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1 NEXT GENERATION 1 ANTIMICROBIAL SUSCEPTIBILITY TESTING 2 3 Alex van Belkum 1 and Wm. Michael Dunne Jr 2 4 5 bioMérieux SA 1 , Unit Microbiology, R&D Microbiology, La Balme Les Grottes, France; 6 bioMérieux Inc. 2 , Unit Microbiology, R&D Microbiology, Durham NC, USA 7 8 9 10 11 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 17 18 19 20 21 22 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 on April 12, 2018 by guest http://jcm.asm.org/ Downloaded from

Transcript of 1 NEXT GENERATION 1 ANTIMICROBIAL SUSCEPTIBILITY ...

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NEXT GENERATION 1

ANTIMICROBIAL SUSCEPTIBILITY TESTING 2

3

Alex van Belkum1 and Wm. Michael Dunne Jr2 4

5

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

8

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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

17

18

19

20

21

22

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

30

Key Words: Antimicrobial Susceptibility Testing (AST) – antibiotics – antibiogram – drug 31

resistance. 32

33

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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

43

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

72

NEAR FUTURE ALTERNATIVES FOR ROUTINE AST 73

74

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

129

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

163

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

196

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

242

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

276

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

291

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

316

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

324

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

330

REFERENCES 331

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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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