Screening of Antibiotic Susceptibility to β-Lactam-Induced Elongation

Article pubs.acs.org/ac

Screening of Antibiotic Susceptibility to β-Lactam-Induced Elongation of Gram-Negative Bacteria Based on Dielectrophoresis Cheng-Che Chung,†,‡ I-Fang Cheng,§ Hung-Mo Chen,∥ Heng-Chuan Kan,⊥ Wen-Horng Yang,*,# and Hsien-Chang Chang*,†,∇,◆,∞ †

Institute of Nanotechnology and Microsystems Engineering, National Cheng Kung University, Tainan, Taiwan Department of Material Science and Engineering, National Cheng Kung University, Tainan, Taiwan § National Nano Device Laboratories, National Applied Research Laboratories, Tainan, Taiwan ∥ Department of Pathology, Medical College and Hospital, National Cheng Kung University, Tainan, Taiwan ⊥ National Center for High-performance Computing, National Applied Research Laboratories, Tainan, Taiwan # Department of Urology, Medical College and Hospital, National Cheng Kung University, Tainan, Taiwan ∇ Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan ◆ Center for Micro/Nano Science and Technology, National Cheng Kung University, Tainan, Taiwan ∞ Medical Device Innovation Center, National Cheng Kung University, Tainan, Taiwan ‡

ABSTRACT: We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the β-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA). The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment. Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively. The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose. According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 μm when the bacterial growth is inhibited by the CEZ treatment. In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly. There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria. Thus, the drug resistance of GNB to β-lactam antibiotics can be rapidly assessed. Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method. Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria.

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obtain the diagnostic results, so the increase in efficiency of the antibiotic susceptibility test (AST) are becoming serious problems that must be improved. More techniques for rapid AST, including genotypic,4,5 optical,6,7 microfluidic,8−10 electrochemical,5,11 and electrokinetic methods,12−15 for use in clinical diagnosis recently have been reported. The approaches of genotypic detection, such as DNA array and real-time PCR, can determine the drug resistance of clinical isolates by the identification of specific mutations. However the complex processes including the cell lysis, DNA extraction, and immobilization of probes cannot still be avoided. The labeling and optical facilities would increase the cost, so the optical method may only be used in the specific laboratories not general institutions. The basic idea of microfluidic biochips is to integrate assay

he issue of hospital- and community-acquired infections caused by drug resistant Gram-negative bacteria (GNB) is growing in importance.1 The Enterobacteriaceae of the GNB family is a prevalent cause of bloodstream infections, intraabdominal infections, pneumonias, and urinary tract infections. Since the late 1990s, Enterobacteriaceae, especially Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae), that produce extended-spectrum β-lactamases (ESBLs) have the ability to cause resistance to extended-spectrum cephalosporins and monobactams.2 Unfortunately, the development of novel antibiotics cannot keep up with mutations in multidrug resistant (MDR) bacteria, in which New Delhi metallo-βlactamase 1 (NDM-1) producing bacteria remain susceptible only to colistin and tigecycline;3 and thus, the appropriate and rational antibiotic usage is important to avoid the drug abuse and reduce the emergence of MDR bacteria. The conventional methods of AST, such as broth dilution, disk diffusion, and E-test, usually take 16−24 h even days to © 2012 American Chemical Society

Received: January 11, 2012 Accepted: March 9, 2012 Published: March 9, 2012 3347

dx.doi.org/10.1021/ac300093w | Anal. Chem. 2012, 84, 3347−3354

Analytical Chemistry

Article

Dielectrophoresis. DEP is the motion of dielectric particles in a nonuniform electric field. If particles are more polarizable than the surrounding medium, they will be attracted to the strong region of the electrical field gradient (positive DEP, pDEP); if particles are less polarizable than the surrounding medium, they will be repelled to the weak region of the electrical field gradient (negative DEP, nDEP). The transition frequency point where the behavior of DEP force switches is called the crossover frequency (cof). A bacillus is shaped like a prolate spheroid, so the DEP force expression resembles that used for a sphere

operations such as sample preparation, sample pretreatment, and detection on one chip, and the electrochemical or electrokinetic techniques are easy to combine with microfluidic chips for improving its functionality in biomedical detections. AC electrokinetic techniques have become tremendously popular in recent years because they lend themselves well to miniaturization, with advantages including portability, sample size, cost, etc. Dielectrophoresis (DEP) is the induced motion of particles in response to a nonuniform electric field, and the forces depend on the magnitude of the local electric field as well as the complex permittivities of the particle and the surrounding medium.16 DEP is induced by the electric field gradient generated by the microelectrodes and has been used widely in biomedical and biotechnological applications,17 such as cell sorting,18−21 cell viability,13,15,22−24 and DNA and virus manipulation.25−27 The recent research, which report the antibiotic susceptibility test (AST) of microbes via screening the changes in their dielectric properties, offer novel approaches to decrease the detection time. Hoettges et al. used a DEP-well system to assess the drug resistance of bacteria and found that significant changes in cell characteristics could be observed after 1 h.13 Peitz and Leeuwen reported the AST of E. coli to polymyxin B by monitoring the growth of bacteria based on DEP-facilitated image analysis in a microfluidic channel,14 and the minimum inhibitory concentration (MIC) can be derived from a dose−response plot. Hawkins et al. utilized the DEP characterization of wild-type and ethambutol-treated Mycobacterium smegmatis populations to measure changes in their cellular dielectric properties.15 We recently described a dielectrophoresis based antibiotic susceptibility test (d-AST) that can assess the antibiotic susceptibility of E. coli to cephalexin by monitoring the DEP behavior of elongated bacilli in which the test period can be shortened from days to hours.12 In this study, wild type GNB, such as E. coli and K. pneumoniae, were treated with cefazolin (CEZ) to determine their antibiotic susceptibilities to obtain their MIC values; the cephalosporin-resistant E. coli (CREC) and cephalosporin-resistant K. pneumoniae (CRKP) isolated from the clinical samples were also tested for rapid discrimination of antibiotic resistance. This reported DEP-enabled AST of GNB not only decreases the time required for tests but also assists physicians in curing the infections. Most importantly, the abuse of antibiotics can be avoided and the emergence of MDR bacteria can be reduced.

FDEP = 2πab2 ε*mRe[fCM (ω)]∇E2

(1)

where a and b are the radii of a prolate spheroid (a > b). The Clausius-Mossotti factor ( f CM), which is a function of the complex permittivity of the particle and medium, is defined as fCM (ω) =

ε*p − ε*m 3[ε*m + (ε*p − ε*m)L]

(2)

where the complex permittivity ε* (ε*= ε − iσ/ω) is related to the permittivity (ε) and conductivity (σ) via the AC field frequency (ω) for both the particle (p) and the medium (m). L is the depolarization factor.35 The elongated bacilli are like needle-shaped particles, thus the major axis of the cell is much longer than that of the minor axis (a≫b) and the depolarization factor can be approximated to L∼b2/a2[ln(2a/b)-1]≪1. Therefore, the CM factor can be simplified to [ε*p−ε*m)/ε*m]. According to our previous paper,12 the induced dipole is proportional to the cell length in

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THEORY We use the change in dielectrophoretic characteristics of the elongated bacteria to assess their antimicrobial susceptibility. The mechanism of cell elongation and the DEP of cell model are introduced in the following sections. Cell Elongation. The cephalosporins, which are a class of β-lactam antibiotics, are used in the treatment of infections caused by bacteria. The β-lactam antibiotics inhibit the penicillin binding proteins involved in the polymerization of the peptidoglycan layer in the cell wall.28 The β-lactams-induced cell elongation happens mostly in GNB because of the composition of its cell wall. The cell wall of GNB is only 1−3 layers of the peptidoglycan, and only one layer exists in most E. coli.29,30 According to previous reports, β-lactams-induced cell elongation can be found in species of Escherichia, Klebsiella, Pseudomonas, and Leptospira.31−33 However, in a recent study Gram-positive bacilli like Lactococcus lactis also form the long filaments because of β-lactam antibiotics.34

Figure 1. (a) The QEA includes 8 sets of ITO electrode arrays and the conductive adhesive will be smeared on the leads of electrodes for connecting electric wires. (b) The schematic configuration of the d-AST chip, which consists of a PDMS film and the QEA, includes a function generator, an inverted microscope with a CCD camera, and a computer. 3348



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microtube and centrifuged at 5000 rpm for 5 min. Finally, the supernatant was taken out, and 500 μL of 0.2 M HEPES buffer (σ = 0.3 S/m) was added for the DEP experiments. The bacterial suspension was shaken by using a vortex mixer (Shin Kwang, Taiwan) for 30 s to avoid aggregation of the bacteria. Broth Dilution Method. The antibiotic susceptibilities of E. coli and K. pneumoniae to CEZ were determined using the broth dilution method. The procedure involved preparing a 2-fold dilution of antibiotic solution with trypticase soy broth and then incubating a bacterial suspension of 105 CFU/mL in each tube. Following overnight incubation at 37 °C, MIC could be determined by the change in the turbidity of the bacterial suspension. Fabrication. The quadruple electrode array (QEA), which compared with other configurations of the interdigitated and castellated electrodes,17 are easily used for detecting samples in different droplets. In the quadruple electrodes, bacteria can be concentrated at one spot to improve the detection rate even if the bacterial concentration is low. The QEA was fabricated on indium tin oxide (ITO) glass slides (76 × 26 and 1 mm thick) using standard photolithography techniques as shown in Figure 1(a). The electrode gap had two dimensions, 15 and 50 μm. The cell length of elongated bacteria were over 20 μm even 30 μm with higher doses of CEZ treatment, so the QEA with the larger gap was used to trap bacteria for measuring cell length. The stronger DEP forces, which are produced in the QEA with the smaller gap, are able to manipulate bacteria more

a nonuniform electric field, and the induced charges of the elongated cells at the ends of major axis are greater than those of normal cells. The distance between the induced charges (accumulating at the ends of the bacterium) increases as the bacterium elongates, and so does the magnitude of the dipole moment. Furthermore, the dipole moment changes from negative to positive as cell length increases.36

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MATERIALS AND METHODS Preparation of Antibiotic Medium and Bacteria. Cefazolin (Sigma, USA) was dissolved in a PBS solution (1X PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, pH = 7.4) to prepare a stock solution with a concentration of 1024 μg/mL. The stock solution was then diluted 2-fold with tryptic soy broth (TSB). The wild type E. coli (WTEC) 23525 and 25922 and wild type K. pneumoniae (WTKP) 9997 were purchased from ATCC. The clinical isolates (CREC and CRKP) were obtained from the Department of Pathology at the NCKU Hospital and isolated as part of a research protocol approved by the NCKU Hospital. Bacteria were incubated in shaken culture at 37 °C in TSB medium, and the bacterial concentration was adjusted to 1 × 106 CFU/mL by a densitometer (VITEK 2, BioMérieux). 100 μL of bacterial suspensions and 900 μL of antibiotic solution were then mixed in a tube and incubated at 37 °C in a shaking incubator for 60, 90, and 120 min treatments. After the treatments, 500 μL of the suspension was transferred to a

Figure 2. The populations of CREC, WTEC 23529, and WTEC 25922 were treated with CEZ for 120 min and pipetted on the QEA to trap via pDEP with 10 Vpp and 5 MHz. 3349



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Figure 3. Changes in the cell length of CREC, WTEC 23529, and WTEC 25922 treated with various doses of CEZ were recorded at incubation times (a) 60, (b) 90, and (c) 120 min, respectively.

not change significantly. After incubating for 90 min the WTEC 23529 and 25922 cells treated with 1 μg/mL of CEZ elongated; however, they became shorter when treated with 8 μg/mL of CEZ and incubated for 120 min. The longer cells lysed under the effect of higher antibiotic doses, so the cell length of the WTEC decreased. The change in the pdfs of E. coli cells versus various doses of CEZ after a 120-min incubation is shown in Figure 4. The pdf of CREC remained the same (∼2 MHz);

easily. The PDMS (Dow Corning, USA) block containing wells, which were loaded with the bacterial suspension, was integrated onto the ITO glass. PDMS was mixed with a curing agent in a 10:1 ratio, poured on the silicon wafer, degassed under a vacuum for 45 min, and cured at 65 °C for 90 min. The cured PDMS was removed from the wafer, cut into a rectangle of 65 × 15 mm, and punched with holes. Experimental System. Ten μL of bacterial suspension was pipetted into the PDMS well on the chip. Then, the electrode array was connected to a function generator (Fluke 284, USA) and set to 10 Vpp. The frequency was slowly increased from 0.1 to 10 MHz in order to discriminate the dielectric properties of the bacteria. The experimental images were observed and recorded through an inverted microscope (Olympus IX70, Japan) and a CCD camera (Optronics MicroFire, USA). The schematic configuration of the d-AST chip is shown in Figure 1(b). Data Processing. To measure cell length bacterial cells were arranged on the QEA using field-induced pDEP force. The cell length was then analyzed using image software (FreePlus32). The means and standard errors were obtained through three repeated experiments and 10 cells were taken for analysis from each experiment. Regarding the determination of cof, the status of the cell elongation of bacteria varies due to biodiversity;37 thus, the positive-DEP frequency (pdf) was considered the frequency at which the entire bacteria migrated from the electrode center (nDEP) to the electrode edge (pDEP) with increasing frequency.

Figure 4. The changes in the pdfs of CREC (diamond), WTEC 23529 (square), and WTEC 25922 (triangle) with various antibiotic doses of CEZ recorded after a 120-min incubation.

RESULTS The β-lactam-induced cell elongation was found mostly in GNB, especially the Enterobacteriaceae family. Accordingly, the most important pathogens, E. coli and K. pneumoniae, were used as test samples to determine their antibiotic susceptibilities to CEZ. E. coli. Figure 2 shows the populations of CREC, WTEC 23529, and WTEC 25922 treated with CEZ dosages of 0, 1, 2, and 4 μg/mL for 120 min. These cells were trapped on the QEA with AC conditions of 10 Vpp and 5 MHz. The higher frequency (≥5 MHz) was operated to induce the stronger pDEP force due to the higher value of Re[f CM(ω)]. The CREC population was not inhibited under the CEZ treatment, so the cell division still happened. Therefore, most CREC cells still maintained their original shape and the population increased. With regard to WTEC 23529 and WTEC 25922, the bacteria became elongated and decreased in number. When the populations of E. coli were treated antibiotics, bacteria were inhibited to divide. Furthermore, the cell lysis happened in some bacteria; therefore, the cell number would decrease. The variation in cell length versus the antibiotic dose and incubation time is shown in Figure 3. Few CREC cells elongated under the CEZ treatment, but the average length of whole population did

however, the pdfs of WTEC 23529 and 25922 went down to 0.8 MHz when treated with 1 and 2 μg/mL of CEZ, respectively. After a 90-min incubation, cell elongation did not occur in all wild type cells. Minority normal sized bacteria still existed, so significant changes in the pdfs of WTEC 23529 and 25922 were not observed. K. pneumoniae. In recent years, Klebsiella has become an important pathogen for nosocomial infections, as they cause a range of clinical diseases includes bloodstream infections, urinary tract infections, wound infections, and pneumonia.38 First, the WTKP bacteria were treated with various doses of CEZ for incubations of 60, 90, and 120 min. Then, the cell length and pdf of WTKP were measured at the QEA. In Figure 5, the bacteria were manipulated at the QEA after being treated with CEZ for 90 min, and the obviously different expressions of DEP forces can be observed at the applied frequency of 1 MHz. The population of WTKP exhibited nDEP when the antibiotic dose fell below 1 μg/mL; in contrast, they exhibited pDEP when the antibiotic dose was above 1 μg/mL. The decline in the number of bacteria could also be seen at the applied frequency of 3 MHz, when the antibiotic dose was increased. The bacteria became obviously elongated after a 90-min incubation when the dose of CEZ exceeded 1 μg/mL [Figure 6(a)].

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Figure 5. The population of WTKP 9997 treated with various doses of CEZ after a 90-min incubation was manipulated at the QEA under the applied voltage of 10 Vpp and frequencies of 1 and 3 MHz.

shown in Figure 7(a). The number of bacterial did not decrease as antibiotic doses increased; in contrast, the number of bacteria increased with increasing incubation times. In addition, the pdf of CRKP did not change significantly and stayed at 2 MHz. The cell length of β-lactam-induced filamentous CRKP was still much shorter than that of WTKP, as shown in Figure 7(b).

We also found that the cell length decreased at a CEZ dose of 8 μg/mL after 120-min of incubation. The pdf began to drop at a CEZ dose of 1 μg/mL and an incubation time of 120 min, and a significant change occurred with a dose of 2 μg/mL and 90 min of incubation [Figure 6(b)]. With regard to CRKP, some of the bacteria would elongate under specific CEZ treatment conditions, but they still divide as 3351



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Figure 6. Variation in (a) cell length and (b) pdf of WTKP 9997 for various antibiotic doses of CEZ were recorded at the initial state and after incubating for 60, 90, and 120 min.

Figure 7. (a) The CRKP cells treated with various doses (μg/mL) of CEZ and incubated for 60, 90, and 120 min were manipulated under the AC condition of 10 Vpp and 1 MHz. (b) Variation in the cell length of CRKP, which was treated with various antibiotic doses of CEZ, were recorded after being incubated for 60, 90, and 120 min.

Comparison with Broth Dilution Method. The results of the antibiotic susceptibilities of E. coli using the broth dilution method are shown in Figure 8(a). The MIC values of WTEC 23529 and 25922 were 2 and 4 μg/mL, and the CEZ treatment was ineffective for inhibiting the growth of CREC. Figure 8(b) shows the antibiotic susceptibilities of K. pneumoniae using the broth dilution method. The MIC value of WTKP 9997 was 2 μg/mL, and the CEZ treatment was ineffective for inhibiting the growth of CRKP. However, the turbidity of CRKP suspension in higher antibiotic doses (4 and 8 μg/mL) was slightly less than that what occurred in the absence of CEZ. This means that the part of the K. pneumoniae cells was affected, but others still grew well under the CEZ treatment. Finally, the results of the d-AST are consistent with the results of the broth dilution method, and the AST period can be shortened to less than 2 h.

bacteria treated with CEZ decreased as the bacteria became longer, so pDEP was exhibited early at lower frequencies. The pdfs of WTEC and WTKP whose cell lengths were more than 10 μm were below 1 MHz when the dose of CEZ reached the MIC. The decrease in bacterial cell length for higher antibiotic doses can be explained as follows. The β-lactam-induced cell lysis took place at high antibiotic doses, which leads to cell death, especially in longer bacteria.40 When a cell dies, its cell envelope becomes permeable.41,42 Consequently, the conductivity of the cell envelope increases, and the difference between the conductivities of the surrounding medium and the cell envelope reduces. Thus, the dead cell exhibits a weaker DEP than a live cell. The change in pdf can be on behalf of the bacterial population, but the cell elongation happened on the individual bacterium would not be the same. Therefore, the MIC determined by the change in the pdf of bacteria may be the appropriate method. The dipole moment can be discussed being caused by two factors: the intrinsic polarization of the bacterium and the polarization of the electrical double layer (EDL).36 When the permittivity of the bacterium is smaller than that of the surrounding medium, the intrinsic polarization of the bacterium is negative. On the other hand, the polarization of the EDL is positive as the conductivity of the surrounding medium is higher than that of the cell envelope. The total

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DISCUSSION The bacteria exhibit nDEP under an applied frequency of several hundreds of kHz, as conductivity of the medium was approximately 0.3 S/m.39 According to Castellarnau’s study, Re[f CM] is dominated by conductivity at the low frequency range (1−100 kHz), and the cell exhibits nDEP due to σmem (∼10−4 S/m) < σmedium (0.3 S/m). Re[f CM] is dominated by permittivity at the frequency range of several hundreds of kHz, and the cell still exhibits nDEP due to εmem (10−20 ε0) < εmedium (80 ε0). When the frequency increases to the order of MHz, σcyto becomes dominative. The DEP force of cell becomes positive due to σcyto (0.3−0.4 S/m) > σmedium (0.3 S/m). The pdf of the wild type 3352



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Figure 8. The results of the broth dilution method show the antibiotic susceptibilities of (a) CREC, WTEC 23529, and WTEC 25922 and (b) CRKP and WTKP 9997 to CEZ (μg/mL). (N: negative control).

but not in the number of wild type bacteria. For further work, more clinical samples will be tested using the d-AST to confirm its usability for the diagnosis of clinical samples. We expect that the d-AST system will improve clinical treatment of bacterial infections with the careful use of antibiotics and reduce the emergence of MDR pathogens.

polarization is negative can be explained by the induced field attracting a smaller number of ions in the EDL because of a shorter cell length. In contrast, the total polarization becomes positive when a cell with a longer cell length attracts a larger number of ions in the EDL. Therefore, the number of available ions is related to the cell length. In addition, the electrothermal effect near the microelectrodes was also taken into consideration. The rise in the local temperature induced by the applied AC electric field can be calculated by △T∼σV2rms/k.43 According to the applied voltage of 10 Vpp, the frequency of 100 kHz −10 MHz, the medium conductivity of 0.3 S/m, and the thermal conductivity of k (1.0 W/mK), the temperature raise can be estimated to be ∼7.5 °C. Moreover, each experimental period of bacteria capture by DEP field did not exceed 3 min. Therefore, bacteria would not be damaged under this environment. Cell elongation induced by β-lactam antibiotics in bacteria has been reported in previous papers.28,31−34 However, the relationship between the cell elongation and antibiotic susceptibility has not been discussed. In the previous study12 we first discovered that the pdf of bacteria would decrease as cell length increased with the antibiotic treatment. According to the experimental results, the change in the pdf of the GNB due to β-lactam-induced elongation can rapidly reflect its antibiotic susceptibility and the screening of drug-resistant bacteria from clinical isolates can also be successfully achieved within a couple of hours. In addition, microfluidics-based approaches, gradient generation,8,44,45 and the bacterial culture9,46,47 can be combined with d-AST to improve the functionality of lab-on-a-chip for automated detection.

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AUTHOR INFORMATION

Corresponding Author

*H.-C.C. Phone: +886-6-2757575 Ext. 63426. Fax: +886-62343270. E-mail: [email protected]. W.-H.Y. Phone: +886-6-2766179. Fax: +886-6-2383678. E-mail: whyang@mail. ncku.edu.tw. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by research grants from National Science Council (NSC 99-2628-B-006-001-MY3 and NSC 1002221-E-006-026-MY3) and Multidisciplinary Center of Excellence for Clinical Trial and Research (DOH101-TD-B-111002), Department of Health, Executive Yuan, Taiwan.

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CONCLUSIONS The rapid screening of antibiotic susceptibility for GNB to CEZ was demonstrated successfully based on the d-AST. The drug susceptibility of the most important pathogens, E. coli and K. pneumoniae, to CEZ can be detected within 2 h. According to the results, the pdfs s of wild type bacteria decrease to the order of hundreds of kHz and their cell lengths are more than 10 μm after CEZ treatment. In addition, the growth of the bacterial number in wild type and drug resistant bacteria differ significantly. There is a rise in the number of drug resistant bacteria 3353



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