porte Michaud, 38390 La Balme Les Grottes, France. 13
Tel: +0033474952656 14
Fax: +0033474952599 15
E-mail: [email protected] 16
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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00313-13 JCM Accepts, published online ahead of print on 13 March 2013
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ABSTRACT 23
Antimicrobial resistance has emerged as one of the most significant healthcare problems of 24
the new millennium and the clinical microbiology laboratory plays a central role in optimizing 25
the therapeutic management of patients with infection. This mini-review explores the 26
potential value of innovative methods for antimicrobial susceptibility testing of 27
microorganisms that could provide valuable alternatives to existing methodologies in the 28
very near future. 29
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Key Words: Antimicrobial Susceptibility Testing (AST) – antibiotics – antibiogram – drug 31
resistance. 32
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TODAY’S GLOBAL AST LANDSCAPE 35
A limited number of methods for antimicrobial susceptibility testing (AST) of medically 36
important microorganisms have survived the maturation of modern diagnostic clinical 37
microbiology. Surprisingly, one of these is the disk diffusion method first published in 1966 38
(1) and the various iterations thereof. Another is broth microdilution (BMD) testing which has 39
attained reference standard status to which all other AST methods are currently compared 40
during development, verification, validation, and clinical trials. As such BMD displaced agar 41
dilution testing, the past “Gold Standard” methodology. 42
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The most important outcome of any AST is the rapid and reliable prediction of antimicrobial 44
success in the treatment of infection. Currently, AST is typically accomplished using either 45
classical manual methods or growth-dependent automated systems such as the Becton 46
Dickinson Phoenix™, the Siemens Micoscan WalkAway™ or the bioMérieux VITEK2™ all of 47
which are based on BMD testing. The major limitations of these methods include the 48
requirement for relatively large numbers of viable organisms, complicated pre-analytical 49
processing, limited organism spectrum, analytical variability, time to results, and cost. Table 50
1 provides a cursory review of current and future technologies and their strengths and 51
weaknesses. 52
At present, non-phenotypic, mostly nucleic acid-based AST methods cannot detect all 53
resistance markers, are expensive, and have not been widely adopted. Multiplex PCR 54
detection of resistance determinants directly from positive blood cultures, however, has 55
been shown to substantially reduce the time to clinically actionable results (2). Further, 56
digital PCR may allow for better quantification of target molecules present in starting 57
material (3) and the refinement of aptamer technology (single stranded short RNA or DNA 58
molecules with antibody-like properties) may further facilitate nucleic acid diagnostics (4). 59
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Clearly, newer generation, transcriptome, and whole genome sequencing will provide near 60
future options to resistance prediction as databases mature. The per strain assessment of 61
detailed minimum inhibitory concentrations (MICs) for all relevant antimicrobials may be 62
confounded by elevated degrees of genetic heterogeneity, as is for instance obvious among 63
many gram-negative bacterial species. This may still frustrate the genomic approach but this 64
would be the subject of an entire review by itself and will not be discussed here. 65
Novel options are available to supplant the existing toolbox, but the timing of such events is 66
hard to foresee. As a disclaimer, we cannot provide a complete survey of all potential AST 67
configurations but will try to highlight a number of phenotypic methods that provide insight 68
into the wealth of the possibilities to come. Below we will briefly describe six technologies 69
that could represent competition for current reference standard methods - nucleic acid 70
amplification and sequencing excluded. 71
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NEAR FUTURE ALTERNATIVES FOR ROUTINE AST 73
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Mass Spectrometry. 75
Matrix Assisted Laser Desorption Ionization – Time Of Flight mass spectrometry (MALDI 76
TOF MS), a powerful tool for the rapid identification of organisms with medical importance, 77
may also prove to be of value as an AST method (5). Several approaches have been 78
explored including: 1) documenting the activity of antibiotic inactivating enzymes (e.g., β-79
lactamases) 2) confirming the presence of a PCR product indicative of antimicrobial 80
resistance (e.g. vanA, mecA, NDM-1) and 3) observing changes in the protein spectrum of 81
an organism in the presence or absence of an antimicrobial agent correlating with 82
susceptibility changes. 83
As an example of the first, carbapenemase activity was detected in a variety of gram-84
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negative organisms using ertapenem as a substrate (6). A bacterial suspension was 85
incubated with ertapenem and serial samples were examined by MALDI-TOF MS. 86
Organisms producing either NDM-1 or IMP-1 completely hydrolyzed ertapenem within 1 87
hour. It should be noted that four distinct peaks were initially observed in the mass spectrum 88
of the parent drug which already included the inactive hydrolyzed form. Other 89
carbapenemases (IMP-2, VIM-1, VIM-2, and KPC-2) hydrolyzed the drug more slowly. 90
Similar data were generated using meropenem as a substrate for enzymes including NDM-91
1, VIM-1, KPC-2, KPC-3, OXA-48 and OXA-162 (7). Applicability of this method using other 92
substrates (penicillin G, ampicillin cefoxitin, and imipenem) was demonstrated with and 93
without clavulanic acid as a means of distinguishing β-lactamase classes such as AmpC or 94
TEM-1 (8). The possibility exists for multiplexing the assay using combinations of β-lactams 95
and inhibitors that would allow classification of extended spectrum β-lactamases as well. 96
The second approach was presented in a study that compared two methods of single 97
nucleotide polymorphism (SNP) analysis for epidemiological typing of 147 strains of 98
methicillin resistant Staphylococcus aureus (MRSA) at 16 distinct loci (9). The predicate 99
method of analysis was a real-time SYBR green PCR assay. The comparator method 100
employed the Sequenom MassARRAY iPLEX SNP typing platform (Sequenom, Brisbane, 101
Australia) which combines multiplexed single-base extension PCR with MALDI-TOF MS of 102
amplicons to determine the location of SNPs. Both methods proved comparable and the 103
mecA PCR amplicon was successfully identified by MALDI-TOF MS for all 147 strains. A 104
combination of a primer extension (PEX) reaction with MALDI-TOF MS also led to the 105
detection of ganciclovir resistance mutations in cytomegalovirus (CMV) among viremic heart 106
transplant patients (10). Compared to a combination of real-time PCR and Sanger 107
sequencing, the PEX/MALDI-TOF MS method disclosed resistance mutations earlier without 108
loss of specificity. Similar analyses of PCR-generated amplicons are the basis for resistance 109
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detection using the more advanced electrospray ionization mass spectrometry (PCR/ESI-110
MS). In one study, the quinolone resistance-determining regions of parC and gyrA of 111
multidrug-resistant strains of Acinetobacter spp were identified with adequate correlation to 112
BMD testing (11). 113
Finally, there are a number of examples of MALDI-TOF MS used to highlight the effects of 114
antimicrobial agents on the protein spectral profile of susceptible organisms. Comparisons 115
of the profiles of Candida albicans grown in the presence of increasing concentrations of 116
fluconazole led to the formulation of a “minimal profile change concentration” or MPCC that 117
was defined as the lowest concentration of the drug at which a change in the profile could 118
be documented (12). The authors found a very high concordance between the MPCC and 119
MIC values obtained by the CLSI broth-based reference method. Similarly, MALDI-TOF MS 120
was used to assess caspofungin resistance secondary to fks mutations in 34 Candida spp 121
and 10 Aspergillus isolates (13). Strains were exposed to increasing concentrations of 122
caspofungin in a BMD format along with a drug-free control well and incubated for 15 hours 123
prior to MALDI-TOF MS. For each drug concentration, a MPCC was calculated for each 124
strain. This group found 100% essential agreement for all of the isolates using CLSI 125
breakpoints for MIC or minimal effective concentration (MEC). Only two Candida isolates 126
were incorrectly interpreted as non-susceptible generating a categorical agreement of 127
94.1%. 128
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Flow cytometry. 130
Flow cytometry (FC) permits changes in morphology, physiological and metabolic activity, 131
and viability of microorganisms to be followed after exposure to antibiotics. Through a 132
process of staining with nucleic acid dyes that do not permeate the cell walls of healthy 133
organisms, the proportion of cells in a dying or dead state (and everything in between) can 134
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be rapidly assessed by examining emission spectra after the cells pass individually through 135
a flow channel and when the dye is excited by a laser (14). In early studies a primitive flow 136
cytometer constructed from a fluorescent microscope was used to assess cellular 137
morphology or DNA after bacteria were exposed to antimicrobial agents. It was concluded 138
that the effects of antimicrobial treatment could be detected within a few hours suggesting a 139
promising application for AST. The earlier studies were also useful for elucidating various 140
dye/fixation combinations that would better differentiate the state of cell viability by FC (15). 141
In 1997, propidium iodide was used to differentiate live/dead C. albicans cells treated with 142
amphotericin B or fluconazole, which could then be rapidly and sensitively quantified by FC 143
(16). Similar assays were developed for the echinocandins, caspofungin and additional 144
azoles (17). The results of these AST strategies were 96-99% concordant with other forms 145
of AST (18). The overall duration of AST could be reduced from overnight incubations to 1-2 146
hr. Also in 1997, bis(1,3-dibutylbarbituric acid) trimethine oxonol or DiBAC4[3] was used to 147
visualize anionic membrane potential changes using fluorescence which proved to be very 148
adequate for AST of E. coli (19). Tests of organisms causing urinary tract infection showed a 149
94% agreement between classical disk diffusion testing and DiBAC4[3]- FC testing (20). FC 150
AST was also described for Mycobacterium tuberculosis. Pyrizinamide susceptibility testing 151
by FC was 93% concordant with the BACTEC MGIT assay and the former was conclusive 152
within 24 hr (21). Also for Yersinia spp significantly faster testing was achieved using FC 153
(22). FC-AST was at least 20% faster than classical methods for E coli, P. aeruginosa and 154
S. aureus and extended spectrum β-lactamases could be reliably detected by FC in 1-2 155
hours (23). Although significant advances in the design and performance of FC 156
instrumentation has occurred, the technology has not yet emerged as a major player in the 157
AST market although commercial assays have been launched. Some of the perceived 158
hurdles for the methodology are, among others, the ability to differentiate cellular damage 159
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caused by cidal versus static antibiotics, autofluorescence of certain bacterial species, and 160
the tremendous amount of work required for verification/validation of the clinical database 161
and the method itself. 162
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Microbial cell weighing by vibrating cantilevers. 164
Cantilevers containing small canals which facilitate microbial passage can be made to 165
vibrate continuously. When bacteria pass through, their weight (in the femtogram range) will 166
cause a change in the frequency of cantilever movement (24). Less dense cells will cause a 167
different change than more dense cells. When cells are treated with antimicrobial agents 168
their buoyant mass density changes and this is measurable (25). The principle has been 169
proven using ampicillin-resistant and -susceptible variants of Citrobacter rodentium. It was 170
also shown that resuscitation of both phenotypes after osmotic shock in the presence or 171
absence of ampicillin allows rapid differentiation in a reduced time-span. Cantilevers can be 172
multiplexed using nanotechnology such that multiple antibiotics in various concentrations 173
could be tested for a single growing culture simultaneously. 174
A rapid biosensor for the detection of bacterial growth was developed using vibrating 175
cantilevers containing a certain number of fixed but still viable bacteria (26). The change in 176
resonance frequency as a function of the increasing mass on the cantilever forms the basis 177
of the detection scheme. The calculated mass sensitivity according to the mechanical 178
properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to 179
about 100 E. coli cells. The sensor was able to detect active growth of E. coli cells within 1 180
hr. The number of E. coli cells initially attached to the cantilever was on average 1,000 cells. 181
Furthermore, the non-inhibited growth of resistant cells could be documented within 2 h after 182
the addition of antibiotics (27). Cantilever technology has also been used to assess 183
vancomycin binding to cell wall precursors (28) and to measure the effects of colistin on P. 184
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aeruginosa (29). 185
Using suspended nano-channel resonators (SNRs), it was demonstrated that the 186
measurements of bacterial mass in solution was even more precise. The SNR consisted of a 187
cantilever with an embedded nano-channel. In addition, a new method was introduced that 188
uses centrifugal force caused by vibration of the cantilever to trap particles at the free end of 189
the SNR (30). This approach eliminates the intrinsic position-dependent error of the SNR 190
and also improves the mass resolution by increasing the average “time of presence” for 191
each particle. In addition, it would facilitate the continuous mass monitoring of a limited 192
number of bacteria during (changing) exposure to antibiotics. Clearly, cantilever systems 193
and precise weight measurements provide an interesting option for the development of 194
multiplexed AST. 195
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Isothermal micro-calorimetry. 197
Isothermal micro-calorimetry (IMC) is a dynamic technique that allows measurement of heat 198
production either as a flow rate (μW/unit time) or total accumulation over time (Joules/unit 199
time) stemming from the metabolism of actively growing cells. Cumulative heat production 200
generally parallels conventional growth curves in that the slope and shape of the 201
accumulating heat production corresponds with classical lag, log, and stationary phases. 202
Maximum heat values represent the total number of cells produced over time (31). The 203
method has been successfully adapted to small culture volumes (e.g. 1-3 ml) and can be 204
used in conjunction with either solid or liquid culture media (32). Using IMC, bacterial 205
species identification from urine specimens was performed even at low bacterial counts 206
within three hours on the basis of dynamic heat flow patterns (33). When adapted to AST, 207
measurements of heat production are made passively from sealed vials containing the 208
organism, growth medium and antimicrobial agents in doubling dilution concentrations. The 209
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minimal heat inhibition concentration (MHIC) can be defined either as the lowest drug 210
concentration to inhibit 50% of total heat production or resulting in a 50% reduction in heat 211
flow rate, depending on the drug being tested. The process requires specific IMC 212
instrumentation (e.g. TAM III, TA Instruments, New Castle, DE) for real-time measurement 213
of heat generation with a detection limit in the 0.2 μW range. The use of IMC for 214
susceptibility testing is not new but has since been successfully adapted for AST of bacteria 215
(31), mycobacteria including M. tuberculosis (32) and fungi (34, 35). The advantages of this 216
technique include that: 1) testing is conducted in sealed ampules alleviating safety concerns 217
when evaluating high-risk organisms such as M. tuberculosis or fungal species; 2) all 218
monitoring during testing is passive and requires no manual manipulation of the test vials; 3) 219
the completed analysis provides information about the maximum growth rate of the 220
organism, the static versus cidal activity of an agent, and delays to log phase growth 221
(extended lag phase) caused by the agent. The latter provides useful information concerning 222
antimicrobial activity at sub-inhibitory concentrations of the drug and can help predict actual 223
MIC when inhibitory concentrations have not been developed (31). This is pronounced with 224
fungal testing where delays or an abbreviation of maximum heat flow can be readily 225
appreciated with sub-inhibitory concentrations of antifungal agents (34, 35). Further, IMC is 226
not prone to subjective interpretations such as trailing MBD wells or the determination of 227
MECs based on morphologic changes at a microscopic level. IMC correlates well with BMD 228
testing and CLSI and/or EUCAST breakpoints when net heat production over time is used 229
as a surrogate for growth. As a bonus, IMC can be used to evaluate the synergistic activity 230
of antimicrobial combinations. Chip calorimetry is a monitoring tool for determining the 231
physiological state of biofilms. Its potential use for the study of the effects of antibiotics was 232
tested using an established model. The real-time monitoring potential of chip calorimetry 233
was successfully demonstrated: a dosage of antibiotics initially increased the heat 234
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production rate probably due to activity of energy-dependent resistance mechanisms (36). 235
The subsequent reduction in heat production was probably due to the loss of activity and the 236
death of the biofilm. This new analytical tool provided fast, quantitative, and mechanistic 237
insights into the effects of antibiotics on biofilm activity. In short, the maximum bacterial 238
growth rate and the start of the lag phase can be quantified by micro-calorimetric technology 239
in an affordable and sensitive manner. Hence, antibiotic-associated changes in these 240
parameters can be efficiently measured as well. 241
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Magnetic bead rotation. 243
When magnetic beads are brought into a revolving magnetic field they self-assemble and 244
assume a specific rotational spin. The frequency of rotation can be influenced by the binding 245
of molecules, viruses or bacteria. So if the beads are equipped with a ligand that specifically 246
captures bacterial cells, the rotation of the beads changes at the moment of capture. This 247
change can be measured. If all beads in a broth culture were paired with one or two cells, 248
something that can be accomplished by incubating ligand-modified beads with a diluted 249
bacterial suspension followed by washing, they will resume a constant rotational frequency. 250
As bacteria start to divide, the rotation frequency changes. If cell division is inhibited or 251
blocked by antimicrobials in case of susceptibility then the change is arrested. If the bacteria 252
are resistant to the antimicrobial applied, then again a change in rotational frequency 253
occurs. In this way, antimicrobial resistance can be detected and precisely quantified. 254
A growth-based antimicrobial susceptibility assay based on asynchronous magnetic bead 255
rotation (AMBR) biosensors has been described (37). In this system, the effects of bacterial 256
growth on the rotation and shape of a cluster of self-assembling magnetic microbeads in a 257
rotating magnetic field can be observed over time. The rotational period (RP) is indirectly 258
proportional to the drag coefficient of the surrounding medium containing bacteria, broth 259
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medium and antimicrobial agents at varying dilutions. RP increases as organisms multiply 260
and attach to the organism-specific antibody-coated beads or if the viscosity of the growth 261
medium is altered. The addition of antimicrobial agents in increasing concentrations 262
prevents an increase in RP over time. This process can be observed directly by illuminating 263
the culture broth (in the form of a hanging drop) with a LED or laser. The hanging drop 264
format also acts as a lens to create magnification of up to 100x such that the structure and 265
rotational rate of the bead aggregates can be observed microscopically or when projected 266
onto a detector. Serial two-fold dilutions of streptomycin and gentamicin were added to 267
Mueller-Hinton broth containing antibody- sensitized magnetic beads pre-bound to a 268
standardized inoculum of E. coli. A hanging drop was formed with the mixture that was 269
subjected to an oscillating magnetic field. Changes in the RP were observed microscopically 270
over time and recorded. As expected, the RP increased relative to bacterial growth with 271
solutions containing higher concentrations of antibiotic demonstrating the lowest increases. 272
This method can be miniaturized to nanoliter volume water-in-oil droplets containing 50 or 273
fewer bacterial cells per droplet (38). These changes substantially reduced the duration of a 274
test. 275
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Testing in microdroplets. 277
Micro- or nano-droplets can be used as small, individual reaction wells. The droplets can be 278
individually manipulated and when they contain bacteria in sufficient numbers, metabolic 279
activity and viability of cells can be monitored. Development of this system became feasible 280
once the emulsification process was refined and the long term stability of the droplets could 281
be guaranteed (39). A system consisting of 100 nl droplets containing 103 bacteria per 282
droplet and differing concentrations of antibiotics was developed (40). By following the 283
droplets over time using epifluorescence, growth curves can be monitored at each drug 284
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concentration. More recently, droplets were prepared that contain a single bacterial cell (41). 285
This technology can be miniaturized and easily multiplexed with respect to the number of 286
antibiotics tested per organism The duration of testing can be as short as a single or a few 287
bacterial replication cycles. Obviously, assessing technical reproducibility and the 288
development of adequate reference MIC databases will be necessary before this approach 289
can be evaluated as a routine AST tool. 290
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TECHNOLOGIES FOR APPLICATION IN THE MORE DISTANT FUTURE 292
Several innovative AST methods have been designed and presented over the past decade 293
(see Long Term alternatives in Table 1), but most are far from introduction into routine 294
clinical microbiology. Some, including bacteriophage-based AST, may be closer than the 295
others. This strategy has been successfully adapted for testing of M. tuberculosis (42) with 296
optimization achieved by including recombinant phages containing luciferase genes (43). It 297
was shown that the luciferase assay could be performed at low cost in two days (44) and 298
could be applied successfully in laboratories in developing countries (45). Unfortunately, this 299
innovative approach has not been broadly accepted in diagnostic microbiology laboratories 300
probably due to contamination (46) or phage resistance issues. As stated above, as 301
promising as some innovative method may seem to be, broad acceptance has not yet been 302
achieved. 303
Real time microscopy is one of the innovative technologies that may be applicable to AST in 304
the not too distant future. High resolution camera-based systems have been commercialized 305
and these show a high degree of efficacy (47). This technology has been extended for use 306
with micro-colonies and serial photography in the presence or absence of antibiotics. Such 307
technologies can generate a “one day AST” for bacteria from positive blood culture bottles 308
(48). It demonstrates that simple “old fashioned” test formats can still be adapted into 309
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systems that better serve the patient’s needs. 310
Several modern technologies have been proposed as being possible long term future 311
alternatives for today’s technologies in the clinical microbiology laboratories (see Table 1). 312
Proof of principle has been demonstrated and in some cases the first trials of feasibility have 313
been published. However, for all of these strategies database development has yet to be 314
completed and until then the prospects of these technologies remain nebulous. 315
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CONCLUSIONS 317
Conventional AST has its diagnostic limitations: it is generally time-consuming and 318
actionable results have a tendency for late arrival. The current methods are very solid and 319
well respected and do generally have CE and FDA certification. Any new technology has to 320
compete with current reference standards and the method that shows significant 321
improvements has yet to be published. As we described here, several technologies may be 322
knocking on the door shortly. 323
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AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST 325
The authors are employees of bioMérieux, a company creating and developing infectious 326
disease diagnostics. No further potential conflicts of interest relevant to this article were 327
reported. The classification of the various innovative AST systems in the three categories 328
suggested in Table 1 represents the author’s personal opinions. 329
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REFERENCES 331
1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility 332
testing by a standardized single disc method. Amer. J. Clin. Pathol. 45:493-496. 333
2. Waldeisen JR, Wang T, Mitra D, Lee LP A real-time PCR antibiogram for drug-resistant 334
on April 12, 2018 by guest
http://jcm.asm
.org/D
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15
sepsis. PLoS One. 2011;6(12):e28528 335
3. Hatch AC, Fisher JS, Tovar AR, Hsieh AT, Lin R, Pentoney SL, et al. 1-Million droplet 336
array with wide-field fluorescence imaging for digital PCR. Lab Chip 2011;11:3838-45. 337
4. DeGrasse JA. A single-stranded DNA aptamer that selectively binds to Staphylococcus 338
aureus enterotoxin B. PLoS One 2012;7:e33410. 339
5. Van Belkum A, Welker M, Erhard M, Chatellier S. Biomedical mass spectrometry in 340
today's and tomorrow's clinical microbiology laboratories. J Clin Microbiol 2012;50:1513-7. 341
6.Burckhardt I, S Zimmermann. 2011. Using matrix-assisted laser desorption ionization –342
time of flight mass spectrometry to detect carbapenem resistance within 1 to 2.5 hours. J. 343
Clin. Microbiol. 49:3321-3324. 344
7. Hrabák J, Walková R, Studentová V, Chudácková E, Bergerová T. Carbapenemase 345
activity detection by matrix-assisted laser desorption ionization-time of flight mass 346
spectrometry. J Clin Microbiol 2011;49:3222-7. 347
8.Hooff GP, JJA van Kampen, RJW Meesters, A van Belkum, WHF Goessens, TM Luider. 348
2012. Characterization of ß-lactamase enzyme activity in bacterial lysates using MALDI-349
mass spectrometry. J. Proteome Res. 11:79-84. 350
9.Syrmis MW, RJ Moser, DM Whiley, V Vaska, GW Coombs, MD Nissen, TP Sloots, GR 351
Nimmo. 2011. Comparison of a multiplexed MassARRAY system with real-time allele-352
specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus. Clin. 353
Microbiol. Infect. 17:1804-1810. 354
10.Zürcher S, C Mooser, AU Lüthi, K Mühlemann, MT Barbani, P Mohacsi, C Garzoni, M 355
Gorgievski-Hrisoho, A Schaller, L Flatz. 2012. Sensitive and rapid detection of ganciclovir 356
resistance by PCR based MALDI-TOF analysis. J. Clin. Virol. 54:359-363. 357
11.Hujer KM, AM Jujer, A Endimiani, JM Thomson, MD Adams, K Goglin, PN Eshoo, R 358
Sampath, LB Blyn, DJ Ecker, RA Bonomo. 2009. Rapid determination of quinolone 359
on April 12, 2018 by guest
http://jcm.asm
.org/D
ownloaded from
16
resistance in Acinetobacter spp. J. Clin. Microbiol. 47:1436-1442. 360
12.Marinach C, A Alanio, M Palous, S Kwesek, A Fekkar, J-Y Brossas, S Brun, G Snounou, 361
C Hennequin, D Sanglard, A Datry J-L Golmard, D Mazier. 2009. MALDI-TOF MS-based 362
drug susceptibility testing of pathogens: The example of Candida albicans and fluconazole. 363
Proteomics 9:4627-4631. 364
13.De Carolis E, A Vella, AR Florio, P Posteraro, DS Perlin, M Sanuinetti, B Posteraro. 365
2012. Use of matrix-assisted laser desorption ionization –time of flight mass spectrometry 366
for caspofungin susceptibility testing of Candida and Aspergillus species. J. Clin. Microbiol. 367
50:2479-2483. 368
14. Soejima T, Minami J, Iwatsuki K. The exclusive use of flow cytometry to evaluate the 369
antibiotic-susceptibility. Biochim Biophys Acta. 2012 Dec;1820(12):1980-6. 370
15. Alvarez-Barrientos A, Arroyo J, Cantón R, Nombela C, Sánchez-Pérez M. Applications 371
of flow cytometry to clinical microbiology. Clin Microbiol Rev. 2000 Apr;13(2):167-95. 372
16.Ramani R, Ramani A, Wong SJ. Rapid flow cytometric susceptibility testing of Candida 373
albicans. J Clin Microbiol. 1997 Sep;35(9):2320-4. 374
17. Rudensky B, Broidie E, Yinnon AM, Weitzman T, Paz E, Keller N, Raveh D. Rapid flow-375
cytometric susceptibility testing of Candida species. J Antimicrob Chemother. 2005 376
Jan;55(1):106-9. Epub 2004 Nov 16. 377
18.Chaturvedi V, Ramani R, Pfaller MA. Collaborative study of the NCCLS and flow 378
cytometry methods for antifungal susceptibility testing of Candida albicans. J Clin Microbiol. 379
2004 May;42(5):2249-51. 380
19. Jepras RI, Paul FE, Pearson SC, Wilkinson MJ. Rapid assessment of antibiotic effects 381
on Escherichia coli by bis-(1,3-dibutylbarbituric acid) trimethine oxonol and flow cytometry. 382
Antimicrob Agents Chemother. 1997 Sep;41(9):2001-5. 383
20. Gauthier C, St-Pierre Y, Villemur R. Rapid antimicrobial susceptibility testing of urinary 384
on April 12, 2018 by guest
http://jcm.asm
.org/D
ownloaded from
17
tract isolates and samples by flow cytometry. J Med Microbiol. 2002 Mar;51(3):192-200. 385
21.Fredricks BA, DeCoster DJ, Kim Y, Sparks N, Callister SM, Schell RF. Rapid 386
pyrazinamide susceptibility testing of Mycobacterium tuberculosis by flow cytometry. J 387
Microbiol Methods. 2006 Nov;67(2):266-72. 388
22.Steinberger-Levy I, Zahavy E, Cohen S, Flashner Y, Mamroud E, Aftalion M, Gur D, Ber 389
R. Enrichment of Yersinia pestis from blood cultures enables rapid antimicrobial 390
susceptibility determination by flow cytometry. Adv Exp Med Biol. 2007;603:339-50. 391
23.Faria-Ramos I, Espinar MJ, Rocha R, Santos-Antunes J, Rodrigues AG, Cantón R, Pina-392
Vaz C. A novel flow cytometric assay for rapid detection of extended-spectrum beta-393
lactamases. Clin Microbiol Infect. 2013 Jan;19(1):E8-E15. 394
24. Godin M, Delgado FF, Son S, Grover WH, Bryan AK, Tzur A, et al. Using buoyant mass 395
to measure the growth of single cells. Nat Methods 2010;7:387-90. 396
25. Knudsen SM, von Muhlen MG, Schauer DB, Manalis SR. Determination of bacterial 397
antibiotic resistance based on osmotic shock response. Anal Chem 2009;81:7087-90. 398
26. Gfeller KY, Nugaeva N, Hegner M. Rapid biosensor for detection of antibiotic-selective 399
growth of Escherichia coli. Appl Environ Microbiol. 2005 May;71(5):2626-31. 400
27. Gfeller KY, Nugaeva N, Hegner M. Micromechanical oscillators as rapid biosensor for 401
the detection of active growth of Escherichia coli. Biosens Bioelectron. 2005 Sep 402
15;21(3):528-33. 403
28.Ndieyira JW, Watari M, Barrera AD, Zhou D, Vögtli M, Batchelor M, Cooper MA, Strunz 404
T, Horton MA, Abell C, Rayment T, Aeppli G, McKendry RA. Nanomechanical detection of 405
antibiotic-mucopeptide binding in a model for superbug drug resistance. Nat Nanotechnol. 406
2008 Nov;3(11):691-6. 407
29.Mortensen NP, Fowlkes JD, Sullivan CJ, Allison DP, Larsen NB, Molin S, Doktycz MJ. 408
Effects of colistin on surface ultrastructure and nanomechanics of Pseudomonas aeruginosa 409
on April 12, 2018 by guest
http://jcm.asm
.org/D
ownloaded from
18
cells. Langmuir. 2009 Apr 9;25(6):3728-33. 410
30. Lee J, Shen W, Payer K, Burg TP, Manalis SR. Toward attogram mass measurements 411
in solution with suspended nanochannel resonators. Nano Lett. 2010 Jul 14;10(7):2537-42 412
31.von Ah U, D Wirz, AU Daniels. 2009. Isothermal micro calorimetry – a new method for 413
MIC determinations: results for 12 antibiotics and reference strains of Escherichia coli and 414
Staphylococcus aureus. BMC Microbiol. 9: 106-120. 415
32.Howell M, D Wirz, AU Daniels, O Braissant. 2012. Application of a microcalorimetric 416
method for determining drug susceptibility in Mycobacterium species. J. Clin. Microbiol. 417
50:16-20. 418
33. Bonkat G, Braissant O, Widmer AF, Frei R, Rieken M, Wyler S, et al. Rapid detection of 419
urinary tract pathogens using micro-calorimetry: principle, technique and first results. BJU Int 420
2012 Feb 7. [Epub ahead of print]. 421
34.Furustrand Tafin U, JF Meis, AJ Trampuz. 2012. Isothermal microcalorimetry for 422
antifungal susceptibility testing of Mucorales, Fusarium spp., and Scedosporium spp. Diagn. 423
Microbiol. Infect. Dis. 73:330-337 424
35.Furustrand Tafin U, M Clauss, PM Hauser, J Bille, JF Meis, A Trampuz. 2012. Isothermal 425
microcalorimetry: a novel method for real-time determination of antifungal susceptibility of 426
Aspergillus species. Clin. Microbiol. Infect. 18:e241-e245. 427
36. Buchholz F, Wolf A, Lerchner J, Mertens F, Harms H, Maskow T. Chip calorimetry for 428
fast and reliable evaluation of bactericidal and bacteriostatic treatments of biofilms. 429
Antimicrob Agents Chemother 2010;54:312–9. 430
37. Kinnunen P, BH McNaughton, T Albertson, I Sinn, S Mofakham, R Elbez, DW Newton, A 431
Hunt, R Kopleman. 2012. Self-assembled magnetic bead biosensor for measuring bacterial 432
growth and antimicrobial susceptibility testing. Small 8:2477-2482. 433
38.Sinn I, T Albertson, P Kinnunen, DN Breslauer, BH McNaughton, MA Burns, R 434
on April 12, 2018 by guest
http://jcm.asm
.org/D
ownloaded from
19
Kopelman. 2012. Asynchronous magnetic bead rotation microviscometer for rapid, sensitive, 435
and label-free studies of bacterial growth and drug sensitivity. Anal Chem. 84: 5250-5256. 436
39. Delmas T, Piraux H, Couffin AC, Texier I, Vinet F, Poulin P, et al. How to prepare and 437
stabilize very small nanoemulsions. Langmuir 2011;27:1683-92. 438
40. Baraban L, Bertholle F, Salverda ML, Bremond N, Panizza P, Baudry J, et al. Millifluidic 439
droplet analyser for microbiology. Lab Chip 2011;11:4057-62. 440
41. Boitard L, Cottinet D, Kleinschmitt C, Bremond N, Baudry J, Yvert G, et al. Monitoring 441
single-cell bioenergetics via the coarsening of emulsion droplets. Proc Natl Acad Sci U S A 442
2012;109:7181-6. 443
42. Wilson SM, al-Suwaidi Z, McNerney R, Porter J, Drobniewski. Evaluation of a new rapid 444
bacteriophage-based method for the drug susceptibility testing of Mycobacterium 445
tuberculosis. Nat Med 1997;3:465-8. 446
43. Banaiee N, Bobadilla-Del-Valle M, Bardarov S Jr, Riska PF, Small PM, Ponce-De-Leon 447
A, et al. Luciferase reporter mycobacteriophages for detection, identification, and antibiotic 448
susceptibility testing of Mycobacterium tuberculosis in Mexico. J Clin Microbiol 449
2001;39:3883-8. 450
44. Smietana M, Bock WJ, Mikulic P, Ng A, Chinnappan R, Zourob M. Detection of bacteria 451
using bacteriophages as recognition elements immobilized on long-period fiber gratings. Opt 452
Express 2011;19:7971-8. 453
45. Traore H, Ogwang S, Mallard K, Joloba ML, Mumbowa F, Narayan K, et al. Low-cost 454
rapid detection of rifampicin resistant tuberculosis using bacteriophage in Kampala, Uganda. 455
Ann Clin Microbiol Antimicrob 2007;6:1. 456
46. Mole R, Trollip A, Abrahams C, Bosman M, Albert H. Improved contamination control for 457
a rapid phage-based rifampicin resistance test for Mycobacterium tuberculosis. J Med 458
Microbiol 2007;56:1334-9. 459
on April 12, 2018 by guest
http://jcm.asm
.org/D
ownloaded from
20
47. Jorgensen JH and Ferraro MJ. Antimicrobial susceptibility testing: a review of general 460
principles and contemporary practices. Clin Infect Dis 2009;49:1749-55. 461
48. Douglas IS, Price C, Overdier K, Thompson K, Wolken B, Metzger S, et al. Rapid 462
Microbiological identification and major drug resistance phenotyping with novel multiplexed 463
automated digital microscopy (MADM) for Ventilator-associated Pneumonia (VAP) 464
Surveillance. Presented at ATS, 2011, Denver, CO. 465
http://www.accelr8.com/docs/ATS_2011a.pdf . 466
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Test Principle
Needs more than 105
cells
Proof of Principle (PoP) or Commer
Avail. (CA)
Costs (High-Low-Inter)
Automor
Manual
TestTime (HR)
ANTIMICROBIAL TESTING
TECHNOLOGY
Detect heteroresist.
Real MIC
CURRENTLY IN
USE
Agar Dilution Testing Growth inhibition on solid media with antibiotics Y CA L M + Y >10 Automated testing VITEK, Phoenix, MicroScan
Monitoring of growth or substrate conversion in a dedicated machine using optics Y CA L A + Y/N <10
Broth Dilution Testing Growth inhibition in liquid media with antibiotics Y CA L M/A + Y >10
Chromogenic Agars Metabolic conversion of chromogenic compounds in agar media Y CA I M + N >10
Disk Diffusion Measurement of growth inhibition around an antibiotic containing disk Y CA L M/A + Y/N >10
Etest Measurement of growth inhibition around a strip containing an antibiotic gradient. Y CA L M/A + Y >10
Fluorescent Life-Dead staining
Microscopy of (non)permeable cells in the presence of fluorescent stains N CA L M - N <1
PCR gene detection DNA amplification N CA I A/M - N <1 Real Time Microscopy Filming bacterial division at the single cell level N CA L M - N <1
NEAR FUTURE ALTERNATIVES
Calorimetrics Detection of heat produced by stressed bacteria N POP NK A - N <5
Cantilever technology Weighing bacterial cells by changes in cantilever vibrations N POP NK A + Y <5
FACS Sizing and measuring differential fluorescence between living and dead cells N CA/POP I A - Y/N <5
Magnetic Bead Spin Changes in spin of beads in a magnetic field as a function of the number of attached bacteria Y POP NK A - N <5
MALDI MS Detection of antibiotic degradation products Y POP L A - N <5 Microdroplets Monitoring of growth or substrate conversion in a nanoliter droplets N POP NK A + Y/N <5 Next Generation Sequencing Sequencing of all cellular DNA and RNA N CA/POP H A - N >10
LONG TERM ALTERNATIVES
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Apoptosis Markers Detection of compounds produced upon programmed cell death Y POP I M/A - N <1 Bacteriophage Amplification Detection of phage reproduction in living cells only N CA I M - N <10
Colorimetric Detection of Cell Respiration
Optical detection of substrate or indicator color change at active cell respiration Y POP L A - N <1
Electronic Noses Direct detection of volatile organic compounds Y POP L M/A - Y <1
Impedance Measurements
Changes in electrical characteristics of suspension with living or dead cells Y POP NK A - N <5
Infrared Spectroscopy Absorption characteristics of bacteria exposed to IR N CA/POP I A + N <5
LC ESI MS Proteomics of living/dead cells and resistance proteins Y POP H A - N <5 Metabolomics (including ROS)
Detection of changes in intracellular composition focused on small molecules Y POP NK A - N <1
Microsound Measurements Measuring vibrational differences between living and dead cells N POP NK A - N <5
NMR Assessment of molecular composition of complex mixtures Y POP NK A - N >10 Raman Spectroscopy Absorption characteristics of bacteria exposed to laser light N CA/POP L A + N <5
RNA Sequencing Definition of gene expression differences by sequencing N POP H A - N >10
Table 1. Partial inventory of contemporary, near and long term alternative methodologies for antimicrobial susceptibility testing.
Legends to Table 1: FACS: Fluorescence Activated Cell Sorting; LC ESI MS: Liquid Chromatography Electron Spray Ionisation Mass Spectrometry; MALDI MS: Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry; NK: Not Known; NMR: Nuclear Magnetic Resonance; ROS: Reactive Oxygen Species.
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AUTHOR BIOGRAPHY 1
2
Professor Alex van Belkum, PhD graduated as a biologist at the University of Leiden, The Netherlands in 3 1983. In 1988, he did his PhD examination in Biochemistry at the same University. In 1996 he received a 4 second PhD in Molecular Microbiology at the Erasmus University, Rotterdam, The Netherlands. Between 5 1988-1990, he was involved in malaria vaccine research as a research scientist at the Biomedical 6 Primate Research Centre BPRC-TNO, Department of Infectious Diseases, Rijswijk, The Netherlands. 7 Between 1990-1991, he was the Head of the Department of Infectious Diseases, MedScand Ingeny B.V., 8 Leiden, The Netherlands after which he joined the Department of Molecular Biology, Diagnostic Centre 9 SSDZ, Delft, The Netherlands (1991-1994) as a staff member. In both positions, his focus was on the 10 development of molecular tests for the detection and characterization of infectious pathogens. From 1994 11 until 2010 he was a staff member at the Erasmus University Medical Center Rotterdam EMCR, 12 Department of Medical Microbiology & Infectious Diseases, Rotterdam, The Netherlands. Between 2002 13 and 2010, van Belkum was the head of the Unit for Research and Development. Since 2003, he been a 14 Professor of Molecular Microbiology at Erasmus MC. From 2010 to 2011 he worked for bioMérieux as 15 R&D Director in the La Balme Microbiology Unit. In 2011, van Belkum became the Corporate Vice 16 President R&D Microbiology at bioMerieux (La Balme les Grottes, France). In his current position at 17 bioMerieux, he heads an international team of microbiology researchers in the field of in vitro diagnostics 18 of bacterial diseases. Professor van Belkum has authored or co-authored more than 440 peer-reviewed 19 publications, 100 chapters in books, and a variety of editorials, letters, etc. Van Belkum is Editor in Chief 20 of the European Journal of Clinical Microbiology and Infectious Diseases 21
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