MINIREVIEW

Next-Generation Antimicrobial Susceptibility Testing

Alex van Belkum,a W. Michael Dunne, Jr.b
bioMérieux SA, Microbiology Unit, R&D Microbiology, La Balme Les Grottes, Francea; bioMérieux, Inc., Microbiology Unit, R&D Microbiology, Durham, North Carolina, USAb

Antimicrobial resistance has emerged as one of the most-significant health care problems of the new millennium, and the clini-
cal microbiology laboratory plays a central role in optimizing the therapeutic management of patients with infection. This mini-
review explores the potential value of innovative methods for antimicrobial susceptibility testing of microorganisms that could
provide valuable alternatives to existing methodologies in the very near future.

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TODAY’S GLOBAL AST LANDSCAPE that provide insight into the wealth of the possibilities to come.
Below, we will briefly describe six technologies that could repre-
A limited number of methods for antimicrobial susceptibility
testing (AST) of medically important microorganisms have
survived the maturation of modern diagnostic clinical microbiol-
sent competition for current reference standard methods—
nucleic acid amplification and sequencing excluded.
ogy. Surprisingly, one of these is the disk diffusion method first
NEAR-FUTURE ALTERNATIVES FOR ROUTINE AST
published in 1966 (1) and the various iterations thereof. Another
is broth microdilution (BMD) testing, which has attained refer- MS. Matrix-assisted laser desorption ionization–time of flight
ence standard status to which all other AST methods are currently mass spectrometry (MALDI-TOF MS), a powerful tool for the
compared during development, verification, validation, and clin- rapid identification of organisms with medical importance, may
ical trials. As such, BMD displaced agar dilution testing, the past also prove to be of value as an AST method (5). Several approaches
gold standard methodology. have been explored, including (i) documenting the activity of an-
The most important outcome of any AST is the rapid and re- tibiotic-inactivating enzymes (e.g., ␤-lactamases), (ii) confirming
liable prediction of antimicrobial success in the treatment of in- the presence of a PCR product indicative of antimicrobial resis-
fection. Currently, AST is typically accomplished using either clas- tance (e.g., vanA, mecA, or NDM-1), and (iii) observing changes
in the protein spectrum of an organism in the presence or absence
sical manual methods or growth-dependent automated systems,
of an antimicrobial agent that correlate with susceptibility
such as the Becton, Dickinson Phoenix, the Siemens Micoscan
changes.
WalkAway, or the bioMérieux Vitek 2, all of which are based on
As an example of the first, carbapenemase activity was detected
BMD testing. The major limitations of these methods include the
in a variety of Gram-negative organisms using ertapenem as a
requirement for relatively large numbers of viable organisms,
substrate (6). A bacterial suspension was incubated with ertap-
complicated preanalytical processing, limited organism spectrum,
enem, and serial samples were examined by MALDI-TOF MS.
analytical variability, time to results, and cost. Table 1 provides a
Organisms producing either NDM-1 or IMP-1 completely hydro-
cursory review of current and future technologies and their lyzed ertapenem within 1 h. It should be noted that four distinct
strengths and weaknesses. peaks were initially observed in the mass spectrum of the parent
At present, nonphenotypic, mostly nucleic acid-based AST drug, which already included the inactive hydrolyzed form. Other
methods cannot detect all resistance markers, are expensive, and carbapenemases (IMP-2, VIM-1, VIM-2, and KPC-2) hydrolyzed
have not been widely adopted. Multiplex PCR detection of resis- the drug more slowly. Similar data were generated using mero-
tance determinants directly from positive blood cultures, how- penem as a substrate for enzymes including NDM-1, VIM-1,
ever, has been shown to substantially reduce the time to clinically KPC-2, KPC-3, OXA-48, and OXA-162 (7). The applicability of
actionable results (2). Furthermore, digital PCR may allow for this method using other substrates (penicillin G, ampicillin,
better quantification of target molecules present in starting mate- cefoxitin, and imipenem) was demonstrated with and without
rial (3), and the refinement of aptamer technology (single- clavulanic acid as a means of distinguishing ␤-lactamase classes
stranded short RNA or DNA molecules with antibody-like prop- such as AmpC or TEM-1 (8). The possibility exists for multiplex-
erties) may further facilitate nucleic acid diagnostics (4). Clearly, ing the assay using combinations of ␤-lactams and inhibitors,
newer-generation, transcriptome, and whole-genome sequencing which would allow classification of extended-spectrum ␤-lacta-
will provide near-future options to resistance prediction as data- mases as well.
bases mature. The per-strain assessment of detailed MICs for all The second approach was presented in a study that compared
relevant antimicrobials may be confounded by elevated degrees of two methods of single-nucleotide polymorphism (SNP) analysis
genetic heterogeneity, as is, for instance, obvious among many for epidemiological typing of 147 strains of methicillin-resistant
Gram-negative bacterial species. This may still frustrate the
genomic approach, but this would be the subject of an entire re-
view by itself and will not be discussed here. Published ahead of print 13 March 2013
Novel options are available to supplant the existing toolbox, Address correspondence to Alex van Belkum, [Link]@[Link].
but the timing of such events is hard to foresee. As a disclaimer, we Copyright © 2013, American Society for Microbiology. All Rights Reserved.
cannot provide a complete survey of all potential AST configura- doi:10.1128/JCM.00313-13
tions but will try to highlight a number of phenotypic methods

2018 [Link] Journal of Clinical Microbiology p. 2018 –2024 July 2013 Volume 51 Number 7
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TABLE 1 Partial inventory of contemporary, near-future, and long-term alternative methodologies for antimicrobial susceptibility testinga
Antimicrobial testing Needs more than POP or Automatic Heteroresistance Test
technology Test principle 105 cells CA Cost or manual detection Real MIC time (h)
Currently in use
Agar dilution testing Growth inhibition on solid medium with Y CA L M ⫹ Y ⬎10
antibiotics
Automated testing (Vitek, Monitoring of growth or substrate conversion Y CA L A ⫾ Y/N ⬍10
Phoenix, MicroScan) in a dedicated machine using optics
Broth dilution testing Growth inhibition in liquid medium with Y CA L M/A ⫾ Y ⬎10
antibiotics
Chromogenic agars Metabolic conversion of chromogenic Y CA I M ⫹ N ⬎10
compounds in agar medium
Disk diffusion Measurement of growth inhibition around an Y CA L M/A ⫹ Y/N ⬎10
antibiotic-containing disk
Etest Measurement of growth inhibition around a Y CA L M/A ⫹ Y ⬎10
strip containing an antibiotic gradient.
Fluorescent live/dead Microscopy of (non)permeable cells in the N CA L M ⫺ N ⬍1

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staining presence of fluorescent stains
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 N POP NK A ⫾ Y ⬍5
cantilever vibrations
FACS Sizing and measuring differential fluorescence N CA/POP I A ⫺ Y/N ⬍5
between living and dead cells
Magnetic bead spin Changes in spin of beads in a magnetic field as Y POP NK A ⫺ N ⬍5
a function of the no. of attached bacteria
MALDI-TOF MS Detection of antibiotic degradation products Y POP L A ⫺ N ⬍5
Microdroplets Monitoring of growth or substrate conversion N POP NK A ⫾ Y/N ⬍5
in nanoliter droplets
Next-generation Sequencing of all cellular DNA and RNA N CA/POP H A ⫺ N ⬎10
sequencing

Long-term alternatives
Apoptosis markers Detection of compounds produced upon Y POP I M/A ⫺ N ⬍1
programmed cell death
Bacteriophage Detection of phage reproduction in living cells N CA I M ⫺ N ⬍10
amplification only
Colorimetric detection of Optical detection of substrate or indicator Y POP L A ⫺ N ⬍1
cell respiration color change at active cell respiration
Electronic noses Direct detection of volatile organic compounds Y POP L M/A ⫺ Y ⬍1
Impedance measurements Changes in electrical characteristics of Y POP NK A ⫺ N ⬍5
suspension with living or dead cells
Infrared spectroscopy Absorption characteristics of bacteria exposed N CA/POP I A ⫾ N ⬍5
to IR
LC-ESI MS Proteomics of living/dead cells and resistance Y POP H A ⫺ N ⬍5
proteins
Metabolomics (including Detection of changes in intracellular Y POP NK A ⫺ N ⬍1
ROS) composition focused on small molecules
Microsound Measuring vibrational differences between N POP NK A ⫺ N ⬍5
measurements living and dead cells
NMR Assessment of molecular composition of Y POP NK A ⫺ N ⬎10
complex mixtures
Raman spectroscopy Absorption characteristics of bacteria exposed N CA/POP L A ⫾ N ⬍5
to laser light
RNA sequencing Definition of gene expression differences by N POP H A ⫺ N ⬎10
sequencing
a
FACS, fluorescence-activated cell sorting; MALDI-TOF MS, matrix-assisted laser desorption ionization–time of flight mass spectrometry; LC-ESI MS, liquid chromatography-
electron spray ionization mass spectrometry; ROS, reactive oxygen species; NMR, nuclear magnetic resonance; Y, yes; N, no; POP, proof of principle; CA, commercially available;
H, high; L, low; I, intermediate; NK, not known; ⫹, detects heteroresistance; ⫾, may detect heteroresistance; ⫺, fails to detect heteroresistance.

Staphylococcus aureus (MRSA) at 16 distinct loci (9). The predi- A combination of a primer extension (PEX) reaction with
cate method of analysis was a real-time SYBR green PCR assay. MALDI-TOF MS also led to the detection of ganciclovir resistance
The comparator method employed the Sequenom MassARRAY mutations in cytomegalovirus (CMV) among viremic heart trans-
iPLEX SNP typing platform (Sequenom, Brisbane, Australia), plant patients (10). Compared to a combination of real-time PCR
which combines multiplexed single-base extension PCR with and Sanger sequencing, the PEX/MALDI-TOF MS method dis-
MALDI-TOF MS of amplicons to determine the location of SNPs. closed resistance mutations earlier without loss of specificity. Sim-
Both methods proved comparable, and the mecA PCR amplicon ilar analyses of PCR-generated amplicons are the basis for resis-
was successfully identified by MALDI-TOF MS for all 147 strains. tance detection using the more-advanced electrospray ionization

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mass spectrometry (PCR/ESI-MS). In one study, the quinolone icant advances in the design and performance of FC instrumenta-
resistance-determining regions of parC and gyrA of multidrug- tion have occurred, the technology has not yet emerged as a major
resistant strains of Acinetobacter spp. were identified with ade- player in the AST market, although commercial assays have been
quate correlation to BMD testing (11). launched. Some of the perceived hurdles for the methodology are,
Finally, there are a number of examples of MALDI-TOF MS among others, the ability to differentiate cellular damage caused
being used to highlight the effects of antimicrobial agents on the by cidal versus static antibiotics, autofluorescence of certain bac-
protein spectral profile of susceptible organisms. Comparisons of terial species, and the tremendous amount of work required for
the profiles of Candida albicans grown in the presence of increas- verification/validation of the clinical database and the method it-
ing concentrations of fluconazole led to the formulation of a min- self.
imal profile change concentration (MPCC) that was defined as the Microbial cell weighing by vibrating cantilevers. Cantilevers
lowest concentration of the drug at which a change in the profile containing small canals which facilitate microbial passage can be
could be documented (12). The authors found a very high con- made to vibrate continuously. When bacteria pass through, their
cordance between the MPCC and the MIC values obtained by the weight (in the femtogram range) will cause a change in the fre-
CLSI broth-based reference method. Similarly, MALDI-TOF MS quency of cantilever movement (24). Less-dense cells will cause a

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was used to assess caspofungin resistance secondary to fks muta- different change than more-dense cells. When cells are treated
tions in 34 Candida species and 10 Aspergillus isolates (13). Strains with antimicrobial agents, their buoyant mass density changes,
were exposed to increasing concentrations of caspofungin in a and this is measurable (25). The principle has been proven using
BMD format, along with a drug-free control well, and incubated ampicillin-resistant and -susceptible variants of Citrobacter roden-
for 15 h prior to MALDI-TOF MS. For each drug concentration, tium. It was also shown that resuscitation of both phenotypes after
an MPCC was calculated for each strain. This group found 100% osmotic shock in the presence or absence of ampicillin allows
essential agreement for all of the isolates using CLSI breakpoints rapid differentiation in a reduced time span. Cantilevers can be
for MIC or minimal effective concentration (MEC). Only two multiplexed using nanotechnology such that multiple antibiotics
Candida isolates were incorrectly interpreted as nonsusceptible, in various concentrations could be tested for a single growing
generating a categorical agreement of 94.1%. culture simultaneously.
FC. Flow cytometry (FC) permits changes in the morphology, A rapid biosensor for the detection of bacterial growth was
physiological and metabolic activity, and viability of microorgan- developed using vibrating cantilevers containing a certain number
isms to be followed after exposure to antibiotics. Through a pro- of fixed but still viable bacteria (26). The change in resonance
cess of staining with nucleic acid dyes that do not permeate the cell frequency as a function of the increasing mass on the cantilever
walls of healthy organisms, the proportion of cells in a dying or forms the basis of the detection scheme. The calculated mass sen-
dead state (and everything in between) can be rapidly assessed by sitivity according to the mechanical properties of the cantilever
examining emission spectra after the cells pass individually sensor is approximately 50 pg/Hz; this mass corresponds to about
through a flow channel and when the dye is excited by a laser (14). 100 E. coli cells. The sensor was able to detect active growth of E.
In early studies, a primitive flow cytometer constructed from a coli cells within 1 h. The number of E. coli cells initially attached to
fluorescence microscope was used to assess cellular morphology the cantilever was on average 1,000 cells. Furthermore, the non-
or DNA after bacteria were exposed to antimicrobial agents. It was inhibited growth of resistant cells could be documented within 2 h
concluded that the effects of antimicrobial treatment could be after the addition of antibiotics (27). Cantilever technology has
detected within a few hours, suggesting a promising application also been used to assess vancomycin binding to cell wall precur-
for AST. The earlier studies were also useful for elucidating vari- sors (28) and to measure the effects of colistin on P. aeruginosa
ous dye/fixation combinations that would better differentiate the (29).
state of cell viability by FC (15). In 1997, propidium iodide was Using suspended nanochannel resonators (SNRs), it was dem-
used to differentiate live/dead C. albicans cells treated with onstrated that the measurement of bacterial mass in solution was
amphotericin B or fluconazole, which could then be rapidly and even more precise. The SNR consisted of a cantilever with an
sensitively quantified by FC (16). Similar assays were developed embedded nanochannel. In addition, a new method was intro-
for the echinocandins, caspofungin, and additional azoles (17). duced that uses centrifugal force caused by vibration of the canti-
The results of these AST strategies were 96 to 99% concordant lever to trap particles at the free end of the SNR (30). This ap-
with other forms of AST (18). The overall duration of AST could proach eliminates the intrinsic position-dependent error of the
be reduced from overnight incubations to 1 to 2 h. Also in 1997, SNR and also improves the mass resolution by increasing the av-
bis(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4[3]) erage “time of presence” for each particle. In addition, it would
was used to visualize anionic membrane potential changes using facilitate the continuous mass monitoring of a limited number of
fluorescence, which proved to be very adequate for AST of Esche- bacteria during (changing) exposure to antibiotics. Clearly, can-
richia coli (19). Tests of organisms causing urinary tract infection tilever systems and precise weight measurements provide an in-
showed 94% agreement between classical disk diffusion testing teresting option for the development of multiplexed AST.
and DiBAC4[3]-FC testing (20). FC AST was also described for IMC. Isothermal microcalorimetry (IMC) is a dynamic tech-
Mycobacterium tuberculosis. Pyrizinamide susceptibility testing by nique that allows the measurement of heat production either as a
FC was 93% concordant with the Bactec MGIT assay, and the flow rate (␮W/unit time) or as total accumulation over time
former was conclusive within 24 h (21). For Yersinia spp. also, (Joules/unit time) stemming from the metabolism of actively
significantly faster testing was achieved using FC (22). FC AST was growing cells. Cumulative heat production generally parallels
at least 20% faster than classical methods for E. coli, Pseudomonas conventional growth curves in that the slope and shape of the
aeruginosa, and S. aureus, and extended-spectrum ␤-lactamases accumulating heat production correspond with classical lag, log,
could be reliably detected by FC in 1 to 2 h (23). Although signif- and stationary phases. Maximum heat values represent the total

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number of cells produced over time (31). The method has been ture. This change can be measured. If all beads in a broth culture
successfully adapted to small culture volumes (e.g., 1 to 3 ml) and are paired with one or two cells, something that can be accom-
can be used in conjunction with either solid or liquid culture me- plished by incubating ligand-modified beads with a diluted bacte-
dium (32). Using IMC, bacterial species identification from urine rial suspension followed by washing, they will resume a constant
specimens was performed even at low bacterial counts within 3 h rotational frequency. As bacteria start to divide, the rotation fre-
on the basis of dynamic heat flow patterns (33). When adapted to quency changes. If cell division is inhibited or blocked by antimi-
AST, measurements of heat production are made passively from crobials due to susceptibility, the change is arrested. If the bacteria
sealed vials containing the organism, growth medium, and anti- are resistant to the antimicrobial applied, again, a change in rota-
microbial agents in doubling-dilution concentrations. The mini- tional frequency occurs. In this way, antimicrobial resistance can
mal heat inhibition concentration (MHIC) can be defined as the be detected and precisely quantified.
lowest drug concentration to either inhibit 50% of total heat pro- A growth-based antimicrobial susceptibility assay based on
duction or result in a 50% reduction in heat flow rate, depending asynchronous magnetic bead rotation (AMBR) biosensors has
on the drug being tested. The process requires specific IMC instru- been described (37). In this system, the effects of bacterial growth
mentation (e.g., TAM III; TA Instruments, New Castle, DE) for
on the rotation and shape of a cluster of self-assembling magnetic

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real-time measurement of heat generation with a detection limit
microbeads in a rotating magnetic field can be observed over time.
in the 0.2-␮W range.
The rotational period (RP) is indirectly proportional to the drag
The use of IMC for susceptibility testing is not new but has
coefficient of the surrounding medium containing bacteria, broth
been successfully adapted for AST of bacteria (31), mycobacteria,
including M. tuberculosis (32), and fungi (34, 35). The advantages medium, and antimicrobial agents at various dilutions. The RP
of this technique include the following: (i) testing is conducted in increases as organisms multiply and attach to the organism-spe-
sealed ampules, alleviating safety concerns when evaluating high- cific antibody-coated beads or if the viscosity of the growth me-
risk organisms, such as M. tuberculosis or fungal species; (ii) all dium is altered. The addition of antimicrobial agents in increasing
monitoring during testing is passive and requires no manual ma- concentrations prevents an increase in RP over time. This process
nipulation of the test vials; and (iii) the completed analysis pro- can be observed directly by illuminating the culture broth (in the
vides information about the maximum growth rate of the organ- form of a hanging drop) with a light-emitting diode (LED) or
ism, static versus cidal activity of an agent, and delays to log-phase laser. The hanging-drop format also acts as a lens to create mag-
growth (extended lag phase) caused by the agent. The latter pro- nification of up to 100⫻ such that the structure and rotational rate
vides useful information concerning antimicrobial activity at sub- of the bead aggregates can be observed microscopically or when
inhibitory concentrations of the drug and can help predict the projected onto a detector. Serial 2-fold dilutions of streptomycin
actual MIC when inhibitory concentrations have not been devel- and gentamicin were added to Mueller-Hinton broth containing
oped (31). This is pronounced with fungal testing, where delays or antibody-sensitized magnetic beads prebound to a standardized
an abbreviation of maximum heat flow can be readily appreciated inoculum of E. coli. A hanging drop was formed with the mixture
with subinhibitory concentrations of antifungal agents (34, 35). that was subjected to an oscillating magnetic field. Changes in the
Furthermore, IMC is not prone to subjective interpretations, such RP were observed microscopically over time and recorded. As
as trailing MBD wells or the determination of MECs based on expected, the RP increased relative to bacterial growth, with solu-
morphological changes at a microscopic level. IMC correlates well tions containing higher concentrations of antibiotic demonstrat-
with BMD testing and CLSI and/or EUCAST breakpoints when ing the lowest increases. This method can be miniaturized to
net heat production over time is used as a surrogate for growth. As nanoliter-volume water-in-oil droplets containing 50 or fewer
a bonus, IMC can be used to evaluate the synergistic activity of bacterial cells per droplet (38). These changes substantially re-
antimicrobial combinations. Chip calorimetry is a monitoring duced the duration of a test.
tool for determining the physiological state of biofilms. Its poten- Testing in microdroplets. Micro- or nanodroplets can be
tial use for the study of the effects of antibiotics was tested using an used as small individual reaction wells. The droplets can be
established model. The real-time monitoring potential of chip cal-
individually manipulated, and when they contain bacteria in
orimetry was successfully demonstrated: a dosage of antibiotics
sufficient numbers, the metabolic activity and viability of cells
initially increased the heat production rate, probably due to activ-
can be monitored. The development of this system became
ity of energy-dependent resistance mechanisms (36). The subse-
feasible once the emulsification process was refined and the
quent reduction in heat production was probably due to the loss of
activity and the death of the biofilm. This new analytical tool pro- long-term stability of the droplets could be guaranteed (39). A
vided fast, quantitative, and mechanistic insights into the effects of system consisting of 100-nl droplets containing 103 bacteria
antibiotics on biofilm activity. In short, the maximum bacterial per droplet and differing concentrations of antibiotics was de-
growth rate and the start of the lag phase can be quantified by veloped (40). By following the droplets over time using epifluo-
microcalorimetric technology in an affordable and sensitive man- rescence, growth curves can be monitored at each drug concen-
ner. Hence, antibiotic-associated changes in these parameters can tration. More recently, droplets were prepared that contain a
be efficiently measured as well. single bacterial cell (41). This technology can be miniaturized
Magnetic bead rotation. When magnetic beads are brought and easily multiplexed with respect to the number of antibiot-
into a revolving magnetic field, they self-assemble and assume a ics tested per organism The duration of testing can be as short
specific rotational spin. The frequency of rotation can be influ- as a single or a few bacterial replication cycles. Obviously, the
enced by the binding of molecules, viruses, or bacteria. So, if the assessment of technical reproducibility and development of
beads are equipped with a ligand that specifically captures bacte- adequate reference MIC databases will be necessary before this
rial cells, the rotation of the beads changes at the moment of cap- approach can be evaluated as a routine AST tool.

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TECHNOLOGIES FOR APPLICATION IN THE DL, Lee AP. 2011. 1-Million droplet array with wide-field fluorescence
MORE-DISTANT FUTURE imaging for digital PCR. Lab Chip 11:3838 –3845.
4. DeGrasse JA. 2012. A single-stranded DNA aptamer that selectively binds
Several innovative AST methods have been designed and pre- to Staphylococcus aureus enterotoxin B. PLoS One 7:e33410. doi:10.1371
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most are far from introduction into routine clinical microbiology. 5. van Belkum A, Welker M, Erhard M, Chatellier S. 2012. Biomedical
Some, including bacteriophage-based AST, may be closer than the mass spectrometry in today’s and tomorrow’s clinical microbiology labo-
ratories. J. Clin. Microbiol. 50:1513–1517.
others. The bacteriophage-based strategy has been successfully 6. Burckhardt I, Zimmermann S. 2011. Using matrix-assisted laser desorp-
adapted for testing of M. tuberculosis (42), with optimization tion ionization–time of flight mass spectrometry to detect carbapenem
achieved by including recombinant phages containing luciferase resistance within 1 to 2.5 hours. J. Clin. Microbiol. 49:3321–3324.
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Carbapenemase activity detection by matrix-assisted laser desorption ion-
formed at low cost in 2 days (44) and could be applied successfully
ization–time of flight mass spectrometry. J. Clin. Microbiol. 49:3222–
in laboratories in developing countries (45). Unfortunately, this 3227.
innovative approach has not been broadly accepted in diagnostic 8. Hooff GP, van Kampen JJA, Meesters RJW, van Belkum A, Goessens
microbiology laboratories, probably due to contamination (46) or WHF, Luider TM. 2012. Characterization of ␤-lactamase enzyme activity

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been achieved. MD, Sloots TP, Nimmo GR. 2011. Comparison of a multiplexed
Real-time microscopy is one of the innovative technologies MassARRAY system with real-time allele-specific PCR technology for
that may be applicable to AST in the not-too-distant future. High- genotyping of methicillin-resistant Staphylococcus aureus. Clin. Micro-
resolution camera-based systems have been commercialized, and biol. Infect. 17:1804 –1810.
10. Zürcher S, Mooser C, Lüthi AU, Mühlemann K, Barbani MT, Mohacsi
these show a high degree of efficacy (47). This technology has been P, Garzoni C, Gorgievski-Hrisoho M, Schaller A, Flatz L. 2012. Sensitive
extended for use with microcolonies and serial photography in the and rapid detection of ganciclovir resistance by PCR based MALDI-TOF
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1980 –1986.
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arrival. The current methods are very solid and well-respected and bility testing of Candida albicans. J. Clin. Microbiol. 35:2320 –2324.
do generally have CE and FDA certification. Any new technology 17. Rudensky B, Broidie E, Yinnon AM, Weitzman T, Paz E, Keller N,
has to compete with current reference standards, and the method Raveh D. 2005. Rapid flow-cytometric susceptibility testing of Candida
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ACKNOWLEDGMENTS
trimethine oxonol and flow cytometry. Antimicrob. Agents Chemother.
The authors are employees of bioMérieux, a company creating and devel- 41:2001–2005.
oping infectious disease diagnostics. No further potential conflicts of in- 20. Gauthier C, St-Pierre Y, Villemur R. 2002. Rapid antimicrobial suscep-
terest relevant to this article are reported. tibility testing of urinary tract isolates and samples by flow cytometry. J.
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July 2013 Volume 51 Number 7 [Link] 2023

Minireview

Alex van Belkum graduated as a biologist at the

University of Leiden, The Netherlands, in 1983.
In 1988, Professor van Belkum did his Ph.D.
examination in Biochemistry at the same uni-
versity. In 1996, he received a second Ph.D. in
Molecular Microbiology at the Erasmus Uni-
versity, Rotterdam, The Netherlands. Between
1988 and 1990, he was involved in malaria vac-
cine research as a research scientist at the Bio-
medical Primate Research Centre (BPRC-
TNO), Department of Infectious Diseases,
Rijswijk, The Netherlands. Between 1990 and 1991, he was the Head of the
Department of Infectious Diseases, MedScand Ingeny B.V., Leiden, The
Netherlands, after which he joined the Department of Molecular Biology,
Diagnostic Centre (SSDZ), Delft, The Netherlands (1991 to 1994), as a staff
member. In both positions, his focus was on the development of molecular

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tests for the detection and characterization of infectious pathogens. From
1994 until 2010, he was a staff member at the Erasmus University Medical
Center Rotterdam (EMCR), Department of Medical Microbiology & Infec-
tious Diseases, Rotterdam, The Netherlands. Between 2002 and 2010, van
Belkum was the head of the Unit for Research and Development. Since 2003,
he has been a Professor of Molecular Microbiology at Erasmus MC. From
2010 to 2011, he worked for bioMérieux as R&D Director in the La Balme
Microbiology Unit. In 2011, Dr. van Belkum became the Corporate Vice
President for Microbiology R&D at bioMérieux (La Balme les Grottes,
France). In his current position at bioMérieux, he heads an international
team of microbiology researchers in the field of in vitro diagnostics of bacte-
rial diseases. Professor van Belkum has authored or coauthored more than
440 peer-reviewed publications, 100 chapters in books, and a variety of edi-
torials, letters, etc. Dr. van Belkum is Editor in Chief of the European Journal
of Clinical Microbiology and Infectious Diseases.

2024 [Link] Journal of Clinical Microbiology