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Headspace gas chromatographic method for antimicrobial screening: Minimum inhibitory concentration determination
Journal of Pharmaceutical and Biomedical Analysis 181 (2020) 113122
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Headspace gas chromatographic method for antimicrobial screening: Minimum inhibitory concentration determination Menghui Li a,b,1 , Chunyun Zhang a,c,d,1 , Guilin Chen a,c,d , Lutfun Nahar e , Satyajit D. Sarker f , Mingquan Guo a,c,d,∗ a CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, China b University of Chinese Academy of Sciences, Beijing, 100049, China c Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, 430074, China d Innovation Academy for Drug Discovery and Development, Chinese Academy of Sciences, Shanghai, 201203, China e Laboratory of Growth Regulators, Institute of Experimental Botany ASCR & Palack´ y University, Sˇ lechtitel˚ u 27, 78371, Olomouc, Czech Republic f Centre for Natural Products Discovery, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool, L3 3AF, United Kingdom
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Article history: Received 5 November 2019 Received in revised form 19 January 2020 Accepted 20 January 2020 Available online 21 January 2020 Keywords: Antimicrobial activity Headspace analysis Metabolic carbon dioxide Natural products Minimum inhibitory concentration
a b s t r a c t The headspace gas chromatographic method has been widely used to detect volatile metabolites to reflect the growth state of microorganisms, however, it has never been used for the determination of the minimum inhibitory concentration in antibacterial drugs. This paper reports a new method for evaluating the antimicrobial activity of drugs by measuring the amount of CO2 produced by bacterial metabolism after treatment with drugs. According to the amount of CO2 produced by bacterial metabolism, a proper amount of bacterial liquid is selected and added to a drug-containing culture medium as compared with bacteria without drugs in parallel. The amount of CO2 produced by bacteria is measured by using a headspace gas chromatograph coupled with a thermal conductivity detector to measure the bacteriostasis rate and the minimum bacteriostasis concentration of the tested drug, so as to evaluate its antibacterial activity. The accuracy of this method was verified by comparison with the standard method (the OD method), which indicated that the precision was less than 3 % (expressed by relative standard deviation), the inhibition rate (R2 = 0.968) was consistent with the reference method above. This method is simple in operation and can avoid the error caused by the properties of the sample such as volatility, solubility and color in the determination of the minimum inhibitory concentration. It is suitable for the determination of antibacterial activity of drugs, especially natural drugs. © 2020 Elsevier B.V. All rights reserved.
1. Introduction In recent years, with the irrational use of antibacterial drugs, excessive and widespread use of antibiotics have led to bacterial resistance to conventional antibacterial agents at an alarmingly rapid rate, especially with the emergence of multi-drug resistant
Abbreviations: CO2 , carbon dioxide; MIC, Minimum inhibitory concentration; INT, The iodonitrotetrazolium violet; TSB, tryptone soy broth; CFU, Colony-Forming Units; HS-GC, Headspace Gas Chromatography; TCD, thermal conductivity detector; PTFE, Polytetrafluoroethylene. ∗ Corresponding author at: CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, China. E-mail address: [email protected] (M. Guo). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jpba.2020.113122 0731-7085/© 2020 Elsevier B.V. All rights reserved.
bacteria [1–3]. The discovery and development of natural products and/or their derivatives, that can interact with some specific targets in cells, has indeed been the source or inspiration for most new drug candidates for many years, especially in the case of oncological and infectious diseases [4,5]. Abundant scaffold diversity of natural products is an ecological defense system generated by the interaction between the organism and the environment during the growth stage. Because of their coevolution with target sites in biological systems, drug discovery clues obtained from natural products are often of better quality and usually more biologically friendly [6]. Antimicrobial susceptibility tests can be used to predict drug treatment, epidemiology, and treatment outcomes, and also to point out the direction for the development of new drugs. Currently, there are many antimicrobial testing methods for various antimicrobial agents in vitro. Disc diffusion susceptibility is simple
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and easy to operate, which make it widely used in screening and clinical application for antibacterial activity [7]. In the disc diffusion method, a filter paper containing a quantitative antibacterial drug is attached to the surface of the agar, which has been inoculated with the test bacteria, or perforates the culture medium with a hole punch, adds the drug into the hole, then the drug is diffused in the agar medium. After a certain period of cultivation, observing and testing the diameter of the antibacterial diameter can reflect the sensitivity of the test bacteria to the drug. However, for new drug candidates, the minimum inhibitory concentration (MIC) determination is one of the first and most important steps to evaluate the antimicrobial potential, which is impossible to quantify the amount of the antimicrobial agent diffused into the agar medium in the disk diffusion method. MIC is the lowest concentration of antimicrobial agent that can be able to visibly inhibit the growth of microorganisms [8]. Therefore, the accuracy of the minimum inhibitory concentration of the drug is required to be higher and higher, thereby evaluating whether the drug has a good value for further development or whether the strain is resistant. Agar dilution method and broth dilution method are the two most commonly used methods to determine the minimum inhibitory concentration, including the bactericidal or bacteriostatic activity of drugs, and it is usually expressed in mg/mL or mg/L [9]. The agar dilution method mainly involves adding different doses of antibacterial drugs to a quantitative agar medium, which is melted and cooled to about 50 ◦ C to make a plate containing different decreasing concentrations of antibiotics, and then inoculated with the test bacteria; after observing the growth of the bacteria after incubation, the minimum drug concentration contained in the agar plate for inhibiting bacterial growth is MIC [10]. However, the chemical components in the natural product are very complicated, especially the crude extract with complex composition may be denatured at high temperature, when preparing drug-containing solid culture medium by agar dilution method, and it is also easy to cause uneven dispersion of drugs in the medium. In the broth dilution method, the microorganism is inoculated into a liquid medium containing a certain concentration of the test drug (usually by a double dilution method), and after incubation under appropriate conditions, the drug inhibition rate was obtained through the naked eye observation or using spectrophotometer to determine the concentration of microorganisms [11]. In order to obtain reproducible results and determine the endpoint of MIC, broth dilution observation devices need to be able to clearly identify and read the endpoint. In addition, several colorimetric methods based on the use of different dyes have been developed to assist the determination of minimum inhibitory concentration in the broth dilution method [12,13]. The iodonitrotetrazolium violet (INT) is a tetrazolium salt widely used in the MIC determination, which can dye the metabolically active bacteria red. And the color depth is proportional to the number of living cells. However, when the sample itself has some turbidity or color (such as crude extracts of natural products), the measurement and observation results will be biased, especially when using spectrophotometer to determine how many bacteria are there. MICs of antibiotics also can be determined by using commercial test strips containing exponential gradient antibiotics (E-test; AB Biodisk). When the E-test strip is placed on an agar plate that has been inoculated with bacteria, the antibacterial agent on the carrier are quickly and efficiently released into the agar, thereby establishing a continuous gradient of the concentration of the antibacterial agent immediately below the test strip. After incubation, when the growth of the bacteria is clearly identifiable, a symmetrical antibacterial oval ring centered on the test strip can be seen, and the scale (in g/mL units) at the boundary between the edge of the oval ring and the test strip indicates the MIC value [14–16]. Although this method is fast and
easy to operate, it can only be used with antibiotics supplied by the manufacturer and is also expensive. The determination of growth-dependent metabolites is considered to be an indirect method for monitoring microbial behavior, that has been widely used as a biomarker for microbial research according to the metabolic characteristics of microorganisms [17,18]. In these studies, carbon dioxide (CO2 ) is one of the main volatile substances of microbial metabolites, that is to say, the monitoring of microbial cell activity and reproductive capacity may be realized by detecting the amount of CO2 released during microbial metabolism [19,20]. The high resolution of the gas chromatograph, coupled with the highly sensitive thermal conductivity detector, allows it to detect CO2 produced by bacteria at low concentrations, so as to achieve rapid determination of bacterial level and growth rate. Previously, our team has made a headspace analysis of CO2 produced by aerobic bacteria metabolism to evaluate the antimicrobial activity of natural products [21]. However, the determination of MIC has not been introduced in more detail, and the experimental scheme also needs to be improved. Due to the different metabolic characteristics of different bacteria, the amount of CO2 produced is also different, and the experimental method needs to be optimized. 2. Experimental 2.1. Chemicals and materials The culture medium was tryptone soybean broth (TSB culture medium), purchased from Hopebiol CO., LTD. (China). The two standard strains of the bacteria, one Gram-positive bacterial strain, Enterococcus faecalis (ATCC29212) and one Gram-negative bacterial strain, Klebsiella pneumoniae [CMCC (B) 46117] were used for demonstrating the method. The testing drugs, including ceftazidime, gentamicin, erythromycin, ciprofloxacin, and chloramphenicol, were of analytical grade and purchased from commercial sources and used without further purification. All the plant extracts were used as testing subjects, including the bark of Warburgia ugandensis, the root of Mondia whitei, and the bark of Cedrelopsis grevei, were made by solvent extraction in the phytochemistry laboratory in Wuhan Botanical Garden, Chinese Academy of Sciences, and the extraction solvents for Warburgia ugandensis and Cedrelopsis grevei were 95 % ethanol, and the extraction solvents of Mondia whitei were 1-butanol and dichloromethane, respectively. Iodonitrotetrazolium chloride was purchased from Shanghai Macklin biochemical technology. It was prepared with sterile water into a solution with a concentration of 0.02 %, protected from light and stored at 4 ◦ C. The inoculum preparation, the bacteria were inoculated in TSB medium, cultured overnight at 37 ◦ C, and its concentration was measured by plate counting method, then stored in a refrigerator at 4 ◦ C until use. The concentration of bacteria was adjusted to 106 CFU/mL upon test. 2.2. Apparatus and operations Headspace gas chromatography is composed of a gas chromatograph (Agilent 7890 A) connected with a headspace sampler (COLIN AutoHS® ). The GC analysis equipped with GS-Q capillary column (0.53 mm × 30 m × 0.2 m, Agilent) and a TCD detector. The column was set at 105 ◦ C and held for 5 min, using nitrogen as carrier gas with a flow rate of 1 mL/min. Headspace sampler (Colin, China) operating conditions were as follows: oven temperature was 60 ◦ C, transfer line temperature was 90 ◦ C, sample loop temperature was 80 ◦ C; using nitrogen as the vial pressurization gas with 2 min pressing, nitrogen was used as the vial pressurization gas.
M. Li, C. Zhang, G. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 181 (2020) 113122
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And microplate reader (Infinite 200 PRO, Switzerland) was used to detect the turbidity of the bacterial culture medium contained in 96-well plate. 2.3. Determination of the ability of bacteria to produce CO2 Two bacterial solutions (106 CFU/mL) of different volumes were placed in a headspace vial(20 mL), sealed with PTFE/silicone membrane and aluminum lid, and placed in the incubator at a temperature of 37 ◦ C, with the oscillation speed at 80 rpm. After 24 h. incubation, the vial was immediately introduced into the oven of the headspace sampler, incubated at 60 ◦ C for another 10 min, and then injected into GC-TCD for analysis. 2.4. Sample preparation using HS-GC method Enterococcus faecalis (1 mL) and 1 mL of TSB medium containing the test drug at the target concentration were added to a vial for sealing, and the cells were cultured and measured. In order to determine the MIC, a set of medium with a series of drug concentrations was prepared and added for cultivation. After the culture, a part of the solution was added with 0.4 mL of INT solution by syringe, incubated at 37 ◦ C for 30 min to observe the bacterial staining, and the other part was determined by gas chromatography. 0.3 mL of Klebsiella pneumoniae and 0.3 mL of TSB medium containing testing drugs were taken for culture, and 0.12 mL INT solution was added for staining observation. For a batch of samples, vials can also be stored at −20 ◦ C after 24 h of incubation, and then rapidly melted to room temperature using a hot air blower, finally introduced into a headspace oven for another 10 min for balance prior to analysis. 2.5. Optical density test After HS-GC-TCD test, the vial was shaken up and opened. The bacterial liquid was added into the 96-well plate with a pipette gun, and repeated for three times with 100 L per well. The absorbance at 625 nm was detected by microplate reader. For plant extracts, in order to avoid the influence of color and turbidity caused by the sample itself, the drug-containing medium with the same concentration was prepared and its optical density was determined for background subtraction. 3. Results and discussion 3.1. The principle of the method This method is based on the determination of the amount of CO2 produced by bacterial metabolism, which is proportional to the number of cells, that is, the amount of CO2 is used to reflect the number of cells. When drugs that inhibit bacterial growth are added, bacteria are killed or microbial activity is reduced, so the amount of CO2 produced by metabolism is reduced. Therefore, this method needs to determine the total amount of CO2 produced by bacteria and the proportional relationship between the number of bacterial cells and the amount of CO2 metabolized. In gas chromatography (GC) analysis, the total amount of CO2 metabolized by microorganisms can be quantified by the peak area of CO2 . At a given temperature, CO2 will reach an equilibrium state in the liquid and gas phases in the headspace vials, which can be detected by GC equipped with automatic headspace sampler and thermal conductivity detector. In order to make the result more accurate, the measured CO2 signal value of the sample needs to be performed within the detection linear range of the instrument [21]. It is a complicated biochemical process that bacteria metabolize nutrients to produce gas. The gas production and gas composition
Fig. 1. The Ability for both Gram-positive and Gram-negative bacteria to produce CO2 by metabolism. Different volumes of bacterial (Enterococcus faecalis and Klebsiella pneumoniae) were cultured for 24 h.
are not only related to bacteria, but also related to the nutrient components on which bacteria grow, especially carbon source, and the growth environment, such as temperature, salinity and pH value. Therefore, in order to increase the application scope and accuracy of this method, we measured the CO2 -producing abilities of one Gram-positive bacterial strain (Enterococcus faecalis) and one Gram-negative bacterial strain (Klebsiella pneumoniae), as shown in Fig. 1. Under the same culture condition, the amount of CO2 produced by Klebsiella pneumoniae was higher than that of Enterococcus faecalis. Therefore, when using this method to determine the antibacterial activity of testing drugs, an appropriate amount of bacterial liquid should be selected according to the linear range of CO2 detection by GC. In order to establish this method, it was necessary to verify the ratio of CO2 produced by microbial metabolism to the number of bacterial cells. Therefore, we used this method to measure the growth curves of Enterococcus faecalis and Klebsiella pneumoniae, and compared them with optical density method for verification. The optical density method reflects the number of cells based on the turbidity of colonies in liquid, the more turbid the bacterial liquid is, the more bacteria it contains. The results are shown in Fig. 2a and b. Between the two detection methods, the growth curve of bacteria showed the same trend, and the growth process of bacteria had gone through the adjustment period, logarithmic growth period and stable growth period, which were clarified in several other studies [22]. At 24 h, the signal was relatively stable, and it was also the time for routine bacteriostatic activity measurement. Therefore, we could establish a proportional relationship between the amount of CO2 metabolized and the number of bacterial cells [19,23]. 3.2. Bacteria growth with and without antimicrobial drug There are many applications for bacterial growth detection by headspace analysis of bacterial metabolites, but no detection of disorders in metabolic system after addition of testing drugs [22,23]. To verify the changes in the growth state of bacteria after the addition of testing drugs, two kinds of bacteria were treated with and without drugs, and measured the carbon dioxide content of timedependent metabolism, as shown in Fig. 3. When 15 g/mL of ceftazidime was added, the increase trend of CO2 of these two bacterial strains was significantly reduced, and after about 18 h of culture, the number of bacteria began to increase rapidly. The results showed that this method could sensitively detect the change of bacterial reaction to drugs. Meanwhile, according to the change
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M. Li, C. Zhang, G. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 181 (2020) 113122 Table 1 The comparison of the precision between the HS-GC method and the OD method for the determination of bacterial activity. Strains
Enterococcus faecalis
Klebsiella pneumoniae
Fig. 2. The growth curve of Gram-positive bacterium (a) and Gram-negative bacterium (b) obtained by headspace gas chromatographic (HS-GC) method and optical density method. 2 mL of Enterococcus faecalis and 0.6 mL of Klebsiella pneumoniae were cultured for different periods of time.
Peak area (CO2 )
34.9 35.8 33.6 34.5 34.3 96.1 100 94 97.6 98
OD (625 nm)
0.1974 0.208 0.2048 0.2124 0.2122 0.7402 0.7476 0.738 0.7598 0.7882
Relative standard deviation, % HS-GC method
OD method
2.34
3.00
2.31
2.72
Fig. 4. Comparison of the inhibition rates obtained for both pure compounds and plant extracts by the present method and the reference method (optical density method). 2 mL of Enterococcus faecalis and 0.6 mL of Klebsiella pneumoniae were cultured with different antibiotics and crude plant extracts for 24 h to determine the bacteriostasis rate, respectively.
time, the optical density method was measured at an optical density value of 625 nm, and each group were made up to 5 repetitions. The results are shown in Table 1. It could be observed that the HSGC had very good precision compared with the reference method (optical density method), and the relative standard deviations of the two strains were less than 3 %.
Fig. 3. The time-dependent metabolic carbon dioxide of the two bacteria (Enterococcus faecalis and Klebsiella pneumoniae) in the headspace vials with and without drug (15 g/mL ceftazidime) treatment. 2 mL of Enterococcus faecalis and 0.6 mL of Klebsiella pneumoniae were added with 15 g/mL of ceftazidime, respectively, and cultured for different periods of time.
of bacterial concentration, the time and dose of clinical medication could be guided. 3.3. Evaluation of the method 3.3.1. Precision To verify the precision of the method, 0.5 g/mL chloramphenicol was added to Enterococcus faecalis, and Klebsiella pneumoniae was treated with 15 g/mL ceftazidime. After 24 h of culture, the amount of CO2 was determined by HS-GC method, at the same
3.3.2. Accuracy The accuracy of the method was verified by comparing the inhibition rates of pure compounds and plant extracts on bacteria by the two methods. As shown in Fig. 4, the inhibition rate (R2 = 0.968) of this method is consistent with that of the reference method. However, when the extract itself had some color or turbidity and was tested by optical density method, there was a significant error, because the color or turbidity only occured in the drug treatment group, but not in the control group. In this work, the comparison between the two methods was based on the results obtained after correcting the optical density method by subtracting the optical density of the background of drug solution. As compared with the traditional method in use, this method used a closed system (headspace vial) for culture, and operated at room temperature, so that the specific components of volatile plant extracts (such as essential oils) could be greatly reduced. Meanwhile, this method also adopted a shaking culture system, in which the microorganisms could fully react with the plant extract, especially for those components with poor solubility and easy to cause precipitation. In addition, some plant extracts, especially crude plant extracts, had relatively complex components, many of which contained pig-
M. Li, C. Zhang, G. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 181 (2020) 113122 Table 2 MICs of antibiotics determined by the present method and reference method (INT staining method). Strains
Klebsiella pneumoniae Enterococcus faecalis
antibiotic
Ceftazidime Ciprofloxacin Gentamicin Chloromycetin
MIC(ug/mL) reference method
present method
GC signal for CO2
32 64 32 16
32 64 32 16
<3 <3 <3 <3
Bacteria were added to the drug culture using a 2-fold gradient dilution method. The reference method was added to INT to determine the minimum inhibitory concentration, and the CO2 signal value was measured by GC.
ments and insoluble components. When measuring antibacterial activity by the traditional optical density method, great background absorbance would be generated, and the background deduction was often inaccurate, especially with high concentration. More recently, the effect of the optical density method on the background absorbance of plant extracts has been experimentally described in our previous work [21]. Since this method was adopted to measure the microbial metabolites (metabolic CO2 ), it could avoid the background error caused by the plant extract itself. Obviously, this method is simplerand with lower possibility of microbial contamination, and the nature of the test sample is more flexible.
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4. Conclusions A new method for the determination of antibacterial activity and the minimum inhibitory concentration of testing drugs by HSGC was developed and verified. The amount of cells was reflected by measuring CO2 produced via microbial metabolism by HS-GC, and the experimental principle of this method was verified. Compared with other standard method in use, this method is proven to have good accuracy and precision in this work. The method is simple to operate and suitable for the determination of antibacterial activity of drugs, especially for natural products. Compared to those traditional methods, this method avoids the error caused by the characteristics of the testing sample itself, such as volatility, solubility and color in the determination of the minimum inhibitory concentration, and can be easily carried out in any microbiology laboratory. Author statement M.Q.G. conceived of, designed and supervised the study. M.H.L. and C.Y. Z. performed the experiments, analyzed the data and wrote the manuscript. G.L.C. participated in the methodological assays and revised the manuscript for submission. In addition to advisory role in the project, L.N. and S.D.S contributed to design, preparation and editing of the manuscript. All authors reviewed and approved the final manuscript. Declaration of Competing Interest
3.4. Determination of minimum inhibitory concentration Studies have shown that tetrazolium salt could form a red compound in the presence of bacteria, which is proportional to the number of active cells and can be used as an indicator for determining the minimum inhibitory concentration of testing drugs. Because of the color and solubility of natural products, it is often difficult to observe the phenomenon above after staining, and in some cases because of bacterial metabolism, indicators cannot make it colored. However, the CO2 produced by the bacterial metabolism can be sensitively detected by GC. The minimum inhibitory concentration (MIC) is the lowest drug concentration that can inhibit bacterial growth, at which the bacteria are killed or its growth is inhibited (the bacterial concentration is the initial concentration). Therefore, at MIC, the bacterial solution was colorless in the method of staining living cells with dyes, while the signals detected by HS-GC were less than 3 because the bacteria did not metabolize CO2 or produced very little CO2 , as shown in Table 2. The MIC value was used to determine the sensitivity of bacteria to drugs, which was also important in evaluating the activity of novel antimicrobial agents [24]. Strong coloring compounds or plant extracts may make MIC value unable to be determined by broth microdilution to exclude the difference between bacterial growth and culture medium [25]. On the other hand, insoluble precipitation caused by some drug samples in the broth microdilution method could also result in a decrease in the contact of the testing compound with the bacteria, thereby limiting their activity. Agar dilution method needs a large amount of drugs, which makes the method limited when the antimicrobial agents are precious or only at the microgram level. At the same time, it is easy to cause the loss of volatile drug components in the process of operation, and in some cases, the insoluble components are difficult to diffuse, resulting in inaccurate results. However, the HS-GC method in this work successfully avoids the hitherto encountered problems associated with testing samples when determining the minimum inhibitory concentration of test drugs to get reliable results, especially for natural products with strong coloring molecules and insolubility.
The authors declare that there is no conflict of interest. Acknowledgements This work is partially supported by Major Project for Special Technology Innovation of Hubei Province (Grant No. 2017AHB054 to M. Guo). In addition, L Nahar also gratefully acknowledges the financial support of the European Regional Development Fund Project ENOCH (No. CZ.02.1.01/0.0/0.0/16 019/0000868). References [1] C. Costelloe, C. Metcalfe, A. Lovering, D. Mant, A.D. Hay, Effect of antibiotic prescribing in primary care on antimicrobial resistance in individual patients: systematic review and meta-analysis, BMJ 340 (2010) c2096. [2] G. Kaneti, H. Sarig, I. Marjieh, Z. Fadia, A. Mor, Simultaneous breakdown of multiple antibiotic resistance mechanisms in S. aureus, FASEB J. 27 (2013) 4834–4843. [3] W. Goettsch, W. van Pelt, N. Nagelkerke, M.G. Hendrix, A.G. Buiting, P.L. Petit, L.J. Sabbe, A.J. van Griethuysen, A.J. de Neeling, Increasing resistance to fluoroquinolones in escherichia coli from urinary tract infections in The Netherlands, J. Antimicrob. Chemother. 46 (2) (2000) 223–228. [4] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs from 1981 to 2014, J. Nat. Prod. 79 (2016) 629–661. [5] D.A. Dias, S. Urban, U. Roessner, A historical overview of natural products in drug discovery, Metabolites 2 (2012) 303–336. [6] B.B. Mishra, V.K. Tiwari, Natural products: an evolving role in future drug discovery, Eur. J. Med. Chem. 46 (2011) 4769–4807. [7] C. McNulty, R. Owen, D. Tompkins, P. Hawtin, K. McColl, A. Price, G. Smith, L. Teare, P.H.W. Grp, Helicobacter pylori susceptibility testing by disc diffusion, J. Antimicrob. Chemoth. 49 (2002) 601–609. [8] K.M. Kuper, D.M. Boles, J.E. Mohr, A. Wanger, Antimicrobial susceptibility testing: a primer for clinicians, Pharmacotherapy. 29 (2009) 1326–1343. [9] I. Wiegand, K. Hilpert, R.E.W. Hancock, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances, Nat. Protoc. 3 (2008) 163–175. [10] J. Golus, R. Sawicki, J. Widelski, G. Ginalska, The agar microdilution method - a new method for antimicrobial susceptibility testing for essential oils and plant extracts, J. Appl. Microbiol. 121 (2016) 1291–1299. [11] B.A. Arthington-Skaggs, W. Lee-Yang, M.A. Ciblak, J.P. Frade, M.E. Brandt, R.A. Hajjeh, L.H. Harrison, A.N. Sofair, D.W. Warnock, C.A.S. Grp, Comparison of visual and spectrophotometric methods of broth microdilution MIC end point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of fluconazole and itraconazole against trailing and
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Headspace gas chromatographic method for antimicrobial screening: Minimum inhibitory concentration determination Menghui Li a,b,1 , Chunyun Zhang a,c,d,1 , Guilin Chen a,c,d , Lutfun Nahar e , Satyajit D. Sarker f , Mingquan Guo a,c,d,∗ a CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, China b University of Chinese Academy of Sciences, Beijing, 100049, China c Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, 430074, China d Innovation Academy for Drug Discovery and Development, Chinese Academy of Sciences, Shanghai, 201203, China e Laboratory of Growth Regulators, Institute of Experimental Botany ASCR & Palack´ y University, Sˇ lechtitel˚ u 27, 78371, Olomouc, Czech Republic f Centre for Natural Products Discovery, School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool, L3 3AF, United Kingdom
a r t i c l e
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Article history: Received 5 November 2019 Received in revised form 19 January 2020 Accepted 20 January 2020 Available online 21 January 2020 Keywords: Antimicrobial activity Headspace analysis Metabolic carbon dioxide Natural products Minimum inhibitory concentration
a b s t r a c t The headspace gas chromatographic method has been widely used to detect volatile metabolites to reflect the growth state of microorganisms, however, it has never been used for the determination of the minimum inhibitory concentration in antibacterial drugs. This paper reports a new method for evaluating the antimicrobial activity of drugs by measuring the amount of CO2 produced by bacterial metabolism after treatment with drugs. According to the amount of CO2 produced by bacterial metabolism, a proper amount of bacterial liquid is selected and added to a drug-containing culture medium as compared with bacteria without drugs in parallel. The amount of CO2 produced by bacteria is measured by using a headspace gas chromatograph coupled with a thermal conductivity detector to measure the bacteriostasis rate and the minimum bacteriostasis concentration of the tested drug, so as to evaluate its antibacterial activity. The accuracy of this method was verified by comparison with the standard method (the OD method), which indicated that the precision was less than 3 % (expressed by relative standard deviation), the inhibition rate (R2 = 0.968) was consistent with the reference method above. This method is simple in operation and can avoid the error caused by the properties of the sample such as volatility, solubility and color in the determination of the minimum inhibitory concentration. It is suitable for the determination of antibacterial activity of drugs, especially natural drugs. © 2020 Elsevier B.V. All rights reserved.
1. Introduction In recent years, with the irrational use of antibacterial drugs, excessive and widespread use of antibiotics have led to bacterial resistance to conventional antibacterial agents at an alarmingly rapid rate, especially with the emergence of multi-drug resistant
Abbreviations: CO2 , carbon dioxide; MIC, Minimum inhibitory concentration; INT, The iodonitrotetrazolium violet; TSB, tryptone soy broth; CFU, Colony-Forming Units; HS-GC, Headspace Gas Chromatography; TCD, thermal conductivity detector; PTFE, Polytetrafluoroethylene. ∗ Corresponding author at: CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, China. E-mail address: [email protected] (M. Guo). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jpba.2020.113122 0731-7085/© 2020 Elsevier B.V. All rights reserved.
bacteria [1–3]. The discovery and development of natural products and/or their derivatives, that can interact with some specific targets in cells, has indeed been the source or inspiration for most new drug candidates for many years, especially in the case of oncological and infectious diseases [4,5]. Abundant scaffold diversity of natural products is an ecological defense system generated by the interaction between the organism and the environment during the growth stage. Because of their coevolution with target sites in biological systems, drug discovery clues obtained from natural products are often of better quality and usually more biologically friendly [6]. Antimicrobial susceptibility tests can be used to predict drug treatment, epidemiology, and treatment outcomes, and also to point out the direction for the development of new drugs. Currently, there are many antimicrobial testing methods for various antimicrobial agents in vitro. Disc diffusion susceptibility is simple
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and easy to operate, which make it widely used in screening and clinical application for antibacterial activity [7]. In the disc diffusion method, a filter paper containing a quantitative antibacterial drug is attached to the surface of the agar, which has been inoculated with the test bacteria, or perforates the culture medium with a hole punch, adds the drug into the hole, then the drug is diffused in the agar medium. After a certain period of cultivation, observing and testing the diameter of the antibacterial diameter can reflect the sensitivity of the test bacteria to the drug. However, for new drug candidates, the minimum inhibitory concentration (MIC) determination is one of the first and most important steps to evaluate the antimicrobial potential, which is impossible to quantify the amount of the antimicrobial agent diffused into the agar medium in the disk diffusion method. MIC is the lowest concentration of antimicrobial agent that can be able to visibly inhibit the growth of microorganisms [8]. Therefore, the accuracy of the minimum inhibitory concentration of the drug is required to be higher and higher, thereby evaluating whether the drug has a good value for further development or whether the strain is resistant. Agar dilution method and broth dilution method are the two most commonly used methods to determine the minimum inhibitory concentration, including the bactericidal or bacteriostatic activity of drugs, and it is usually expressed in mg/mL or mg/L [9]. The agar dilution method mainly involves adding different doses of antibacterial drugs to a quantitative agar medium, which is melted and cooled to about 50 ◦ C to make a plate containing different decreasing concentrations of antibiotics, and then inoculated with the test bacteria; after observing the growth of the bacteria after incubation, the minimum drug concentration contained in the agar plate for inhibiting bacterial growth is MIC [10]. However, the chemical components in the natural product are very complicated, especially the crude extract with complex composition may be denatured at high temperature, when preparing drug-containing solid culture medium by agar dilution method, and it is also easy to cause uneven dispersion of drugs in the medium. In the broth dilution method, the microorganism is inoculated into a liquid medium containing a certain concentration of the test drug (usually by a double dilution method), and after incubation under appropriate conditions, the drug inhibition rate was obtained through the naked eye observation or using spectrophotometer to determine the concentration of microorganisms [11]. In order to obtain reproducible results and determine the endpoint of MIC, broth dilution observation devices need to be able to clearly identify and read the endpoint. In addition, several colorimetric methods based on the use of different dyes have been developed to assist the determination of minimum inhibitory concentration in the broth dilution method [12,13]. The iodonitrotetrazolium violet (INT) is a tetrazolium salt widely used in the MIC determination, which can dye the metabolically active bacteria red. And the color depth is proportional to the number of living cells. However, when the sample itself has some turbidity or color (such as crude extracts of natural products), the measurement and observation results will be biased, especially when using spectrophotometer to determine how many bacteria are there. MICs of antibiotics also can be determined by using commercial test strips containing exponential gradient antibiotics (E-test; AB Biodisk). When the E-test strip is placed on an agar plate that has been inoculated with bacteria, the antibacterial agent on the carrier are quickly and efficiently released into the agar, thereby establishing a continuous gradient of the concentration of the antibacterial agent immediately below the test strip. After incubation, when the growth of the bacteria is clearly identifiable, a symmetrical antibacterial oval ring centered on the test strip can be seen, and the scale (in g/mL units) at the boundary between the edge of the oval ring and the test strip indicates the MIC value [14–16]. Although this method is fast and
easy to operate, it can only be used with antibiotics supplied by the manufacturer and is also expensive. The determination of growth-dependent metabolites is considered to be an indirect method for monitoring microbial behavior, that has been widely used as a biomarker for microbial research according to the metabolic characteristics of microorganisms [17,18]. In these studies, carbon dioxide (CO2 ) is one of the main volatile substances of microbial metabolites, that is to say, the monitoring of microbial cell activity and reproductive capacity may be realized by detecting the amount of CO2 released during microbial metabolism [19,20]. The high resolution of the gas chromatograph, coupled with the highly sensitive thermal conductivity detector, allows it to detect CO2 produced by bacteria at low concentrations, so as to achieve rapid determination of bacterial level and growth rate. Previously, our team has made a headspace analysis of CO2 produced by aerobic bacteria metabolism to evaluate the antimicrobial activity of natural products [21]. However, the determination of MIC has not been introduced in more detail, and the experimental scheme also needs to be improved. Due to the different metabolic characteristics of different bacteria, the amount of CO2 produced is also different, and the experimental method needs to be optimized. 2. Experimental 2.1. Chemicals and materials The culture medium was tryptone soybean broth (TSB culture medium), purchased from Hopebiol CO., LTD. (China). The two standard strains of the bacteria, one Gram-positive bacterial strain, Enterococcus faecalis (ATCC29212) and one Gram-negative bacterial strain, Klebsiella pneumoniae [CMCC (B) 46117] were used for demonstrating the method. The testing drugs, including ceftazidime, gentamicin, erythromycin, ciprofloxacin, and chloramphenicol, were of analytical grade and purchased from commercial sources and used without further purification. All the plant extracts were used as testing subjects, including the bark of Warburgia ugandensis, the root of Mondia whitei, and the bark of Cedrelopsis grevei, were made by solvent extraction in the phytochemistry laboratory in Wuhan Botanical Garden, Chinese Academy of Sciences, and the extraction solvents for Warburgia ugandensis and Cedrelopsis grevei were 95 % ethanol, and the extraction solvents of Mondia whitei were 1-butanol and dichloromethane, respectively. Iodonitrotetrazolium chloride was purchased from Shanghai Macklin biochemical technology. It was prepared with sterile water into a solution with a concentration of 0.02 %, protected from light and stored at 4 ◦ C. The inoculum preparation, the bacteria were inoculated in TSB medium, cultured overnight at 37 ◦ C, and its concentration was measured by plate counting method, then stored in a refrigerator at 4 ◦ C until use. The concentration of bacteria was adjusted to 106 CFU/mL upon test. 2.2. Apparatus and operations Headspace gas chromatography is composed of a gas chromatograph (Agilent 7890 A) connected with a headspace sampler (COLIN AutoHS® ). The GC analysis equipped with GS-Q capillary column (0.53 mm × 30 m × 0.2 m, Agilent) and a TCD detector. The column was set at 105 ◦ C and held for 5 min, using nitrogen as carrier gas with a flow rate of 1 mL/min. Headspace sampler (Colin, China) operating conditions were as follows: oven temperature was 60 ◦ C, transfer line temperature was 90 ◦ C, sample loop temperature was 80 ◦ C; using nitrogen as the vial pressurization gas with 2 min pressing, nitrogen was used as the vial pressurization gas.
M. Li, C. Zhang, G. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 181 (2020) 113122
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And microplate reader (Infinite 200 PRO, Switzerland) was used to detect the turbidity of the bacterial culture medium contained in 96-well plate. 2.3. Determination of the ability of bacteria to produce CO2 Two bacterial solutions (106 CFU/mL) of different volumes were placed in a headspace vial(20 mL), sealed with PTFE/silicone membrane and aluminum lid, and placed in the incubator at a temperature of 37 ◦ C, with the oscillation speed at 80 rpm. After 24 h. incubation, the vial was immediately introduced into the oven of the headspace sampler, incubated at 60 ◦ C for another 10 min, and then injected into GC-TCD for analysis. 2.4. Sample preparation using HS-GC method Enterococcus faecalis (1 mL) and 1 mL of TSB medium containing the test drug at the target concentration were added to a vial for sealing, and the cells were cultured and measured. In order to determine the MIC, a set of medium with a series of drug concentrations was prepared and added for cultivation. After the culture, a part of the solution was added with 0.4 mL of INT solution by syringe, incubated at 37 ◦ C for 30 min to observe the bacterial staining, and the other part was determined by gas chromatography. 0.3 mL of Klebsiella pneumoniae and 0.3 mL of TSB medium containing testing drugs were taken for culture, and 0.12 mL INT solution was added for staining observation. For a batch of samples, vials can also be stored at −20 ◦ C after 24 h of incubation, and then rapidly melted to room temperature using a hot air blower, finally introduced into a headspace oven for another 10 min for balance prior to analysis. 2.5. Optical density test After HS-GC-TCD test, the vial was shaken up and opened. The bacterial liquid was added into the 96-well plate with a pipette gun, and repeated for three times with 100 L per well. The absorbance at 625 nm was detected by microplate reader. For plant extracts, in order to avoid the influence of color and turbidity caused by the sample itself, the drug-containing medium with the same concentration was prepared and its optical density was determined for background subtraction. 3. Results and discussion 3.1. The principle of the method This method is based on the determination of the amount of CO2 produced by bacterial metabolism, which is proportional to the number of cells, that is, the amount of CO2 is used to reflect the number of cells. When drugs that inhibit bacterial growth are added, bacteria are killed or microbial activity is reduced, so the amount of CO2 produced by metabolism is reduced. Therefore, this method needs to determine the total amount of CO2 produced by bacteria and the proportional relationship between the number of bacterial cells and the amount of CO2 metabolized. In gas chromatography (GC) analysis, the total amount of CO2 metabolized by microorganisms can be quantified by the peak area of CO2 . At a given temperature, CO2 will reach an equilibrium state in the liquid and gas phases in the headspace vials, which can be detected by GC equipped with automatic headspace sampler and thermal conductivity detector. In order to make the result more accurate, the measured CO2 signal value of the sample needs to be performed within the detection linear range of the instrument [21]. It is a complicated biochemical process that bacteria metabolize nutrients to produce gas. The gas production and gas composition
Fig. 1. The Ability for both Gram-positive and Gram-negative bacteria to produce CO2 by metabolism. Different volumes of bacterial (Enterococcus faecalis and Klebsiella pneumoniae) were cultured for 24 h.
are not only related to bacteria, but also related to the nutrient components on which bacteria grow, especially carbon source, and the growth environment, such as temperature, salinity and pH value. Therefore, in order to increase the application scope and accuracy of this method, we measured the CO2 -producing abilities of one Gram-positive bacterial strain (Enterococcus faecalis) and one Gram-negative bacterial strain (Klebsiella pneumoniae), as shown in Fig. 1. Under the same culture condition, the amount of CO2 produced by Klebsiella pneumoniae was higher than that of Enterococcus faecalis. Therefore, when using this method to determine the antibacterial activity of testing drugs, an appropriate amount of bacterial liquid should be selected according to the linear range of CO2 detection by GC. In order to establish this method, it was necessary to verify the ratio of CO2 produced by microbial metabolism to the number of bacterial cells. Therefore, we used this method to measure the growth curves of Enterococcus faecalis and Klebsiella pneumoniae, and compared them with optical density method for verification. The optical density method reflects the number of cells based on the turbidity of colonies in liquid, the more turbid the bacterial liquid is, the more bacteria it contains. The results are shown in Fig. 2a and b. Between the two detection methods, the growth curve of bacteria showed the same trend, and the growth process of bacteria had gone through the adjustment period, logarithmic growth period and stable growth period, which were clarified in several other studies [22]. At 24 h, the signal was relatively stable, and it was also the time for routine bacteriostatic activity measurement. Therefore, we could establish a proportional relationship between the amount of CO2 metabolized and the number of bacterial cells [19,23]. 3.2. Bacteria growth with and without antimicrobial drug There are many applications for bacterial growth detection by headspace analysis of bacterial metabolites, but no detection of disorders in metabolic system after addition of testing drugs [22,23]. To verify the changes in the growth state of bacteria after the addition of testing drugs, two kinds of bacteria were treated with and without drugs, and measured the carbon dioxide content of timedependent metabolism, as shown in Fig. 3. When 15 g/mL of ceftazidime was added, the increase trend of CO2 of these two bacterial strains was significantly reduced, and after about 18 h of culture, the number of bacteria began to increase rapidly. The results showed that this method could sensitively detect the change of bacterial reaction to drugs. Meanwhile, according to the change
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M. Li, C. Zhang, G. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 181 (2020) 113122 Table 1 The comparison of the precision between the HS-GC method and the OD method for the determination of bacterial activity. Strains
Enterococcus faecalis
Klebsiella pneumoniae
Fig. 2. The growth curve of Gram-positive bacterium (a) and Gram-negative bacterium (b) obtained by headspace gas chromatographic (HS-GC) method and optical density method. 2 mL of Enterococcus faecalis and 0.6 mL of Klebsiella pneumoniae were cultured for different periods of time.
Peak area (CO2 )
34.9 35.8 33.6 34.5 34.3 96.1 100 94 97.6 98
OD (625 nm)
0.1974 0.208 0.2048 0.2124 0.2122 0.7402 0.7476 0.738 0.7598 0.7882
Relative standard deviation, % HS-GC method
OD method
2.34
3.00
2.31
2.72
Fig. 4. Comparison of the inhibition rates obtained for both pure compounds and plant extracts by the present method and the reference method (optical density method). 2 mL of Enterococcus faecalis and 0.6 mL of Klebsiella pneumoniae were cultured with different antibiotics and crude plant extracts for 24 h to determine the bacteriostasis rate, respectively.
time, the optical density method was measured at an optical density value of 625 nm, and each group were made up to 5 repetitions. The results are shown in Table 1. It could be observed that the HSGC had very good precision compared with the reference method (optical density method), and the relative standard deviations of the two strains were less than 3 %.
Fig. 3. The time-dependent metabolic carbon dioxide of the two bacteria (Enterococcus faecalis and Klebsiella pneumoniae) in the headspace vials with and without drug (15 g/mL ceftazidime) treatment. 2 mL of Enterococcus faecalis and 0.6 mL of Klebsiella pneumoniae were added with 15 g/mL of ceftazidime, respectively, and cultured for different periods of time.
of bacterial concentration, the time and dose of clinical medication could be guided. 3.3. Evaluation of the method 3.3.1. Precision To verify the precision of the method, 0.5 g/mL chloramphenicol was added to Enterococcus faecalis, and Klebsiella pneumoniae was treated with 15 g/mL ceftazidime. After 24 h of culture, the amount of CO2 was determined by HS-GC method, at the same
3.3.2. Accuracy The accuracy of the method was verified by comparing the inhibition rates of pure compounds and plant extracts on bacteria by the two methods. As shown in Fig. 4, the inhibition rate (R2 = 0.968) of this method is consistent with that of the reference method. However, when the extract itself had some color or turbidity and was tested by optical density method, there was a significant error, because the color or turbidity only occured in the drug treatment group, but not in the control group. In this work, the comparison between the two methods was based on the results obtained after correcting the optical density method by subtracting the optical density of the background of drug solution. As compared with the traditional method in use, this method used a closed system (headspace vial) for culture, and operated at room temperature, so that the specific components of volatile plant extracts (such as essential oils) could be greatly reduced. Meanwhile, this method also adopted a shaking culture system, in which the microorganisms could fully react with the plant extract, especially for those components with poor solubility and easy to cause precipitation. In addition, some plant extracts, especially crude plant extracts, had relatively complex components, many of which contained pig-
M. Li, C. Zhang, G. Chen et al. / Journal of Pharmaceutical and Biomedical Analysis 181 (2020) 113122 Table 2 MICs of antibiotics determined by the present method and reference method (INT staining method). Strains
Klebsiella pneumoniae Enterococcus faecalis
antibiotic
Ceftazidime Ciprofloxacin Gentamicin Chloromycetin
MIC(ug/mL) reference method
present method
GC signal for CO2
32 64 32 16
32 64 32 16
<3 <3 <3 <3
Bacteria were added to the drug culture using a 2-fold gradient dilution method. The reference method was added to INT to determine the minimum inhibitory concentration, and the CO2 signal value was measured by GC.
ments and insoluble components. When measuring antibacterial activity by the traditional optical density method, great background absorbance would be generated, and the background deduction was often inaccurate, especially with high concentration. More recently, the effect of the optical density method on the background absorbance of plant extracts has been experimentally described in our previous work [21]. Since this method was adopted to measure the microbial metabolites (metabolic CO2 ), it could avoid the background error caused by the plant extract itself. Obviously, this method is simplerand with lower possibility of microbial contamination, and the nature of the test sample is more flexible.
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4. Conclusions A new method for the determination of antibacterial activity and the minimum inhibitory concentration of testing drugs by HSGC was developed and verified. The amount of cells was reflected by measuring CO2 produced via microbial metabolism by HS-GC, and the experimental principle of this method was verified. Compared with other standard method in use, this method is proven to have good accuracy and precision in this work. The method is simple to operate and suitable for the determination of antibacterial activity of drugs, especially for natural products. Compared to those traditional methods, this method avoids the error caused by the characteristics of the testing sample itself, such as volatility, solubility and color in the determination of the minimum inhibitory concentration, and can be easily carried out in any microbiology laboratory. Author statement M.Q.G. conceived of, designed and supervised the study. M.H.L. and C.Y. Z. performed the experiments, analyzed the data and wrote the manuscript. G.L.C. participated in the methodological assays and revised the manuscript for submission. In addition to advisory role in the project, L.N. and S.D.S contributed to design, preparation and editing of the manuscript. All authors reviewed and approved the final manuscript. Declaration of Competing Interest
3.4. Determination of minimum inhibitory concentration Studies have shown that tetrazolium salt could form a red compound in the presence of bacteria, which is proportional to the number of active cells and can be used as an indicator for determining the minimum inhibitory concentration of testing drugs. Because of the color and solubility of natural products, it is often difficult to observe the phenomenon above after staining, and in some cases because of bacterial metabolism, indicators cannot make it colored. However, the CO2 produced by the bacterial metabolism can be sensitively detected by GC. The minimum inhibitory concentration (MIC) is the lowest drug concentration that can inhibit bacterial growth, at which the bacteria are killed or its growth is inhibited (the bacterial concentration is the initial concentration). Therefore, at MIC, the bacterial solution was colorless in the method of staining living cells with dyes, while the signals detected by HS-GC were less than 3 because the bacteria did not metabolize CO2 or produced very little CO2 , as shown in Table 2. The MIC value was used to determine the sensitivity of bacteria to drugs, which was also important in evaluating the activity of novel antimicrobial agents [24]. Strong coloring compounds or plant extracts may make MIC value unable to be determined by broth microdilution to exclude the difference between bacterial growth and culture medium [25]. On the other hand, insoluble precipitation caused by some drug samples in the broth microdilution method could also result in a decrease in the contact of the testing compound with the bacteria, thereby limiting their activity. Agar dilution method needs a large amount of drugs, which makes the method limited when the antimicrobial agents are precious or only at the microgram level. At the same time, it is easy to cause the loss of volatile drug components in the process of operation, and in some cases, the insoluble components are difficult to diffuse, resulting in inaccurate results. However, the HS-GC method in this work successfully avoids the hitherto encountered problems associated with testing samples when determining the minimum inhibitory concentration of test drugs to get reliable results, especially for natural products with strong coloring molecules and insolubility.
The authors declare that there is no conflict of interest. Acknowledgements This work is partially supported by Major Project for Special Technology Innovation of Hubei Province (Grant No. 2017AHB054 to M. Guo). In addition, L Nahar also gratefully acknowledges the financial support of the European Regional Development Fund Project ENOCH (No. CZ.02.1.01/0.0/0.0/16 019/0000868). References [1] C. Costelloe, C. Metcalfe, A. Lovering, D. Mant, A.D. Hay, Effect of antibiotic prescribing in primary care on antimicrobial resistance in individual patients: systematic review and meta-analysis, BMJ 340 (2010) c2096. [2] G. Kaneti, H. Sarig, I. Marjieh, Z. Fadia, A. Mor, Simultaneous breakdown of multiple antibiotic resistance mechanisms in S. aureus, FASEB J. 27 (2013) 4834–4843. [3] W. Goettsch, W. van Pelt, N. Nagelkerke, M.G. Hendrix, A.G. Buiting, P.L. Petit, L.J. Sabbe, A.J. van Griethuysen, A.J. de Neeling, Increasing resistance to fluoroquinolones in escherichia coli from urinary tract infections in The Netherlands, J. Antimicrob. Chemother. 46 (2) (2000) 223–228. [4] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs from 1981 to 2014, J. Nat. Prod. 79 (2016) 629–661. [5] D.A. Dias, S. Urban, U. Roessner, A historical overview of natural products in drug discovery, Metabolites 2 (2012) 303–336. [6] B.B. Mishra, V.K. Tiwari, Natural products: an evolving role in future drug discovery, Eur. J. Med. Chem. 46 (2011) 4769–4807. [7] C. McNulty, R. Owen, D. Tompkins, P. Hawtin, K. McColl, A. Price, G. Smith, L. Teare, P.H.W. Grp, Helicobacter pylori susceptibility testing by disc diffusion, J. Antimicrob. Chemoth. 49 (2002) 601–609. [8] K.M. Kuper, D.M. Boles, J.E. Mohr, A. Wanger, Antimicrobial susceptibility testing: a primer for clinicians, Pharmacotherapy. 29 (2009) 1326–1343. [9] I. Wiegand, K. Hilpert, R.E.W. Hancock, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances, Nat. Protoc. 3 (2008) 163–175. [10] J. Golus, R. Sawicki, J. Widelski, G. Ginalska, The agar microdilution method - a new method for antimicrobial susceptibility testing for essential oils and plant extracts, J. Appl. Microbiol. 121 (2016) 1291–1299. [11] B.A. Arthington-Skaggs, W. Lee-Yang, M.A. Ciblak, J.P. Frade, M.E. Brandt, R.A. Hajjeh, L.H. Harrison, A.N. Sofair, D.W. Warnock, C.A.S. Grp, Comparison of visual and spectrophotometric methods of broth microdilution MIC end point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of fluconazole and itraconazole against trailing and
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