Next Generation Antimicrobial Susceptibility Testing

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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.

TODAY’S GLOBAL AST LANDSCAPE

A

limited number of methods for antimicrobial susceptibility

testing (AST) of medically important microorganisms have survived the maturation of modern diagnostic clinical microbiol-ogy. Surprisingly, one of these is the disk diffusion method first

published in 1966 (1) and the various iterations thereof. Another

is broth microdilution (BMD) testing, which has attained refer-ence standard status to which all other AST methods are currently compared during development, verification, validation, and clin-ical trials. As such, BMD displaced agar dilution testing, the past gold standard methodology.

The most important outcome of any AST is the rapid and re-liable prediction of antimicrobial success in the treatment of in-fection. Currently, AST is typically accomplished using either clas-sical manual methods or growth-dependent automated systems, such as the Becton, Dickinson Phoenix, the Siemens Micoscan WalkAway, or the bioMérieux Vitek 2, all of which are based on BMD testing. The major limitations of these methods include the requirement for relatively large numbers of viable organisms, complicated preanalytical processing, limited organism spectrum,

analytical variability, time to results, and cost.Table 1provides a

cursory review of current and future technologies and their strengths and weaknesses.

At present, nonphenotypic, mostly nucleic acid-based AST methods cannot detect all resistance markers, are expensive, and have not been widely adopted. Multiplex PCR detection of resis-tance determinants directly from positive blood cultures, how-ever, has been shown to substantially reduce the time to clinically

actionable results (2). Furthermore, digital PCR may allow for

better quantification of target molecules present in starting

mate-rial (3), and the refinement of aptamer technology

(single-stranded short RNA or DNA molecules with antibody-like

prop-erties) may further facilitate nucleic acid diagnostics (4). Clearly,

newer-generation, transcriptome, and whole-genome sequencing will provide near-future options to resistance prediction as data-bases mature. The per-strain assessment of detailed MICs for all relevant antimicrobials may be confounded by elevated degrees of genetic heterogeneity, as is, for instance, obvious among many 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.

Novel options are available to supplant the existing toolbox, but the timing of such events is hard to foresee. As a disclaimer, we cannot provide a complete survey of all potential AST configura-tions but will try to highlight a number of phenotypic methods

that provide insight into the wealth of the possibilities to come. Below, we will briefly describe six technologies that could repre-sent competition for current reference standard methods— nucleic acid amplification and sequencing excluded.

NEAR-FUTURE ALTERNATIVES FOR ROUTINE AST

MS. Matrix-assisted laser desorption ionization–time of flight

mass spectrometry (MALDI-TOF MS), a powerful tool for the rapid identification of organisms with medical importance, may

also prove to be of value as an AST method (5). Several approaches

have been explored, including (i) documenting the activity of

an-tibiotic-inactivating enzymes (e.g.,␤-lactamases), (ii) confirming

the presence of a PCR product indicative of antimicrobial

resis-tance (e.g.,vanA,mecA, or NDM-1), and (iii) observing changes

in the protein spectrum of an organism in the presence or absence of an antimicrobial agent that correlate with susceptibility changes.

As an example of the first, carbapenemase activity was detected in a variety of Gram-negative organisms using ertapenem as a

substrate (6). A bacterial suspension was incubated with

ertap-enem, and serial samples were examined by MALDI-TOF MS. Organisms producing either NDM-1 or IMP-1 completely hydro-lyzed ertapenem within 1 h. It should be noted that four distinct peaks were initially observed in the mass spectrum of the parent drug, which already included the inactive hydrolyzed form. Other carbapenemases (IMP-2, VIM-1, VIM-2, and KPC-2) hydrolyzed the drug more slowly. Similar data were generated using mero-penem as a substrate for enzymes including NDM-1, VIM-1,

KPC-2, KPC-3, OXA-48, and OXA-162 (7). The applicability of

this method using other substrates (penicillin G, ampicillin, cefoxitin, and imipenem) was demonstrated with and without

clavulanic acid as a means of distinguishing␤-lactamase classes

such as AmpC or TEM-1 (8). The possibility exists for

multiplex-ing the assay usmultiplex-ing combinations of␤-lactams and inhibitors,

which would allow classification of extended-spectrum␤

-lacta-mases as well.

The second approach was presented in a study that compared two methods of single-nucleotide polymorphism (SNP) analysis for epidemiological typing of 147 strains of methicillin-resistant

Published ahead of print13 March 2013

doi:10.1128/JCM.00313-13

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Staphylococcus aureus(MRSA) at 16 distinct loci (9). The predi-cate method of analysis was a real-time SYBR green PCR assay. The comparator method employed the Sequenom MassARRAY iPLEX SNP typing platform (Sequenom, Brisbane, Australia), which combines multiplexed single-base extension PCR with MALDI-TOF MS of amplicons to determine the location of SNPs.

Both methods proved comparable, and themecAPCR amplicon

was successfully identified by MALDI-TOF MS for all 147 strains.

A combination of a primer extension (PEX) reaction with MALDI-TOF MS also led to the detection of ganciclovir resistance mutations in cytomegalovirus (CMV) among viremic heart

trans-plant patients (10). Compared to a combination of real-time PCR

[image:2.585.44.543.73.582.2]

and Sanger sequencing, the PEX/MALDI-TOF MS method dis-closed resistance mutations earlier without loss of specificity. Sim-ilar analyses of PCR-generated amplicons are the basis for resis-tance detection using the more-advanced electrospray ionization

TABLE 1Partial inventory of contemporary, near-future, and long-term alternative methodologies for antimicrobial susceptibility testinga

Antimicrobial testing

technology Test principle

Needs more than 105

cells

POP or CA Cost

Automatic or manual

Heteroresistance

detection Real MIC Test time (h) Currently in use

Agar dilution testing Growth inhibition on solid medium 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 medium with antibiotics

Y CA L M/A ⫾ Y ⬎10

Chromogenic agars Metabolic conversion of chromogenic compounds in agar medium

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 live/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 no. of attached bacteria

Y POP NK A ⫺ N ⬍5

MALDI-TOF MS Detection of antibiotic degradation products Y POP L A ⫺ N ⬍5 Microdroplets Monitoring of growth or substrate conversion

in 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

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

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

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

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.

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mass spectrometry (PCR/ESI-MS). In one study, the quinolone

resistance-determining regions ofparCandgyrAof

multidrug-resistant strains ofAcinetobacter spp. were identified with

ade-quate correlation to BMD testing (11).

Finally, there are a number of examples of MALDI-TOF MS being used to highlight the effects of antimicrobial agents on the protein spectral profile of susceptible organisms. Comparisons of

the profiles ofCandida albicansgrown in the presence of

increas-ing concentrations of fluconazole led to the formulation of a min-imal profile change concentration (MPCC) that was defined as the lowest concentration of the drug at which a change in the profile

could be documented (12). The authors found a very high

con-cordance between the MPCC and the MIC values obtained by the CLSI broth-based reference method. Similarly, MALDI-TOF MS

was used to assess caspofungin resistance secondary tofks

muta-tions in 34Candidaspecies and 10Aspergillusisolates (13). Strains

were exposed to increasing concentrations of caspofungin in a BMD format, along with a drug-free control well, and incubated for 15 h prior to MALDI-TOF MS. For each drug concentration, an MPCC was calculated for each strain. This group found 100% essential agreement for all of the isolates using CLSI breakpoints for MIC or minimal effective concentration (MEC). Only two

Candidaisolates were incorrectly interpreted as nonsusceptible, generating a categorical agreement of 94.1%.

FC.Flow cytometry (FC) permits changes in the morphology,

physiological and metabolic activity, and viability of microorgan-isms to be followed after exposure to antibiotics. Through a pro-cess of staining with nucleic acid dyes that do not permeate the cell walls of healthy organisms, the proportion of cells in a dying or dead state (and everything in between) can be rapidly assessed by examining emission spectra after the cells pass individually

through a flow channel and when the dye is excited by a laser (14).

In early studies, a primitive flow cytometer constructed from a fluorescence microscope was used to assess cellular morphology or DNA after bacteria were exposed to antimicrobial agents. It was concluded that the effects of antimicrobial treatment could be detected within a few hours, suggesting a promising application for AST. The earlier studies were also useful for elucidating vari-ous dye/fixation combinations that would better differentiate the

state of cell viability by FC (15). In 1997, propidium iodide was

used to differentiate live/dead C. albicans cells treated with

amphotericin B or fluconazole, which could then be rapidly and

sensitively quantified by FC (16). Similar assays were developed

for the echinocandins, caspofungin, and additional azoles (17).

The results of these AST strategies were 96 to 99% concordant

with other forms of AST (18). The overall duration of AST could

be reduced from overnight incubations to 1 to 2 h. Also in 1997, bis(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4[3]) was used to visualize anionic membrane potential changes using

fluorescence, which proved to be very adequate for AST of

Esche-richia coli(19). Tests of organisms causing urinary tract infection showed 94% agreement between classical disk diffusion testing

and DiBAC4[3]-FC testing (20). FC AST was also described for

Mycobacterium tuberculosis. Pyrizinamide susceptibility testing by FC was 93% concordant with the Bactec MGIT assay, and the

former was conclusive within 24 h (21). ForYersiniaspp. also,

significantly faster testing was achieved using FC (22). FC AST was

at least 20% faster than classical methods forE. coli,Pseudomonas

aeruginosa, andS. aureus, and extended-spectrum␤-lactamases

could be reliably detected by FC in 1 to 2 h (23). Although

signif-icant advances in the design and performance of FC instrumenta-tion have occurred, the technology has not yet emerged as a major player in the AST market, although commercial assays have been launched. Some of the perceived hurdles for the methodology are, among others, the ability to differentiate cellular damage caused by cidal versus static antibiotics, autofluorescence of certain bac-terial species, and the tremendous amount of work required for verification/validation of the clinical database and the method it-self.

Microbial cell weighing by vibrating cantilevers.Cantilevers containing small canals which facilitate microbial passage can be made to vibrate continuously. When bacteria pass through, their weight (in the femtogram range) will cause a change in the

fre-quency of cantilever movement (24). Less-dense cells will cause a

different change than more-dense cells. When cells are treated with antimicrobial agents, their buoyant mass density changes,

and this is measurable (25). The principle has been proven using

ampicillin-resistant and -susceptible variants ofCitrobacter

roden-tium. It was also shown that resuscitation of both phenotypes after

osmotic shock in the presence or absence of ampicillin allows rapid differentiation in a reduced time span. Cantilevers can be multiplexed using nanotechnology such that multiple antibiotics in various concentrations could be tested for a single growing culture simultaneously.

A rapid biosensor for the detection of bacterial growth was developed using vibrating cantilevers containing a certain number

of fixed but still viable bacteria (26). The change in resonance

frequency as a function of the increasing mass on the cantilever forms the basis of the detection scheme. The calculated mass sen-sitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to about 100E. colicells. The sensor was able to detect active growth ofE.

colicells within 1 h. The number ofE. colicells initially attached to

the cantilever was on average 1,000 cells. Furthermore, the non-inhibited growth of resistant cells could be documented within 2 h

after the addition of antibiotics (27). Cantilever technology has

also been used to assess vancomycin binding to cell wall

precur-sors (28) and to measure the effects of colistin onP. aeruginosa

(29).

Using suspended nanochannel resonators (SNRs), it was dem-onstrated that the measurement of bacterial mass in solution was even more precise. The SNR consisted of a cantilever with an embedded nanochannel. In addition, a new method was intro-duced that uses centrifugal force caused by vibration of the

canti-lever to trap particles at the free end of the SNR (30). This

ap-proach eliminates the intrinsic position-dependent error of the SNR and also improves the mass resolution by increasing the av-erage “time of presence” for each particle. In addition, it would facilitate the continuous mass monitoring of a limited number of bacteria during (changing) exposure to antibiotics. Clearly, can-tilever systems and precise weight measurements provide an in-teresting option for the development of multiplexed AST.

IMC.Isothermal microcalorimetry (IMC) is a dynamic

tech-nique that allows the measurement of heat production either as a

flow rate (␮W/unit time) or as total accumulation over time

(Joules/unit time) stemming from the metabolism of actively growing cells. Cumulative heat production generally parallels conventional growth curves in that the slope and shape of the accumulating heat production correspond with classical lag, log, and stationary phases. Maximum heat values represent the total

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number of cells produced over time (31). The method has been successfully adapted to small culture volumes (e.g., 1 to 3 ml) and can be used in conjunction with either solid or liquid culture

me-dium (32). Using IMC, bacterial species identification from urine

specimens was performed even at low bacterial counts within 3 h

on the basis of dynamic heat flow patterns (33). When adapted to

AST, measurements of heat production are made passively from sealed vials containing the organism, growth medium, and anti-microbial agents in doubling-dilution concentrations. The mini-mal heat inhibition concentration (MHIC) can be defined as the lowest drug concentration to either inhibit 50% of total heat pro-duction or result in a 50% repro-duction in heat flow rate, depending on the drug being tested. The process requires specific IMC instru-mentation (e.g., TAM III; TA Instruments, New Castle, DE) for real-time measurement of heat generation with a detection limit

in the 0.2-␮W range.

The use of IMC for susceptibility testing is not new but has

been successfully adapted for AST of bacteria (31), mycobacteria,

includingM. tuberculosis(32), and fungi (34,35). The advantages

of this technique include the following: (i) testing is conducted in sealed ampules, alleviating safety concerns when evaluating

high-risk organisms, such asM. tuberculosisor fungal species; (ii) all

monitoring during testing is passive and requires no manual ma-nipulation of the test vials; and (iii) the completed analysis pro-vides information about the maximum growth rate of the organ-ism, static versus cidal activity of an agent, and delays to log-phase growth (extended lag phase) caused by the agent. The latter pro-vides useful information concerning antimicrobial activity at sub-inhibitory concentrations of the drug and can help predict the actual MIC when inhibitory concentrations have not been

devel-oped (31). This is pronounced with fungal testing, where delays or

an abbreviation of maximum heat flow can be readily appreciated

with subinhibitory concentrations of antifungal agents (34,35).

Furthermore, IMC is not prone to subjective interpretations, such as trailing MBD wells or the determination of MECs based on morphological changes at a microscopic level. IMC correlates well with BMD testing and CLSI and/or EUCAST breakpoints when net heat production over time is used as a surrogate for growth. As a bonus, IMC can be used to evaluate the synergistic activity of antimicrobial combinations. Chip calorimetry is a monitoring tool for determining the physiological state of biofilms. Its poten-tial use for the study of the effects of antibiotics was tested using an established model. The real-time monitoring potential of chip cal-orimetry was successfully demonstrated: a dosage of antibiotics initially increased the heat production rate, probably due to

activ-ity of energy-dependent resistance mechanisms (36). The

subse-quent reduction in heat production was probably due to the loss of activity and the death of the biofilm. This new analytical tool pro-vided fast, quantitative, and mechanistic insights into the effects of antibiotics on biofilm activity. In short, the maximum bacterial growth rate and the start of the lag phase can be quantified by microcalorimetric technology in an affordable and sensitive man-ner. Hence, antibiotic-associated changes in these parameters can be efficiently measured as well.

Magnetic bead rotation.When magnetic beads are brought into a revolving magnetic field, they self-assemble and assume a specific rotational spin. The frequency of rotation can be influ-enced by the binding of molecules, viruses, or bacteria. So, if the beads are equipped with a ligand that specifically captures bacte-rial cells, the rotation of the beads changes at the moment of

cap-ture. This change can be measured. If all beads in a broth culture are paired with one or two cells, something that can be accom-plished by incubating ligand-modified beads with a diluted bacte-rial suspension followed by washing, they will resume a constant rotational frequency. As bacteria start to divide, the rotation fre-quency changes. If cell division is inhibited or blocked by antimi-crobials due to susceptibility, the change is arrested. If the bacteria are resistant to the antimicrobial applied, again, a change in rota-tional frequency occurs. In this way, antimicrobial resistance can be detected and precisely quantified.

A growth-based antimicrobial susceptibility assay based on asynchronous magnetic bead rotation (AMBR) biosensors has

been described (37). In this system, the effects of bacterial growth

on the rotation and shape of a cluster of self-assembling magnetic microbeads in a rotating magnetic field can be observed over time. The rotational period (RP) is indirectly proportional to the drag coefficient of the surrounding medium containing bacteria, broth medium, and antimicrobial agents at various dilutions. The RP increases as organisms multiply and attach to the organism-spe-cific antibody-coated beads or if the viscosity of the growth me-dium is altered. The addition of antimicrobial agents in increasing concentrations prevents an increase in RP over time. This process can be observed directly by illuminating the culture broth (in the form of a hanging drop) with a light-emitting diode (LED) or laser. The hanging-drop format also acts as a lens to create

mag-nification of up to 100⫻such that the structure and rotational rate

of the bead aggregates can be observed microscopically or when projected onto a detector. Serial 2-fold dilutions of streptomycin and gentamicin were added to Mueller-Hinton broth containing antibody-sensitized magnetic beads prebound to a standardized

inoculum ofE. coli. A hanging drop was formed with the mixture

that was subjected to an oscillating magnetic field. Changes in the RP were observed microscopically over time and recorded. As expected, the RP increased relative to bacterial growth, with solu-tions containing higher concentrasolu-tions of antibiotic demonstrat-ing the lowest increases. This method can be miniaturized to nanoliter-volume water-in-oil droplets containing 50 or fewer

bacterial cells per droplet (38). These changes substantially

re-duced the duration of a test.

Testing in microdroplets.Micro- or nanodroplets can be used as small individual reaction wells. The droplets can be individually manipulated, and when they contain bacteria in sufficient numbers, the metabolic activity and viability of cells can be monitored. The development of this system became feasible once the emulsification process was refined and the

long-term stability of the droplets could be guaranteed (39). A

system consisting of 100-nl droplets containing 103 _bacteria

per droplet and differing concentrations of antibiotics was

de-veloped (40). By following the droplets over time using

epifluo-rescence, growth curves can be monitored at each drug concen-tration. More recently, droplets were prepared that contain a

single bacterial cell (41). This technology can be miniaturized

and easily multiplexed with respect to the number of antibiot-ics tested per organism The duration of testing can be as short as a single or a few bacterial replication cycles. Obviously, the assessment of technical reproducibility and development of adequate reference MIC databases will be necessary before this approach can be evaluated as a routine AST tool.

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TECHNOLOGIES FOR APPLICATION IN THE MORE-DISTANT FUTURE

Several innovative AST methods have been designed and

pre-sented over the past decade (Table 1, Long-term alternatives), but

most are far from introduction into routine clinical microbiology. Some, including bacteriophage-based AST, may be closer than the others. The bacteriophage-based strategy has been successfully

adapted for testing of M. tuberculosis (42), with optimization

achieved by including recombinant phages containing luciferase

genes (43). It was shown that the luciferase assay could be

per-formed at low cost in 2 days (44) and could be applied successfully

in laboratories in developing countries (45). Unfortunately, this

innovative approach has not been broadly accepted in diagnostic

microbiology laboratories, probably due to contamination (46) or

phage resistance issues. As stated above, as promising as some innovative methods may seem to be, broad acceptance has not yet been achieved.

Real-time microscopy is one of the innovative technologies that may be applicable to AST in the not-too-distant future. High-resolution camera-based systems have been commercialized, and

these show a high degree of efficacy (47). This technology has been

extended for use with microcolonies and serial photography in the presence or absence of antibiotics. Such technologies can generate a “1-day AST” for bacteria from positive blood culture bottles

(48). This demonstrates that simple, seemingly old-fashioned test

formats can still be adapted into systems that better serve the pa-tient’s needs.

Several modern technologies have been proposed as being pos-sible long-term future alternatives to today’s technologies in the

clinical microbiology laboratory; these are briefly described in

Ta-ble 1. Proof of principle has been demonstrated, and in some cases, the first trials of feasibility have been published. However, for all of these strategies, database development has yet to be com-pleted, and until then, the prospects of these technologies remain nebulous.

CONCLUSIONS

Conventional AST has its diagnostic limitations: it is generally time-consuming, and actionable results have a tendency for late arrival. The current methods are very solid and well-respected and do generally have CE and FDA certification. Any new technology has to compete with current reference standards, and the method that shows significant improvements has yet to be published. As we described here, several technologies may be knocking on the door shortly.

ACKNOWLEDGMENTS

The authors are employees of bioMérieux, a company creating and devel-oping infectious disease diagnostics. No further potential conflicts of in-terest relevant to this article are reported.

The classification of the various innovative AST systems in the three categories suggested inTable 1represents the authors’ personal opinions. REFERENCES

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Alex van Belkumgraduated 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 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 ofin vitrodiagnostics 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 theEuropean Journal of Clinical Microbiology and Infectious Diseases.