Journal of Microbiological Methods 105 (2014) 134–140
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Optimization of tetrazolium salt assay for Pseudomonas aeruginosa biofilm using microtiter plate method Parastoo Sabaeifard a, Ahya Abdi-Ali a,⁎, Mohammad Reza Soudi a, Rasoul Dinarvand b a b
Department of Biology, Faculty of Science, Alzahra University, Tehran, Iran Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Iran
a r t i c l e
i n f o
Article history: Received 7 April 2014 Received in revised form 22 July 2014 Accepted 23 July 2014 Available online 30 July 2014 Keywords: Biofilm Crystal violet Microtiter plate Optimization TTC XTT
a b s t r a c t Pseudomonas aeruginosa is one of the most important pathogenic bacteria related to biofilm infections. Due to the biofilm multi-drug resistance, methods of biofilm formation enumeration are of interest for assessment of efficient drug regimen development for biofilm inhibition or eradication. There are many different assay methods to determine the biofilm formation, using vital or non-vital dyes. The primary aim of the current study was to develop an assay using a member of tetrazolium salts family, 2,3,5-triphenyl-tetrazolium chloride (TTC), for detection of P. aeruginosa biofilm formation in 96-well microtiter plates and also a method of Minimum Biofilm Inhibitory Concentration (MBIC) determination of antibiotics against P. aeruginosa PAO1. Furthermore, the assay was optimized for TTC concentration, wavelength and period of incubation for 4 different antibiotics. The optimized condition was then compared with two other prevalent methods: the crystal violet (CV) assay and the 2,3-bis (2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay. In general, the optimized TTC assay (0.5% TTC, 6 h of incubation and absorbance measurement at 405 nm for biofilm assay and 1% TTC, 5 h of incubation and absorbance measurement at 490 nm for MBIC determination) distinguished between biofilms formed by different concentrations of bacteria and also was able to detect lower amounts of biofilm formed in contrast to the other two assay methods suggesting that TTC assay is more sensitive and also less expensive than other vital staining methods. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bacterial biofilm can be defined as attached communities of bacteria surrounded by a matrix which undergo morphological and physiological changes during transition from planktonic (free-swimming) cells to sessile (surface-attached) ones (Drenkard, 2003; O'Toole G. et al., 2000). Resistance to antimicrobial agents and components of the host immune system is the most important characteristic of biofilms and the major cause of recalcitrant infections (Brooun et al., 2000). Diseases involving biofilms are generally chronic and hard to eradicate (Drenkard, 2003). Biofilms contribute to about 60% of human infections (Spoering and Lewis, 2001) Pseudomonas aeruginosa is one of the most important opportunistic human pathogens with the ability of biofilm formation on different abiotic surfaces, including artificial implants, endotracheal tubes, urinary catheters and contact lenses (Knezevic and Petrovic, 2008). Also, P. aeruginosa is one of the most important nosocomial pathogens which can lead to death especially among patients suffering from immunosuppression, cystic fibrosis, malignancy and burns or traumatic wounds (Karakoç and Gerçeker, 2001). ⁎ Corresponding author. Tel.: +98 2188044052; fax: +98 2188058912. E-mail address: [email protected] (A. Abdi-Ali).
http://dx.doi.org/10.1016/j.mimet.2014.07.024 0167-7012/© 2014 Elsevier B.V. All rights reserved.
Over the past years, several assay methods for biofilm quantification in microtiter plates have been described (Peeters et al., 2008). The most common method of biofilm formation evaluation is crystal violet (CV) staining (Merritt et al., 2005). CV is a basic dye which binds to negatively charged surface molecules in the extracellular matrix. Since all cells (including living and dead ones), as well as matrix are stained by CV, it is poorly suited to evaluate killing of biofilm cells (Peeters et al., 2008). CV assay has been in use for a number of years, due to its ease of use and the adaptability of the protocol to a variety of applications. But, due to the indirect nature of biofilm assessment in this method, it is desirable to pair this assay with another method (Merritt et al., 2005). Tetrazolium salts have become one of the most widely used tools in biology for measuring the metabolic activity of cells (Berridge et al., 2005). The prototype tetrazolium salt, 2,3,5-triphenyl-tetrazolium chloride (TTC), developed more than a century ago. Then, various tetrazolium salts such as 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) have been synthesized on the base of the structure of TTC (Tsukatani et al., 2008). The tetrazolium salt-based assay has been used extensively for the quantification of viable cells in planktonic cultures (Gabrielson et al., 2002; Kuhn et al., 2003) and for the quantification of bacterial (Cerca
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Table 1 Antibiotics MBICs (μg/ml) determined with Different TTC concentrations and wavelengths at different hours of incubation with TTC. Antibiotics
Amikacin
Cefixime
Levofloxacin
Piperacillin
TTC concentration (%)
0.1
Wavelength (nm)
5th hour
6th hour
7th hour
5th hour
0.5 6th hour
7th hour
5th hour
1 6th hour
7th hour
2 5th hour
6th hour
7th hour
405 450 490 540 405 450 490 540 405 450 490 540 405 450 490 540
16 16 16 16 128 512 512 128 2 2 2 ≤0.25 8 16 16 ≤0.25
32 32 16 16 256 512 256 128 2 2 1 0.5 8 8 8 8
8 8 8 8 512 512 512 512 0.5 0.5 0.5 0.5 8 8 8 8
8 16 16 8 128 512 512 128 2 2 2 2 8 16 16 16
V V V V 256 512 N1024 256 ≤0.25 2 0.5 0.5 0.5 8 8 8
16 16 16 16 512 512 512 512 1 1 1 1 8 8 8 8
4 16 16 8 256 512 512 256 2 2 2 2 16 16 16 16
V V V V 256 N1024 N1024 256 ≤0.25 ND ND ND 1 N16 N16 16
8 8 8 8 512 512 512 512 ND ND ND ND 16 16 16 16
16 16 64 32 256 N1024 512 256 ≤0.25 ≤0.25 ≤0.25 ≤0.25 16 16 16 16
V
16 8 8 4 128 128 128 128 ND ND ND ND OVRFLW OVRFLW OVRFLW OVRFLW
8 8 8 512 N1024 512 256 ≤0.25 ≤0.25 ≤0.25 ≤0.25 0.5 16 16 16
V: Not determined due to variation in different experiments. ND: The MBIC could not be determined due to biofilm detection in MBIC higher concentrations. In almost all cases, the lower concentrations of antibiotic inhibited biofilm formation while the higher concentrations could not inhibit the biofilm formation so that MBIC determination was not accurate. OVRFLW: The MBIC could not be determined due to high absorbance level.
et al., 2005; Johansen et al., 1997; Rändler et al., 2010; Shakeri et al., 2007; Xu et al., 2000) and yeast biofilms (Nett et al., 2011; Ramage et al., 2001; Taff et al., 2012). To date, the most commonly used tetrazolium salt among colorimetric assays for antimicrobial susceptibility testing of bacteria and fungi is XTT which after reduction yields a water-soluble formazan derivative that can be easily quantified colorimetrically (Tsukatani et al., 2009). Despite its popularity, XTT is expensive and problems regarding intraand interspecies variability have been reported (Honraet et al., 2005; Kuhn et al., 2003). In present study, a colorimetric assay was developed and optimized on the basis of TTC. In this assay, metabolically active cells convert TTC to a colored formazan derivative which can be easily quantified colorimetrically to measure P. aeruginosa PAO1 biofilm formation. The results were then compared with CV and XTT assay methods. 2. Materials and methods 2.1. Bacterial strain and culture condition The bacterial strains used in this study were P. aeruginosa PAO1 and P. aeruginosa strain 48 a clinical biofilm forming P. aeruginosa strain isolated in a previous study. Tryptic soy agar (TSA; Merck, Germany) supplemented with 0.2% glucose was used to culture bacterial cells prior to each experiment. The bacteria were cultured aerobically on the medium at 37 °C. Also, tryptic soy broth (TSB; Merck, Germany) supplemented with 0.2% glucose was used to form biofilm in microtiter plates (Abdi-Ali et al., 2006). 2.2. Antibiotics The most common agents used to inhibit biofilm formation are antibiotics. In case of P. aeruginosa due to high resistance to antibiotics, there are limited antibiotics which are in use today. To study the effect of antibiotics on P. aeruginosa PAO1 biofilm, four effective antibiotics of different common classes (aminoglycosides, beta-lactams, fluoroquinolones and cephalosporins) used against the strains of P. aeruginosa were tested. Cefixime and levofloxacin were received as a gift from Loghman Pharmaceutical & Hygienic Co. (Tehran, Iran). Piperacillin and amikacin were obtained from Sigma and Eczacibasi, respectively. Piperacillin (64 μg/ml) and amikacin (256 μg/ml) solutions were prepared in distilled water and cefixime (4096 μg/ml) and
levofloxacin (64 μg/ml) solutions were made by addition of the least amount of methanol and dimethyl sulfoxide (DMSO), respectively. The final volume of the latter two antibiotics was reached by distilled water addition (Lorian, 2005).
2.3. Biofilm formation In each experiment, a flat-bottomed 96-well cell culture microtiter plate (SPL Plastic Labware, Korea) was used. 100 μl of TSB medium supplemented with 0.2% glucose was added to each well. Four different inoculum concentrations (105 to 108 CFU/ml) were used to form biofilm by P. aeruginosa PAO1 and P. aeruginosa strain 48. Using 24 hour bacterial culture, the suspension was adjusted to McFarland standard 0.5 in TSB supplemented with 0.2% glucose. 10fold dilutions were made to reach 105 CFU/ml bacterial cell concentrations. 100 μl of each suspension was inoculated to each well. The test was performed in 6 replicates for each inoculum concentrations. The microplates were incubated at 37 °C for 20 h.
2.4. Antibiotics MBIC determination The wells were filled with medium as mentioned in Section 2.3. Two-fold antibiotic serial dilutions were made in wells in a way that for each antibiotic seven different concentrations were tested. The last row of each microtiter plate was used as controls (H1–H6 as medium control and H7–H12 as bacterial growth control). Using 24 h P. aeruginosa PAO1 culture, the suspension was adjusted to McFarland standard 0.5 in TSB supplemented with 0.2% glucose. 100 μl of the suspension was inoculated to each well except for the medium controls. The microplates were incubated at 37 °C for 20 h (Abdi-Ali et al., 2006).
2.5. Biofilm assay Following 20 h of incubation, the planktonic cells were removed from each well and plates were rinsed thrice using prewarmed (37 °C) physiological saline. Excess moisture was removed by tapping the microplates on sterile napkins. The plates were dried for 15 min in upside-down position in a sterile setting. Then, the prepared microplates were assayed as below.
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2.5.1. TTC assay To determine the optimum conditions for the TTC assay, the TTC (Merck, Germany) concentration, the assay period and the wavelength in which the absorbance was measured, were tested in 4, 3 and 4 different levels, respectively. TTC solutions in distilled water were prepared by dissolving TTC in distilled water in concentrations of 2, 1, 0.5 and 0.1% and then sterilized by 0.22 μm cellulose acetate filters. 50 μL of each solution was added to 3 different set of biofilms formed in the presence of two-fold serial dilution of the aforementioned antibiotics. 200 μl of TSB supplemented with 0.2% glucose was added to all the wells (final TTC concentration: 0.4, 0.2, 0.1 and 0.02% respectively). Plates were incubated in the dark for 5, 6 and 7 h at 37 °C and 120 rpm. After the incubation period, 200 μl of the well contents were moved to a new flat-bottomed microplate and the absorbance was measured at 405, 450, 490 and 540 nm (Epoch Microplate Spectrophotometer, BioTek) (Daniel, 1997; Gabrielson et al., 2002; Knezevic and Petrovic, 2008; Rahman et al., 2004; Shakeri et al., 2007). The assay was performed in 6 replicates in 2 different days for each experiment.
2.5.2. XTT assay XTT assay was performed following the method used by Peeters et al. (2008) with some modifications. Fresh XTT solution was made by dissolving 4 mg XTT (Sigma) in 10 ml prewarmed (37 °C) physiological saline before each assay. The solution was filter- sterilized and then supplemented with 100 μl menadione solution, containing 55 mg menadione (Sigma) in 100 ml acetone. 100 μl of XTT-menadione solution and 100 μl of fresh TSB medium supplemented with 0.2% glucose were added to each well. Plates were incubated in the dark for 7 h at 37 °C and 120 rpm. After 7 h of incubation, 200 μl of well contents were transfered to a new flat-bottomed microplate and the absorbance
was measured at 486 nm with microplate spectrophotometer. The assay was performed in 6 replicates for each antibiotic serial dilution. 2.5.3. Crystal violet assay (CV assay) 250 μl of 0.1% crystal violet (Merck, Germany) solution was added to each well. After 10 min at room temperature, the wells were emptied and the microplates were carefully washed under running tap water to remove excess CV dye. Subsequently, the microplates were vigorously tapped on napkins to remove any excess liquid and air-dried. After an hour, 250 μl of 30% glacial acetic acid (Merck, Germany) was added to all wells. Following a ten minute period, 200 μl of the well contents were transferred to a new flat-bottomed microplate and the absorbance was measured at 550 nm using a microplate spectrophotometer. The assay was performed in 6 replicates for each antibiotic serial dilution (Merritt et al., 2005). 2.6. Statistical analysis To find the optimum condition of biofilm assay, the absorbance amounts were statistically analyzed. Significant differences were determined by one-way analysis of variance (ANOVA One-way) with pairwise comparisons using Tukey's method. A P value of b0.05 was considered statistically significant. For MBIC determination, well contents treated with drugs were compared to treatment controls without drugs. Data are presented here as a percentage of biofilm growth (biofilm remaining after treatment to untreated control). Significant differences were determined by a two-way analysis of variance (ANOVA Two-way) with pairwise comparisons using Tukey's method. A P value of b0.05 was considered statistically significant.
A
B
C
D
Fig. 1. Impact of the TTC concentration on the susceptibility determination of P. aeruginosa PAO1 biofilm to amikacin (A), cefixime (B), levofloxacin (C) and piperacillin (D). The amount of biofilm formed after 20 h of incubation with each antibiotic was estimated. Absorbance at 490 nm was measured following incubation with TTC (0.1, 0.5, 1 and 2%) for 5 h. ANOVA with pairwise comparison using Tukey's method was used to compare TTC concentrations at each drug concentration. *, P b 0.05. Error bars indicate standard errors of mean of six experiments.
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A
137
B
Fig. 2. Comparison of 2 P. aeruginosa strains' biofilm detected by optimized TTC, XTT and CV assays. The amount of biofilm formed after 20 h of incubation was estimated. Absorbance was measured at 405 nm and 486 nm following incubation with TTC (1%) and XTT, respectively. Absorbance was measured at 550 nm for CV assay. ANOVA with pairwise comparison using Tukey's method was used to compare 3 methods for each inoculum concentration. *, P b 0.05. Error bars indicate standard errors of mean of six experiments.
The repeatability of results in both cases were estimated by coefficient of variance percentage (CV%) calculation. The statistical analysis was performed using Minitab 15 (Minitab, Inc.) 3. Results and discussion 3.1. TTC assay A convenient way to estimate the amount of biofilm formed in microtiter plates is to use a colorimetric assay. Various colorimetric assays based on viability assays, using vital dyes have been established (Honraet et al., 2005; Johansen et al., 1997; Kuhn et al., 2003; Peeters et al., 2008; Ramage et al., 2001). The unique chemical and biological properties of tetrazolium salts have led to their widespread application in biology. The reduction of tetrazolium salts from colorless or weakly colored, aqueous solutions to colored derivatives, formazans, is the basis of their use (Berridge et al., 2005). Some studies have reported the use of TTC to detect the microbial biofilm formed (Kim et al., 2010; Shakeri et al., 2007), but our experiments showed that the methods used is not suitable for P. aeruginosa biofilm detection since there were no detectable color changes even after 24 h of incubation with TTC. Therefore, there was a need to develop and optimize a method to use TTC as a vital dye to identify the amount of P. aeruginosa biofilm formation. To this end, the presented method has been developed and optimized for three important parameters including TTC concentration (4 different concentrations), incubation time (5, 6 and 7 h of incubation) and wavelength (4 distinct wavelengths) in which the formed formazan has been detected. MBICs of the tested antibiotics determined under the conditions mentioned are summarized in Table 1. 3.1.1. Effects of incubation time The amount of incubation time is likely to be different in the case of each bacterial strain since formazan formation from TTC is the result of Table 2 Comparison of MBIC determined by TTC, XTT and CV biofilm assay methods. Antibiotic
MBIC determined by the use of TTC
Amikacin Cefixime Levofloxacin Piperacillin
16 512 2 16
XTT μg/ml μg/ml μg/ml μg/ml
8 256 1 8
CV μg/ml μg/ml μg/ml μg/ml
ND ND b0.25 μg/ml 8 μg/ml
ND: The MBIC could not be determined since the lower concentrations of antibiotic inhibited biofilm formation while the higher concentrations could not inhibit the biofilm formation so that MBIC could not be determined.
dye reduction by living cells. Statistical analysis showed that the formed biofilm with the 2 strains were best detected after 6 h of incubation, since the reproducibility of the results were more in contrast with the other incubation periods. The MBIC results show that among 3 different incubation times, in case of MBIC determination, 5 h of incubation with TTC is optimum for P. aeruginosa PAO1 strain, since before this period the color change is limited and is difficult to measure (data not shown) correctly. After 7 h of incubation with TTC (and in some cases even after 6 h), the absorbance exceed amounts that can be measured correctly with the instrument used. On the other hand, in almost all cases, the MBIC after 5 h is favorable since after 6 h of incubation the differences are insignificant. Although, in some other cases, the 5th hour of incubation showed a significant difference with the 6th hour and could show the higher concentration as MBIC (Table 1).
3.1.2. Impact of wavelength used to measure the absorbance Formazan resulted from TTC reduction has been assayed by various wavelengths (Daniel, 1997; Gabrielson et al., 2002; Knezevic and Petrovic, 2008; Rahman et al., 2004; Shakeri et al., 2007). The formed biofilms were best detected in 405 nm. The reproducibility of the results were less in case of 450 nm and 490 nm (lower CV%), yet, the absorbance amount in 540 nm in some cases were as high as it could not be detected by the instrument. So, the best wavelength to detect the non-treated formed biofilm is 405 nm. In case of MBIC determination, absorbance at 405 and 540 nm is significantly less than the other wavelengths (P b 0.05). In some few cases absorbance at 540 nm is as high as it could not be measured. In some other cases absorbance measurement in 490 nm at the 5th hour of incubation with TTC was significantly better than 450 nm at the same period so that the selected wavelength for the rest of the experiments is 490 nm.
3.1.3. Impact of TTC concentration It was shown that the lowest effective concentration of TTC to detect planktonic cells of some bacteria including P. aeruginosa is 0.2% (Eloff, 1998). Also, it has been reported that TTC could be toxic to bacteria in high concentrations (Weinberg, 1953). To find the nontoxic and also the lowest effective concentration of the TTC salt, 4 concentrations have been studied. Using 4 TTC concentrations showed that the formed biofilm could be better detected using 0.5% TTC. The absorbance amounts were significantly different for 108 to 106 CFU/ml inoculum concentrations (P b 0.05). As a result, the biofilms formed by less than 106 CFU/ml initial bacterial concentrations could not be truly detected using the presented optimum TTC assay.
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A
B
C
D
Fig. 3. Comparison of 3 different methods of susceptibility determination of P. aeruginosa PAO1 biofilm to amikacin (A), cefixime (B), levofloxacin (C) and piperacillin (D). The amount of biofilm formed after 20 h of incubation with each antibiotic was estimated. Absorbance was measured at 490 nm and 486 nm following incubation with TTC (1%) and XTT, respectively. Absorbance was measusured at 550 nm for CV assay. ANOVA with pairwise comparison using Tukey's method was used to compare 3 methods at each drug concentration. *, P b 0.05. Error bars indicate standard errors of mean of six experiments.
The MBIC could be better detected using 1% TTC. Yet, 0.5% TTC concentration in almost all the sub-MBIC concentrations showed significant differences with other TTC tested concentrations to determine the biofilm formation (P b 0.05) but the accuracy of 1% TTC is more. Using 2% TTC could lead to a decrease in biofilm growth absorbance (Fig. 1) possibly due to its toxicity for the tested bacterium.
Fig. 4. Biofilm formed in the presence of cefixime (1024–16 μg/ml). Assays were performed in columns 1–4 with CV, in columns 5–8 with TTC (after 5 h) and in columns 9–12 with XTT (10 times diluted) (after 7 h). The MBIC could not be detected by CV but the two other assays shows the raw C (256 μg/ml) as MBIC. H row contains control cells: H3, H4, H7, H8, H11, H12: Controls without bacteria and the rest are controls of bacteria biofilm formation.
3.2. Comparison of XTT, CV and TTC assay methods Among the methods which use non-vital dyes for biofilm detection, CV assay is the most common for various bacteria including P. aeruginosa (Borucki et al., 2003; Djordjevic et al., 2002; Kaplan et al., 2004; Merritt et al., 2005; Mireles et al., 2001; O'Toole G.A. et al., 2000; Pratt and Kolter, 1998; Stepanović et al., 2000; Watnick and Kolter, 1999). Although common, the main drawback of crystal violet assay is the fact that only the biofilm biomass can be measured which is less informative in comparison to viability (Pan et al., 2010; Skogman et al., 2012). This problem seems more prominent when testing microorganisms with high extracellular polymeric substances. Furthermore, Peeters et al. (2008) showed that this method, due to its low repeatability, is not suitable for P. aeruginosa strains which the idea is supported by the results of this study. On the other hand, XTT assay is one of the most common methods using vital dyes to assay the biofilm formation for different microorganisms (Cerca et al., 2005; da SILVA et al., 2008; Nett et al., 2011; Peeters et al., 2008; Pettit et al., 2005; Taff et al., 2012). Comparison of optimized TTC assay for biofilm detection with the two other assay methods showed that the optimized TTC assay was able to detect biofilms formed by different inoculum concentrations (108 to 106 CFU/ml), while the two other methods were not able to distinguish between biofilms formed by different initial bacterial concentrations (Fig. 2). On the other hand, comparison of optimized TTC assay with the two other assay methods for MBIC determination indicated that TTC assay was able to detect the less amounts of biofilm formed which was not detectable by XTT and CV assays (Table 2). Also the accuracy and reproducibility of the results, especially in the lower amount of biofilm which is supposed to form in higher antibiotic concentrations, is more in TTC assay in contrast with the other methods (lower CV%) (Fig. 3).
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It was shown that subinhibitory concentrations of antibiotics are able to induce biofilm formation (Hoffman et al., 2005; Kaplan, 2011; Kaplan et al., 2012). In the case of piperacillin, TTC assay showed the induction of biofilm formation in the presence of piperacillin sub-MBIC concentrations (175–358% due to different concentrations) while the other two methods were not able to detect this kind of induction (Fig. 3D). Interpretation of results of the CV assay with cefixime and amikacin were problematic. As shown in Fig. 4, biofilm formation was detected at the two highest concentrations but the lower concentrations of the antibiotic inhibited biofilm formation. The two other methods did not show such results. Authors believed that since CV attached the EPS of biofilm, it can detect even the cell-free EPS, but the two other methods are able to detect live cells, so that the highest concentrations detected by CV could be cell free matrices which could be produced due to antibiotic stress or any other unknown reasons and not a real biofilm. These findings also support the idea that CV assay is by itself not a reliable assay and the assay results need to be confirmed with another assay which uses a vital dye. 4. Conclusion In the present study, the TTC assay was optimized for P. aeruginosa PAO1 biofilm detection followed by comparison to the two other methods commonly used to identify biofilm formation. All assays were evaluated for their accuracy and repeatability for quantification of biofilm formation in a microtiter plate. All assays showed applicability for determination and quantification of biofilms. However, the repeatability of TTC assays was higher than the two other assays. Although CV assay is simple, fast and cheap, it is not as accurate as the two other methods as it can be seen in the case of amikacin and cefixime. On the other hand, among the three assays used, the XTT assay is the most expensive method, though not the most accurate one. As results indicate, TTC assay (0.5% TTC, 6 h of incubation and absorbance measurement at 405 nm) could be a suitable method to detect P. aeruginosa biofilm formation and 1% TTC, 5 h of incubation and absorbance measurement at 490 nm could be an accurate method to define MBIC in P. aeruginosa PAO1 since it is not very expensive but is sufficiently repeatable and accurate. It is worth mentioning that the method was optimized for factors which the authors hypothesized or observed and effected the TTC assay for P. aeruginosa PAO1 biofilm detection. The factors studied here are not necessarily the same for any other Pseudomonas strains since the ability of biofilm formation, the biofilm growth period, the ability to reduce tetrazolium salts and many other known and unknown parameters may affect the assay results. Acknowledgments Authors thank Dr. Sedigheh Shams for her excellent assistance in the statistical analysis and Dr. Sara Gharavi for editing of this article. References Abdi-Ali, A.,Mohammadi-Mehr, M.,Agha Alaei, Y., 2006. Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa. Int. J. Antimicrob. Agents 27, 196–200. Berridge, M.V., Herst, P.M., Tan, A.S., 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 11, 127–152. Borucki, M.K.,Peppin, J.D.,White, D.,Loge, F.,Call, D.R., 2003. Variation in biofilm formation among strains of Listeria monocytogenes. Appl. Environ. Microbiol. 69, 7336–7342. Brooun, A., Liu, S., Lewis, K., 2000. A dose–response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 44, 640–646. Cerca, N., Martins, S., Cerca, F., Jefferson, K.K., Pier, G.B., Oliveira, R., Azeredo, J., 2005. Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry. J. Antimicrob. Chemother. 56, 331–336.
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Bacteriol. 182, 425–431. Pan, Y.,Breidt, F.,Gorski, L., 2010. Synergistic effects of sodium chloride, glucose, and temperature on biofilm formation by Listeria monocytogenes serotype 1/2a and 4b strains. Appl. Environ. Microbiol. 76, 1433–1441. Peeters, E.,Nelis, H.J.,Coenye, T., 2008. Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J. Microbiol. Methods 72, 157–165. Pettit, R.K.,Weber, C.A.,Kean, M.J.,Hoffmann, H.,Pettit, G.R.,Tan, R.,Franks, K.S.,Horton, M.L., 2005. Microplate alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing. Antimicrob. Agents Chemother. 49, 2612–2617. Pratt, L.A., Kolter, R., 1998. Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol. Microbiol. 30, 285–293. Rahman, M., Kühn, I., Rahman, M., Olsson-Liljequist, B., Möllby, R., 2004. Evaluation of a scanner-assisted colorimetric MIC method for susceptibility testing of gramnegative fermentative bacteria. Appl. Environ. Microbiol. 70, 2398–2403. Ramage, G., Vande Walle, K., Wickes, B.L., López-Ribot, J.L., 2001. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob. Agents Chemother. 45, 2475–2479. Rändler, C., Matthes, R., McBain, A., Giese, B., Fraunholz, M., Sietmann, R., Kohlmann, T., Hübner, N.-O., Kramer, A., 2010. A three-phase in-vitro system for studying Pseudomonas aeruginosa adhesion and biofilm formation upon hydrogel contact lenses. BMC Microbiol. 10, 282. Shakeri, S.,Kermanshahi, R.K.,Moghaddam, M.M.,Emtiazi, G., 2007. Assessment of biofilm cell removal and killing and biocide efficacy using the microtiter plate test. Biofouling 23, 79–86. Skogman, M.E.,Vuorela, P.M.,Fallarero, A., 2012. 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Optimization of tetrazolium salt assay for Pseudomonas aeruginosa biofilm using microtiter plate method Parastoo Sabaeifard a, Ahya Abdi-Ali a,⁎, Mohammad Reza Soudi a, Rasoul Dinarvand b a b
Department of Biology, Faculty of Science, Alzahra University, Tehran, Iran Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Iran
a r t i c l e
i n f o
Article history: Received 7 April 2014 Received in revised form 22 July 2014 Accepted 23 July 2014 Available online 30 July 2014 Keywords: Biofilm Crystal violet Microtiter plate Optimization TTC XTT
a b s t r a c t Pseudomonas aeruginosa is one of the most important pathogenic bacteria related to biofilm infections. Due to the biofilm multi-drug resistance, methods of biofilm formation enumeration are of interest for assessment of efficient drug regimen development for biofilm inhibition or eradication. There are many different assay methods to determine the biofilm formation, using vital or non-vital dyes. The primary aim of the current study was to develop an assay using a member of tetrazolium salts family, 2,3,5-triphenyl-tetrazolium chloride (TTC), for detection of P. aeruginosa biofilm formation in 96-well microtiter plates and also a method of Minimum Biofilm Inhibitory Concentration (MBIC) determination of antibiotics against P. aeruginosa PAO1. Furthermore, the assay was optimized for TTC concentration, wavelength and period of incubation for 4 different antibiotics. The optimized condition was then compared with two other prevalent methods: the crystal violet (CV) assay and the 2,3-bis (2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay. In general, the optimized TTC assay (0.5% TTC, 6 h of incubation and absorbance measurement at 405 nm for biofilm assay and 1% TTC, 5 h of incubation and absorbance measurement at 490 nm for MBIC determination) distinguished between biofilms formed by different concentrations of bacteria and also was able to detect lower amounts of biofilm formed in contrast to the other two assay methods suggesting that TTC assay is more sensitive and also less expensive than other vital staining methods. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bacterial biofilm can be defined as attached communities of bacteria surrounded by a matrix which undergo morphological and physiological changes during transition from planktonic (free-swimming) cells to sessile (surface-attached) ones (Drenkard, 2003; O'Toole G. et al., 2000). Resistance to antimicrobial agents and components of the host immune system is the most important characteristic of biofilms and the major cause of recalcitrant infections (Brooun et al., 2000). Diseases involving biofilms are generally chronic and hard to eradicate (Drenkard, 2003). Biofilms contribute to about 60% of human infections (Spoering and Lewis, 2001) Pseudomonas aeruginosa is one of the most important opportunistic human pathogens with the ability of biofilm formation on different abiotic surfaces, including artificial implants, endotracheal tubes, urinary catheters and contact lenses (Knezevic and Petrovic, 2008). Also, P. aeruginosa is one of the most important nosocomial pathogens which can lead to death especially among patients suffering from immunosuppression, cystic fibrosis, malignancy and burns or traumatic wounds (Karakoç and Gerçeker, 2001). ⁎ Corresponding author. Tel.: +98 2188044052; fax: +98 2188058912. E-mail address: [email protected] (A. Abdi-Ali).
http://dx.doi.org/10.1016/j.mimet.2014.07.024 0167-7012/© 2014 Elsevier B.V. All rights reserved.
Over the past years, several assay methods for biofilm quantification in microtiter plates have been described (Peeters et al., 2008). The most common method of biofilm formation evaluation is crystal violet (CV) staining (Merritt et al., 2005). CV is a basic dye which binds to negatively charged surface molecules in the extracellular matrix. Since all cells (including living and dead ones), as well as matrix are stained by CV, it is poorly suited to evaluate killing of biofilm cells (Peeters et al., 2008). CV assay has been in use for a number of years, due to its ease of use and the adaptability of the protocol to a variety of applications. But, due to the indirect nature of biofilm assessment in this method, it is desirable to pair this assay with another method (Merritt et al., 2005). Tetrazolium salts have become one of the most widely used tools in biology for measuring the metabolic activity of cells (Berridge et al., 2005). The prototype tetrazolium salt, 2,3,5-triphenyl-tetrazolium chloride (TTC), developed more than a century ago. Then, various tetrazolium salts such as 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) have been synthesized on the base of the structure of TTC (Tsukatani et al., 2008). The tetrazolium salt-based assay has been used extensively for the quantification of viable cells in planktonic cultures (Gabrielson et al., 2002; Kuhn et al., 2003) and for the quantification of bacterial (Cerca
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Table 1 Antibiotics MBICs (μg/ml) determined with Different TTC concentrations and wavelengths at different hours of incubation with TTC. Antibiotics
Amikacin
Cefixime
Levofloxacin
Piperacillin
TTC concentration (%)
0.1
Wavelength (nm)
5th hour
6th hour
7th hour
5th hour
0.5 6th hour
7th hour
5th hour
1 6th hour
7th hour
2 5th hour
6th hour
7th hour
405 450 490 540 405 450 490 540 405 450 490 540 405 450 490 540
16 16 16 16 128 512 512 128 2 2 2 ≤0.25 8 16 16 ≤0.25
32 32 16 16 256 512 256 128 2 2 1 0.5 8 8 8 8
8 8 8 8 512 512 512 512 0.5 0.5 0.5 0.5 8 8 8 8
8 16 16 8 128 512 512 128 2 2 2 2 8 16 16 16
V V V V 256 512 N1024 256 ≤0.25 2 0.5 0.5 0.5 8 8 8
16 16 16 16 512 512 512 512 1 1 1 1 8 8 8 8
4 16 16 8 256 512 512 256 2 2 2 2 16 16 16 16
V V V V 256 N1024 N1024 256 ≤0.25 ND ND ND 1 N16 N16 16
8 8 8 8 512 512 512 512 ND ND ND ND 16 16 16 16
16 16 64 32 256 N1024 512 256 ≤0.25 ≤0.25 ≤0.25 ≤0.25 16 16 16 16
V
16 8 8 4 128 128 128 128 ND ND ND ND OVRFLW OVRFLW OVRFLW OVRFLW
8 8 8 512 N1024 512 256 ≤0.25 ≤0.25 ≤0.25 ≤0.25 0.5 16 16 16
V: Not determined due to variation in different experiments. ND: The MBIC could not be determined due to biofilm detection in MBIC higher concentrations. In almost all cases, the lower concentrations of antibiotic inhibited biofilm formation while the higher concentrations could not inhibit the biofilm formation so that MBIC determination was not accurate. OVRFLW: The MBIC could not be determined due to high absorbance level.
et al., 2005; Johansen et al., 1997; Rändler et al., 2010; Shakeri et al., 2007; Xu et al., 2000) and yeast biofilms (Nett et al., 2011; Ramage et al., 2001; Taff et al., 2012). To date, the most commonly used tetrazolium salt among colorimetric assays for antimicrobial susceptibility testing of bacteria and fungi is XTT which after reduction yields a water-soluble formazan derivative that can be easily quantified colorimetrically (Tsukatani et al., 2009). Despite its popularity, XTT is expensive and problems regarding intraand interspecies variability have been reported (Honraet et al., 2005; Kuhn et al., 2003). In present study, a colorimetric assay was developed and optimized on the basis of TTC. In this assay, metabolically active cells convert TTC to a colored formazan derivative which can be easily quantified colorimetrically to measure P. aeruginosa PAO1 biofilm formation. The results were then compared with CV and XTT assay methods. 2. Materials and methods 2.1. Bacterial strain and culture condition The bacterial strains used in this study were P. aeruginosa PAO1 and P. aeruginosa strain 48 a clinical biofilm forming P. aeruginosa strain isolated in a previous study. Tryptic soy agar (TSA; Merck, Germany) supplemented with 0.2% glucose was used to culture bacterial cells prior to each experiment. The bacteria were cultured aerobically on the medium at 37 °C. Also, tryptic soy broth (TSB; Merck, Germany) supplemented with 0.2% glucose was used to form biofilm in microtiter plates (Abdi-Ali et al., 2006). 2.2. Antibiotics The most common agents used to inhibit biofilm formation are antibiotics. In case of P. aeruginosa due to high resistance to antibiotics, there are limited antibiotics which are in use today. To study the effect of antibiotics on P. aeruginosa PAO1 biofilm, four effective antibiotics of different common classes (aminoglycosides, beta-lactams, fluoroquinolones and cephalosporins) used against the strains of P. aeruginosa were tested. Cefixime and levofloxacin were received as a gift from Loghman Pharmaceutical & Hygienic Co. (Tehran, Iran). Piperacillin and amikacin were obtained from Sigma and Eczacibasi, respectively. Piperacillin (64 μg/ml) and amikacin (256 μg/ml) solutions were prepared in distilled water and cefixime (4096 μg/ml) and
levofloxacin (64 μg/ml) solutions were made by addition of the least amount of methanol and dimethyl sulfoxide (DMSO), respectively. The final volume of the latter two antibiotics was reached by distilled water addition (Lorian, 2005).
2.3. Biofilm formation In each experiment, a flat-bottomed 96-well cell culture microtiter plate (SPL Plastic Labware, Korea) was used. 100 μl of TSB medium supplemented with 0.2% glucose was added to each well. Four different inoculum concentrations (105 to 108 CFU/ml) were used to form biofilm by P. aeruginosa PAO1 and P. aeruginosa strain 48. Using 24 hour bacterial culture, the suspension was adjusted to McFarland standard 0.5 in TSB supplemented with 0.2% glucose. 10fold dilutions were made to reach 105 CFU/ml bacterial cell concentrations. 100 μl of each suspension was inoculated to each well. The test was performed in 6 replicates for each inoculum concentrations. The microplates were incubated at 37 °C for 20 h.
2.4. Antibiotics MBIC determination The wells were filled with medium as mentioned in Section 2.3. Two-fold antibiotic serial dilutions were made in wells in a way that for each antibiotic seven different concentrations were tested. The last row of each microtiter plate was used as controls (H1–H6 as medium control and H7–H12 as bacterial growth control). Using 24 h P. aeruginosa PAO1 culture, the suspension was adjusted to McFarland standard 0.5 in TSB supplemented with 0.2% glucose. 100 μl of the suspension was inoculated to each well except for the medium controls. The microplates were incubated at 37 °C for 20 h (Abdi-Ali et al., 2006).
2.5. Biofilm assay Following 20 h of incubation, the planktonic cells were removed from each well and plates were rinsed thrice using prewarmed (37 °C) physiological saline. Excess moisture was removed by tapping the microplates on sterile napkins. The plates were dried for 15 min in upside-down position in a sterile setting. Then, the prepared microplates were assayed as below.
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2.5.1. TTC assay To determine the optimum conditions for the TTC assay, the TTC (Merck, Germany) concentration, the assay period and the wavelength in which the absorbance was measured, were tested in 4, 3 and 4 different levels, respectively. TTC solutions in distilled water were prepared by dissolving TTC in distilled water in concentrations of 2, 1, 0.5 and 0.1% and then sterilized by 0.22 μm cellulose acetate filters. 50 μL of each solution was added to 3 different set of biofilms formed in the presence of two-fold serial dilution of the aforementioned antibiotics. 200 μl of TSB supplemented with 0.2% glucose was added to all the wells (final TTC concentration: 0.4, 0.2, 0.1 and 0.02% respectively). Plates were incubated in the dark for 5, 6 and 7 h at 37 °C and 120 rpm. After the incubation period, 200 μl of the well contents were moved to a new flat-bottomed microplate and the absorbance was measured at 405, 450, 490 and 540 nm (Epoch Microplate Spectrophotometer, BioTek) (Daniel, 1997; Gabrielson et al., 2002; Knezevic and Petrovic, 2008; Rahman et al., 2004; Shakeri et al., 2007). The assay was performed in 6 replicates in 2 different days for each experiment.
2.5.2. XTT assay XTT assay was performed following the method used by Peeters et al. (2008) with some modifications. Fresh XTT solution was made by dissolving 4 mg XTT (Sigma) in 10 ml prewarmed (37 °C) physiological saline before each assay. The solution was filter- sterilized and then supplemented with 100 μl menadione solution, containing 55 mg menadione (Sigma) in 100 ml acetone. 100 μl of XTT-menadione solution and 100 μl of fresh TSB medium supplemented with 0.2% glucose were added to each well. Plates were incubated in the dark for 7 h at 37 °C and 120 rpm. After 7 h of incubation, 200 μl of well contents were transfered to a new flat-bottomed microplate and the absorbance
was measured at 486 nm with microplate spectrophotometer. The assay was performed in 6 replicates for each antibiotic serial dilution. 2.5.3. Crystal violet assay (CV assay) 250 μl of 0.1% crystal violet (Merck, Germany) solution was added to each well. After 10 min at room temperature, the wells were emptied and the microplates were carefully washed under running tap water to remove excess CV dye. Subsequently, the microplates were vigorously tapped on napkins to remove any excess liquid and air-dried. After an hour, 250 μl of 30% glacial acetic acid (Merck, Germany) was added to all wells. Following a ten minute period, 200 μl of the well contents were transferred to a new flat-bottomed microplate and the absorbance was measured at 550 nm using a microplate spectrophotometer. The assay was performed in 6 replicates for each antibiotic serial dilution (Merritt et al., 2005). 2.6. Statistical analysis To find the optimum condition of biofilm assay, the absorbance amounts were statistically analyzed. Significant differences were determined by one-way analysis of variance (ANOVA One-way) with pairwise comparisons using Tukey's method. A P value of b0.05 was considered statistically significant. For MBIC determination, well contents treated with drugs were compared to treatment controls without drugs. Data are presented here as a percentage of biofilm growth (biofilm remaining after treatment to untreated control). Significant differences were determined by a two-way analysis of variance (ANOVA Two-way) with pairwise comparisons using Tukey's method. A P value of b0.05 was considered statistically significant.
A
B
C
D
Fig. 1. Impact of the TTC concentration on the susceptibility determination of P. aeruginosa PAO1 biofilm to amikacin (A), cefixime (B), levofloxacin (C) and piperacillin (D). The amount of biofilm formed after 20 h of incubation with each antibiotic was estimated. Absorbance at 490 nm was measured following incubation with TTC (0.1, 0.5, 1 and 2%) for 5 h. ANOVA with pairwise comparison using Tukey's method was used to compare TTC concentrations at each drug concentration. *, P b 0.05. Error bars indicate standard errors of mean of six experiments.
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A
137
B
Fig. 2. Comparison of 2 P. aeruginosa strains' biofilm detected by optimized TTC, XTT and CV assays. The amount of biofilm formed after 20 h of incubation was estimated. Absorbance was measured at 405 nm and 486 nm following incubation with TTC (1%) and XTT, respectively. Absorbance was measured at 550 nm for CV assay. ANOVA with pairwise comparison using Tukey's method was used to compare 3 methods for each inoculum concentration. *, P b 0.05. Error bars indicate standard errors of mean of six experiments.
The repeatability of results in both cases were estimated by coefficient of variance percentage (CV%) calculation. The statistical analysis was performed using Minitab 15 (Minitab, Inc.) 3. Results and discussion 3.1. TTC assay A convenient way to estimate the amount of biofilm formed in microtiter plates is to use a colorimetric assay. Various colorimetric assays based on viability assays, using vital dyes have been established (Honraet et al., 2005; Johansen et al., 1997; Kuhn et al., 2003; Peeters et al., 2008; Ramage et al., 2001). The unique chemical and biological properties of tetrazolium salts have led to their widespread application in biology. The reduction of tetrazolium salts from colorless or weakly colored, aqueous solutions to colored derivatives, formazans, is the basis of their use (Berridge et al., 2005). Some studies have reported the use of TTC to detect the microbial biofilm formed (Kim et al., 2010; Shakeri et al., 2007), but our experiments showed that the methods used is not suitable for P. aeruginosa biofilm detection since there were no detectable color changes even after 24 h of incubation with TTC. Therefore, there was a need to develop and optimize a method to use TTC as a vital dye to identify the amount of P. aeruginosa biofilm formation. To this end, the presented method has been developed and optimized for three important parameters including TTC concentration (4 different concentrations), incubation time (5, 6 and 7 h of incubation) and wavelength (4 distinct wavelengths) in which the formed formazan has been detected. MBICs of the tested antibiotics determined under the conditions mentioned are summarized in Table 1. 3.1.1. Effects of incubation time The amount of incubation time is likely to be different in the case of each bacterial strain since formazan formation from TTC is the result of Table 2 Comparison of MBIC determined by TTC, XTT and CV biofilm assay methods. Antibiotic
MBIC determined by the use of TTC
Amikacin Cefixime Levofloxacin Piperacillin
16 512 2 16
XTT μg/ml μg/ml μg/ml μg/ml
8 256 1 8
CV μg/ml μg/ml μg/ml μg/ml
ND ND b0.25 μg/ml 8 μg/ml
ND: The MBIC could not be determined since the lower concentrations of antibiotic inhibited biofilm formation while the higher concentrations could not inhibit the biofilm formation so that MBIC could not be determined.
dye reduction by living cells. Statistical analysis showed that the formed biofilm with the 2 strains were best detected after 6 h of incubation, since the reproducibility of the results were more in contrast with the other incubation periods. The MBIC results show that among 3 different incubation times, in case of MBIC determination, 5 h of incubation with TTC is optimum for P. aeruginosa PAO1 strain, since before this period the color change is limited and is difficult to measure (data not shown) correctly. After 7 h of incubation with TTC (and in some cases even after 6 h), the absorbance exceed amounts that can be measured correctly with the instrument used. On the other hand, in almost all cases, the MBIC after 5 h is favorable since after 6 h of incubation the differences are insignificant. Although, in some other cases, the 5th hour of incubation showed a significant difference with the 6th hour and could show the higher concentration as MBIC (Table 1).
3.1.2. Impact of wavelength used to measure the absorbance Formazan resulted from TTC reduction has been assayed by various wavelengths (Daniel, 1997; Gabrielson et al., 2002; Knezevic and Petrovic, 2008; Rahman et al., 2004; Shakeri et al., 2007). The formed biofilms were best detected in 405 nm. The reproducibility of the results were less in case of 450 nm and 490 nm (lower CV%), yet, the absorbance amount in 540 nm in some cases were as high as it could not be detected by the instrument. So, the best wavelength to detect the non-treated formed biofilm is 405 nm. In case of MBIC determination, absorbance at 405 and 540 nm is significantly less than the other wavelengths (P b 0.05). In some few cases absorbance at 540 nm is as high as it could not be measured. In some other cases absorbance measurement in 490 nm at the 5th hour of incubation with TTC was significantly better than 450 nm at the same period so that the selected wavelength for the rest of the experiments is 490 nm.
3.1.3. Impact of TTC concentration It was shown that the lowest effective concentration of TTC to detect planktonic cells of some bacteria including P. aeruginosa is 0.2% (Eloff, 1998). Also, it has been reported that TTC could be toxic to bacteria in high concentrations (Weinberg, 1953). To find the nontoxic and also the lowest effective concentration of the TTC salt, 4 concentrations have been studied. Using 4 TTC concentrations showed that the formed biofilm could be better detected using 0.5% TTC. The absorbance amounts were significantly different for 108 to 106 CFU/ml inoculum concentrations (P b 0.05). As a result, the biofilms formed by less than 106 CFU/ml initial bacterial concentrations could not be truly detected using the presented optimum TTC assay.
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A
B
C
D
Fig. 3. Comparison of 3 different methods of susceptibility determination of P. aeruginosa PAO1 biofilm to amikacin (A), cefixime (B), levofloxacin (C) and piperacillin (D). The amount of biofilm formed after 20 h of incubation with each antibiotic was estimated. Absorbance was measured at 490 nm and 486 nm following incubation with TTC (1%) and XTT, respectively. Absorbance was measusured at 550 nm for CV assay. ANOVA with pairwise comparison using Tukey's method was used to compare 3 methods at each drug concentration. *, P b 0.05. Error bars indicate standard errors of mean of six experiments.
The MBIC could be better detected using 1% TTC. Yet, 0.5% TTC concentration in almost all the sub-MBIC concentrations showed significant differences with other TTC tested concentrations to determine the biofilm formation (P b 0.05) but the accuracy of 1% TTC is more. Using 2% TTC could lead to a decrease in biofilm growth absorbance (Fig. 1) possibly due to its toxicity for the tested bacterium.
Fig. 4. Biofilm formed in the presence of cefixime (1024–16 μg/ml). Assays were performed in columns 1–4 with CV, in columns 5–8 with TTC (after 5 h) and in columns 9–12 with XTT (10 times diluted) (after 7 h). The MBIC could not be detected by CV but the two other assays shows the raw C (256 μg/ml) as MBIC. H row contains control cells: H3, H4, H7, H8, H11, H12: Controls without bacteria and the rest are controls of bacteria biofilm formation.
3.2. Comparison of XTT, CV and TTC assay methods Among the methods which use non-vital dyes for biofilm detection, CV assay is the most common for various bacteria including P. aeruginosa (Borucki et al., 2003; Djordjevic et al., 2002; Kaplan et al., 2004; Merritt et al., 2005; Mireles et al., 2001; O'Toole G.A. et al., 2000; Pratt and Kolter, 1998; Stepanović et al., 2000; Watnick and Kolter, 1999). Although common, the main drawback of crystal violet assay is the fact that only the biofilm biomass can be measured which is less informative in comparison to viability (Pan et al., 2010; Skogman et al., 2012). This problem seems more prominent when testing microorganisms with high extracellular polymeric substances. Furthermore, Peeters et al. (2008) showed that this method, due to its low repeatability, is not suitable for P. aeruginosa strains which the idea is supported by the results of this study. On the other hand, XTT assay is one of the most common methods using vital dyes to assay the biofilm formation for different microorganisms (Cerca et al., 2005; da SILVA et al., 2008; Nett et al., 2011; Peeters et al., 2008; Pettit et al., 2005; Taff et al., 2012). Comparison of optimized TTC assay for biofilm detection with the two other assay methods showed that the optimized TTC assay was able to detect biofilms formed by different inoculum concentrations (108 to 106 CFU/ml), while the two other methods were not able to distinguish between biofilms formed by different initial bacterial concentrations (Fig. 2). On the other hand, comparison of optimized TTC assay with the two other assay methods for MBIC determination indicated that TTC assay was able to detect the less amounts of biofilm formed which was not detectable by XTT and CV assays (Table 2). Also the accuracy and reproducibility of the results, especially in the lower amount of biofilm which is supposed to form in higher antibiotic concentrations, is more in TTC assay in contrast with the other methods (lower CV%) (Fig. 3).
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It was shown that subinhibitory concentrations of antibiotics are able to induce biofilm formation (Hoffman et al., 2005; Kaplan, 2011; Kaplan et al., 2012). In the case of piperacillin, TTC assay showed the induction of biofilm formation in the presence of piperacillin sub-MBIC concentrations (175–358% due to different concentrations) while the other two methods were not able to detect this kind of induction (Fig. 3D). Interpretation of results of the CV assay with cefixime and amikacin were problematic. As shown in Fig. 4, biofilm formation was detected at the two highest concentrations but the lower concentrations of the antibiotic inhibited biofilm formation. The two other methods did not show such results. Authors believed that since CV attached the EPS of biofilm, it can detect even the cell-free EPS, but the two other methods are able to detect live cells, so that the highest concentrations detected by CV could be cell free matrices which could be produced due to antibiotic stress or any other unknown reasons and not a real biofilm. These findings also support the idea that CV assay is by itself not a reliable assay and the assay results need to be confirmed with another assay which uses a vital dye. 4. Conclusion In the present study, the TTC assay was optimized for P. aeruginosa PAO1 biofilm detection followed by comparison to the two other methods commonly used to identify biofilm formation. All assays were evaluated for their accuracy and repeatability for quantification of biofilm formation in a microtiter plate. All assays showed applicability for determination and quantification of biofilms. However, the repeatability of TTC assays was higher than the two other assays. Although CV assay is simple, fast and cheap, it is not as accurate as the two other methods as it can be seen in the case of amikacin and cefixime. On the other hand, among the three assays used, the XTT assay is the most expensive method, though not the most accurate one. As results indicate, TTC assay (0.5% TTC, 6 h of incubation and absorbance measurement at 405 nm) could be a suitable method to detect P. aeruginosa biofilm formation and 1% TTC, 5 h of incubation and absorbance measurement at 490 nm could be an accurate method to define MBIC in P. aeruginosa PAO1 since it is not very expensive but is sufficiently repeatable and accurate. It is worth mentioning that the method was optimized for factors which the authors hypothesized or observed and effected the TTC assay for P. aeruginosa PAO1 biofilm detection. The factors studied here are not necessarily the same for any other Pseudomonas strains since the ability of biofilm formation, the biofilm growth period, the ability to reduce tetrazolium salts and many other known and unknown parameters may affect the assay results. Acknowledgments Authors thank Dr. Sedigheh Shams for her excellent assistance in the statistical analysis and Dr. Sara Gharavi for editing of this article. References Abdi-Ali, A.,Mohammadi-Mehr, M.,Agha Alaei, Y., 2006. Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa. Int. J. Antimicrob. Agents 27, 196–200. Berridge, M.V., Herst, P.M., Tan, A.S., 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 11, 127–152. Borucki, M.K.,Peppin, J.D.,White, D.,Loge, F.,Call, D.R., 2003. Variation in biofilm formation among strains of Listeria monocytogenes. Appl. Environ. Microbiol. 69, 7336–7342. Brooun, A., Liu, S., Lewis, K., 2000. A dose–response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 44, 640–646. Cerca, N., Martins, S., Cerca, F., Jefferson, K.K., Pier, G.B., Oliveira, R., Azeredo, J., 2005. Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry. J. Antimicrob. Chemother. 56, 331–336.
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