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Engineered colorimetric detection of Staphylococcus aureus extracellular proteases
Talanta 198 (2019) 30–38
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Engineered colorimetric detection of Staphylococcus aureus extracellular proteases
T
Ghadeer A.R.Y. Suaifana, , Samah W.A. Al Nobania, Mayadah B. Shehadeha, Rula M. Darwishb ⁎
a b
Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan
ARTICLE INFO
ABSTRACT
Keywords: Colorimetric assay Staphylococcus aureus Extracellular proteases PH indicators Microorganism Proteolytic activity
This work describes the engineering of a feasible, sensitive, and specific colorimetric assay for the detection of Staphylococcus aureus (S. aureus) extracellular protease production, using a tailored agar-based readout platform. Firstly, S. aureus protease production was evaluated using different culture media. Maximum proteolytic activity was achieved after 24 h of incubation in brain heart infusion (BHI) broth at 37 °C, as confirmed using azo-casein and Sigma’s non-specific protease activity assays. Secondly, to enhance proteolysis detection, novel agar-based platform readouts for S. aureus proteases were developed. Clear proteolytic zones were observed after 48 h of incubation at 37 °C, using an agar-agar platform supplemented with 3% skim milk as a non-specific protease substrate. Thirdly, the colorimetric visualization of S. aureus proteolytic zones was evaluated using three different techniques, namely, the use of chromogenic media, dye flooding over the platform, and protease staining prior to testing. Colorimetric sensing of proteolysis was achieved by applying a Bromocresol Purple-colored protease solution on a skim milk plate count agar–PEG 4000 readout platform. Color change (yellow to burgundy) indicated a positive readout after 15 min of incubation. The lowest limit of S. aureus detection was 102 CFU mL−1. Moreover, direct detection of S. aureus BHI culture was achieved by sensing the pH change because of protease production, using bromothymol blue dye. A lightgreen color represented a positive readout. A direct relationship between S. aureus culture concentration and color change was observed, with a detection limit as low as 103 CFU mL−1. Examination of blind samples of Escherichia coli and S. aureus showed the potential of this assay to specifically detect S. aureus in situ. Therefore, the developed approach is anticipated to help detect S. aureus for biomedical applications.
1. Introduction Staphylococcus aureus (S. aureus) is a versatile pathogenic bacterium responsible for a significant number of acquired infections in communities and hospitals [1]. S. aureus causes broad-spectrum infections ranging from superficial lesions, such as wound infections and abscesses, to systemic life-threatening conditions such as bacteremia, endocarditis, meningitis, osteomyelitis, pneumonia, and brain abscesses [2,3]. The National Institutes of Health and Centers for Disease Control and Prevention proclaimed S. aureus as one of the major human pathogens, causing more than 500,000 infections in the United States of America (USA) annually, of which more than 90,000 cases are antibiotic-resistant [4]. Currently, less than 10% of S. aureus infections can be treated with penicillin [5]. Furthermore, S. aureus has been found to be responsible for toxinmediated diseases such as Staphylococcal foodborne diseases (SFDs) [6]. SFDs are considered the second most common reported cause of
⁎
food-borne illness, as 10% of the affected patients visited or were admitted to hospitals [7]. Hence, S. aureus infection control is of major importance. Hitherto, no vaccine is available against S. aureus infections in humans. Hyperimmune serums or monoclonal antibodies directed toward surface components like capsular polysaccharides or surface protein adhesions could theoretically prevent bacterial adherence, and initiate phagocytosis; however, they have not been applied yet [8,9]. Hence, there is a need for a rapid and reliable detection method, in order to start the appropriate therapy without delay, which is unacceptable in emergencies. Furthermore, early detection of S. aureus reduces mortality, the length of hospitalization, and therefore, elevated costs [10,11]. Definitive S. aureus detection methods include the conventional culturing technique [12]. However, the heterogeneous nature of S. aureus, especially methicillin-resistant S. aureus (MRSA), limits the method’s accuracy, sensitivity, and reliability [12,13]. In addition, the
Correspondence to: Department of Pharmaceutical Sciences, Faculty of Pharmacy, Amman 11942, Jordan. E-mail address: [email protected] (G.A.R.Y. Suaifan).
https://doi.org/10.1016/j.talanta.2019.01.067 Received 22 January 2019; Accepted 23 January 2019 Available online 23 January 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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technique is laborious and time-consuming [12–14]. Other selective in vitro detection methods include ELISA, oligonucleotide-embedded agar matrix application [15], PCR, and liquid chromatography–tandem mass spectrometry. However, these strategies require overnight culturing or sophisticated instrumentation [12,14,16,17]. All these methods have their own advantages and disadvantages. S. aureus produces a group of extracellular proteases that have been recognized to play an important role in its pathogenicity [18]. Proteolytic activity of S. aureus proteases was previously assessed on a solid agar medium. This method was a simple, inexpensive, and straightforward approach with great freedom for substrate selection. However, clear results necessitate incubation for at least 48–72 h, and sometimes up to a week, causing delayed result acquisition, which is by all means not acceptable in severe S. aureus infections. Recently, Suaifan et al. developed a diagnostic colorimetric biosensor for the specific detection of Escherichia coli (E. coli) and Listeria, by utilizing their extracellular proteases as biomarkers [19–21]. This biosensor was easy to operate and rapid, but expensive. In recent years, colorimetric analysis techniques have shown good development prospects due to their high specificity and sensitivity. Foremost among them is the use of a fluorogenic substrate [22]. Other strategies include zymographic techniques [23–26]. All these are promising breakthrough methods, but the need for sophisticated operation and complicated instruments limits their application. Hence, presently, researchers devote more time and energy to improve the simplicity, safety, and accuracy of colorimetric methods for implementation under a wide variety of settings in monitoring devices, following disinfection and sterilization processes in hospitals and nursing homes, as this notorious pathogen leads to serious infections. This study was aimed at the development of a simple, rapid, and specific colorimetric assay for the detection of S. aureus extracellular proteases. An agar-based platform supplemented with a peptide substrate was constructed to visually detect the proteolytic activity of stained S. aureus extracellular protease solutions. Upon substrate digestion, liberated tyrosine fragments alter the media ion concentration and result in a dye color change (Scheme 1).
sensitive S. aureus (MSSA) (ATCC 25923), methicillin-resistant S. aureus (MRSA) (ATCC 33591), S. aureus (ATCC 6539), E. coli O157:H7, E. coli (ATCC 51446), E. coli (ATCC 4157), and E. coli (ATCC 8739). The detection media used included brain heart infusion (BHI) agar and broth, Mueller-Hinton (MH) agar and broth, and malt extract (ME) agar, and were purchased from HiMedia (India). Todd Hewitt (TH) broth was obtained from Sigma Aldrich (India). Skim milk (SK) powder and polyethylene glycol (PEG) were purchased from Fluka–Sigma Aldrich (Germany). Agar-agar (AA), MacConkey (MAC) agar, and Sabouraud dextrose agar (SDA) agar were purchased from Oxoid (England). Nutrient agar (NA) was obtained from Scharlau (European Union). Gelatin from Biotech (USA) and skim milk plate count agar were obtained from Biolab (Hungary). Sterile syringe filters (0.22 µm) were purchased from Millipore (Amman, Jordan). 2.2. Chemicals L-cysteine, azo-casein, tris-buffered saline (pH 7.2), hydrochloric acid (HCl), trichloroacetic acid, potassium phosphate buffer (pH 7.5), casein from bovine milk, Folin & Ciocalteu’s phenol reagent, and anhydrous sodium acetate were purchased from Sigma-Aldrich (USA). Sodium hydroxide (NaOH) was obtained from Scharlau (Spain), and anhydrous sodium carbonate (Na2CO3) was purchased from SDFCL (India). L-tyrosine was purchased from Hopkin and Williams Ltd. (England). Methylene Blue (MB) was obtained from Gurr-BDH chemicals (England). Coomassie Brilliant Blue (CBB), naphthol blue black (NBB), bromothymol blue (BTB), and brilliant green (BG) were purchased from Fluka–Sigma Aldrich (Germany). Bromocresol green (BCG) and bromocresol purple (BCP) were purchased from Xilong (China). Neutral Red (NR) from Koch-Light (England) and pH-Fix test strips 0–14 were purchased from Macherey-Nagel (USA). 2.3. Bacterial counting and calibration curves Bacterial growth in BHI, MH, and TH broth media was determined by measuring the optical density using a microtiter plate reader spectrophotometer (Epoch-Biotec, California, USA). Cell growth was monitored using the viable plate count method. Blank samples (broth only) were included as a sterility control. Three replications were carried out on different days. The degree of correlation between S. aureus counts and optical densities (ODs) was investigated by linear regression of
2. Materials and methods 2.1. Bacterial strains and detection media The microorganisms used in this study included methicillin-
Scheme 1. Mechanism of colorimetric detection of S. aureus proteinase proteolytic activity using a skim milk plate count agar–PEG 4000 sensing platform. 31
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Table 1 Proteolytic activity of S. aureus culture supernatant proteases assayed using azo-casein assay and Sigma's non-specific protease activity assay. Proteolytic activity a
Azo-casein assay (U) Sigma's non-specific assay (U mL−1)b a b
ATCC 25932 BHI
ATCC 33591 BHI
ATCC 25932 MH
ATCC 33591 MH
ATCC 25932 TH
ATCC 33591 TH
25.5 0.227
26.7 0.234
23.8 0.157
24.1 0.123
24.8 0.168
25.1 0.170
One unit of activity was defined as an increase in optical density of 0.01 after 30 min of incubation at 37 °C. One unit of activity was defined as the amount in micromoles of tyrosine equivalents released from casein per minute.
continuous data. For the viable count, 100 µL of each S. aureus primary broth culture (PBC) was spread over the appropriate agar plate and incubated for 24 h at 37 °C.
Table 3 Detection of S. aureus culture supernatant protease proteolytic activity using agar-based platform supplemented with either skim milk or gelatin as substrates.
2.4. Optimization of S. aureus extracellular protease activity One colony each of S. aureus (ATCC 25923) and S. aureus (ATCC 33591) was isolated, inoculated onto BHI, MH, and TH broths, and incubated for 24 h at 37 °C. Each bacterial PBC was serially diluted to determine the bacterial colony forming unit (CFU) value of the original stock, using the viable count spread dilution method. Next, the S. aureus PBCs were centrifuged at 14,000 rpm for 10 min at RT to sediment the bacteria and filtered through a 0.22 µm sterile filter membrane to obtain the S. aureus extracellular protease solutions [19]. The pH value of the extracellular protease solutions was measured using pH-Fix test strips 0–14 (Table 1), and the solutions were stored at −80 °C for later use. The proteolytic activity of proteases from each bacterial culture supernatant was measured using the azo-casein assay [27] and Sigma's non-specific protease activity assay [28] (Table 2). The bacterial protease concentration was associated with the bacterial concentration and was referred to as a colony forming unit (CFU).
2.6.1. Chromogenic media Dye solutions of BCG (0.0015% w/v) and CBB (0.25% w/v) in methanol-acetic acid-water 5:1:4 (v/v/v) were used to stain 3% SK-AA media. Then, a 50 µL aliquot of S. aureus extracellular protease solution was injected into the well punched at the center of the solid media. Thereafter, the plates were incubated at 37 °C for 48 h [29]. The protease proteolytic activity was evaluated by observing the change in color of the zone around the well (Fig. 2I).
BHI BHI MH MH TH TH
6 5.5 7 8 7 7
25932 33591 25932 33591 25932 33591
-ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve
+vea +ve +ve +ve -ve +ve -ve +ve -ve -ve -ve +ve
Most clear proteolytic zone developed after incubation at 37 °C for 48 h.
2.7. Dye-based quantitative detection of S. aureus culture supernatant protease proteolytic activity To gain an insight into the applicability of the developed readout platform based on plate count skim milk supplemented with PEG 4000, different concentrations of BCP-colored S. aureus protease solutions prepared from a series of PBC concentrations (101 to 109 CFU mL−1) were tested, and the lower limit of detection (LOD) was determined (Fig. 4).
Table 2 pH values of S. aureus culture supernatant proteases prepared from different broth media.
ATCC ATCC ATCC ATCC ATCC ATCC
SK-AA SK-NA SK-MH SK-BHI SK-MEA SK-MAC G-AA G-NA G-MH G-BHI G-ME G -MAC
2.6.3. Protease solution dying S. aureus culture supernatant was spiked with a number of coloring agents. Then, 50 µL of the colored supernatant solution was injected into a well punched at the center of the biosensing platform (Fig. 2III). Proteolysis was evaluated by observing the color change of the proteolytic zone around the well. In this approach, combinations of pH indicators [BCP (0.6% w/v), BTB (0.4% w/v), BCG (0.1% w/v), NBB (0.1% w/v), BG (0.1% w/v), CBB (0.1% w/v), NR (0.1% w/v), MB (0.1% w/v), and TB (0.1% w/v)] and diffusivity agents [(PEG 400 (5%), PEG 4000 (5%), and PEG 6000 (5%)] were used (Fig. 2). Proteolysis was evaluated by visual observation of the change in color zone intensity versus the control (dyed broth only) (Fig. 3).
2.6. Optimization of dye-based qualitative visualization of S. aureus culture supernatant protease proteolytic activity
Protease solution pH value
48 h incubation period
2.6.2. Dye flooding technique S. aureus culture supernatant (50 µL) was injected into a well punched at the center of the biosensing platform. Thereafter, the plates were incubated at 37 °C for 48 h. Dye solutions BCG (0.0015% w/v) or CBB (0.25% w/v) were poured over the plates, which were re-incubated for 20–30 min at RT [29]. The protease activity was evaluated by visually observing the change in proteolytic zone color around the well (Fig. 2II).
To obtain an optimal agar-based readout platform, proteases from different S. aureus concentrations in CFU were screened using different platforms, including plate count skim milk agar, AA, NA, MH agar, BHI agar, MAC agar, and ME agar supplemented with one of the following non-specific protease substrates: gelatin (0.5% w/v) and SK (1%, 3%, 5%, and 7% w/v). Fifty microliters of S. aureus culture supernatant were added to the punch well (6 mm), and the plate was incubated at 37 °C for 24 and 48 h (Table 3, Fig. 1).
Culture broth medium
24 h incubation period
a
2.5. Optimization of agar-based readout platform
S. aureus
Substrate-agar platform
2.8. Qualitative and semi-quantitative assessment of S. aureus cultures In situ assessment of 2 h incubated S. aureus cultures (101 to 109 CFU mL−1) was performed using BTB (0.4% w/v). The change in color intensity as a function of pH change following protease production was 32
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Fig. 1. S. aureus protease agar–based biosensing platform supplemented with different SK percentages after incubation for 48 h at 37 °C. (A) 1% SK-AA; (B) 3% SK-AA (optimum percentage); (C) 5% SK-AA; and (D) 7% SK-AA.
observed and compared to the negative blank (dyed broth only) (Fig. 5).
3.2. Development of S. aureus extracellular protease agar–based biosensing platform Extracellular proteases of S. aureus (ATCC 25923 and ATCC 33591) cultivated in the optimized culture medium (BHI) preferentially hydrolyzed SK-containing agar platforms. Proteolytic zones were visually detected as a clear halo after inoculation on the SK-AA platform, followed by incubation for 48 h at 37 °C (Table 3). On the other hand, no clear zones were detected in the AA, MH agar, BHI agar, and ME agar platforms supplemented with gelatin. However, when proteases were inoculated on gelatin-NA and gelatin-MAC agar platforms, hazy proteolytic zones were observed. Accordingly, SK-AA is the favorable biosensing platform for the detection of S. aureus extracellular protease proteolytic activity. Visualization of S. aureus proteolysis using different percentages of SK-supplemented media (1%, 3%, 5%, and 7%) revealed a clear proteolytic zone in the 3% SK platform (Fig. 1I). Hence, the 3% SK-supplemented medium was designated as the optimal agar-based biosensing platform.
2.9. Preparation of blind control samples PCBs of different strains of S. aureus (ATCC 25932, ATCC 33591, and ATCC 6539) and E. coli (ATCC 156, ATCC 51446, ATCC 4157, and ATCC 8739) were prepared and labeled with a number to hide the identity of each bacterium for blind testing. Samples were incubated for 2 h at 37 °C, spiked with BTB (0.4% w/v), and assessed (Fig. 6). 3. Results and discussion 3.1. Effect of culture media composition on S. aureus extracellular protease activity The tested S. aureus cultures (ATCC 25923 and ATCC 33591) were cultivated in BHI (pH = 7.5), MH (pH = 7.5), and TH (pH = 8) broth media, and incubated at 37 °C for 48 h. BHI was the most appropriate culture medium for the production of active extracellular proteases, which were detected using the azo-casein assay (26.7 U) and Sigma's non-specific protease activity assay (0.234 U mL−1) [27,28]; the results are shown in Table 1. Additionally, it is to be mentioned that the S. aureus extracellular protease solutions prepared from BHI, MH, and TH broth media varied in pH, as indicated in Table 2. This variation is an indicative biomarker for the effect of media composition on the type of proteases produced, and consequently, the protease activity [30].
3.3. Dye-based qualitative assessment of S. aureus protease activity Two coloring agents (BCG and CBB) were used to visually assess the proteolytic zones of S. aureus proteinases, by dying the solid agar platform to produce a chromogenic medium (Fig. 2I), or by flooding the dye solution over the detecting platform (Fig. 2II). Unfortunately, the proteinase proteolytic zones were not clear. Therefore, a new approach using stained S. aureus protease solutions was adopted, wherein different coloring agents (BCG, NBB, BG, CBB, NR, MB, TB, and BCP) were 33
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Fig. 2. Visualization of S. aureus extracellular proteases cultivated in BHI broth using: (I) Chromogenic media dye incorporated into 3% SK-AA platform; (II) Dye flooding over the 3% SK-AA platform, followed by incubation for 20–30 min at RT. (III) In situ dying of S. aureus protease solution with different dyes (Coomassie Brilliant Blue (CBB), Bromocresol Green (BCG), Brilliant Green (BG), Naphthol Blue Black (NBB), Methylene Blue (MB), Thymol Blue (TB), Neutral Red (NR) (0.1% w/v) prior to application over the 3% SK-AA platform; and the chemical structures of the dyes. Positive visual readout for red-colored chemical structures. *C stands for control sample (no protease) and T stands for S. aureus culture supernatant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
added. Positive visual readout results with BCG (0.1% w/v), BG (0.1% w/v), and NBB (0.1% w/v) were observed, as shown in Fig. 2III. The color intensity difference between the stained test protease sample and the control (stained media only) could be correlated to the electrostatic interactions between dye sulfate (SOзˉ and HSO₄ˉ) groups and tyrosine amino groups [31]. Tyrosine liberated upon SK substrate digestion (proteolysis) may alter the chemical structure conjugation system and result in a color change. The low water solubility of the MB, TB, NR, and CBB dyes counteracted ionic interaction and resulted in negative readout results (Fig. 2III).
Interestingly, in situ staining of S. aureus extracellular protease solutions with the BCG, BG, and NBB visualizing agents shortened the time needed to detect proteinase activity to 18 h (Fig. 3I), compared with 48 h when unstained (Fig. 1B). To speed up the visual detection, simultaneous dying of the biosensing platform and S. aureus culture supernatant was conducted. Accordingly, PEG was incorporated into the 3% SK-AA platform to promote dye diffusion. Three different grades of PEG, 400, 4000, and 6000, were used. However, the presence of PEG in SK-AA prevented the formation of homogenous media after autoclaving. Therefore, other agar-based media were tested. Interestingly, 34
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Fig. 3. Qualitative detection of S. aureus extracellular proteases using different coloring agents and different biosensing platforms: (I) BG-colored protease over 3% skim milk agar-agar platform; (II) BG-colored protease over skim milk plate count agar–PEG 4000 platform at different time intervals. A 2 h; B 18 h; C 24 h; and D 48 h over black background. (III) A BCP pH color chart; B S. aureus and E. coli BCP-colored protease over skim milk plate count agar–PEG 4000 biosensing platform after 15 min of incubation. *C stands for control sample (no protease) and T stands for S. aureus culture supernatant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
homogeneity was achieved when PEG 4000 was incorporated into the skim milk plate count agar medium. Following the application of BGcolored S. aureus culture supernatant on the PEG 4000–skim milk plate count agar platform, visual detection of proteolysis was tracked at different time intervals (2–48 h) (Fig. 3II(A–D)). A difference in proteolytic zone color intensity was detected visually after 2 h of incubation at 37 °C (Fig. 3IIA). The inclusion of PEG 4000 in the medium improved dye diffusion through the solid agar platform and triggered water retention, which enabled protease proteolytic ability that manifested as a clear difference in proteolytic zone color intensity for the test sample when compared to that of the control (Fig. 3IIA). To optimize detection rapidity over the developed optical readout platform, the BCP dye reagent was used to track the change in pH following proteolysis, due to tyrosine amino acid liberation. The change in hydrogen ion concentration at the digested area resulted in a color shift from yellow to burgundy for S. aureus, compared to a purplish color for E. coli, following incubation at 37 °C for 15 min, as shown in
Fig. 3III. The pH of the negative control (BHI broth only) was adjusted using glacial acetic acid to match that for the tested protease at zero time. All the experiments were carried out in triplicates. 3.4. Quantitative detection of S. aureus culture supernatant proteolytic activity To gain an insight into the ability of the optical readout platform to semi-quantitatively detect S. aureus proteases, different concentrations of S. aureus (ATCC 25932) culture supernatants (109, 108, 107, 106, 105, 104, 103, 102, and 101 CFU mL−1) were prepared and examined. A direct relationship between different S. aureus proteinase culture densities and proteolytic zone burgundy chromophore intensities was observed, as shown in (Fig. 4I). At higher proteolytic activity (as measured using Sigma's non-specific protease activity assay (Fig. 4II(A and B)) more tyrosine residues could be released; thus, the media pH increased in association with an increase in chromophore generation. 35
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Fig. 4. Quantitative detection of proteolytic activity of different S. aureus ATCC 25932 culture supernatants prepared from different PBC concentrations: (I) Skim milk plate count agar–PEG 4000 biosensing platform under the effect of different BCP-colored S. aureus ATCC 25932 protease concentrations; (II) Average proteolytic activity of different S. aureus culture supernatants measured using Sigma's non-specific protease activity assay ± CV. The protease activity was determined in terms of Units, which are the amounts in micromoles of tyrosine equivalents released from casein per minute. *C stands for control sample (no protease) and T stands for S. aureus culture supernatant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A lower LOD of 100 CFU mL−1 was achieved without the use of any instrumentation and within 15 min of incubation (Fig. 4I). The LOD was based on visual detection of the lowest protease concentration incapable of developing a distinct proteolytic zone color change. It is to be mentioned that the solid agar biosensing platform showed an adequate long-term stability of up to three months if stored in a sterile container away from direct light at 2–8 °C.
3.6. Qualitative and semi-quantitative detection of S. aureus culture In accordance with our findings, the S. aureus PBC pH value varied with the incubation period at 37 °C. At zero time, the culture pH was > 7; however, after 24 h of incubation, the culture pH dropped to 5.5–6. Hence, the BTB coloring reagent (0.4% w/v) was used for in situ detection of S. aureus (ATCC 25932, ATCC 33591, and ATCC 6539) PBCs. This reagent has a yellow chromophore below pH 6.0 and a blue chromophore above pH 7.6. A light-green color indicated a positive visual readout result, whereas the negative control sample (BHI broth) produced a dark green–bluish color. Fig. 5 shows the semi-quantitative detection of different S. aureus culture concentrations (109, 108, 107, 106, 105, 104, 103, 102, and 101 CFU mL−1). A proportional decrease in culture media green chromophore intensity was observed with higher PBC concentrations. This may be attributed to the increase in media ion concentration with higher
3.5. Comparison with literature The colorimetric assay presented herein is simple and can be used hand-held by untrained personnel; it is cheap, with no need for any instrumentation to read out the results. At present, a number of detection methods have been applied [31–42]. A summary of their advantages and limitations is presented in Table 4. 36
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Fig. 5. Qualitative and semi-quantitative detection of S. aureus PBCs. Correlation between different S. aureus PBC concentrations (CFU mL−1) and BTB color intensity, illustrating a lower limit of detection of 103. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 6. Dye-based S. aureus culture assay specificity. Positive green color for S. aureus spp. and yellow color for E. coli spp.: (C) control, (1) E. coli (ATCC 8739), (2) S. aureus (ATCC 25932), (3) E. coli (ATCC 156), (4) E. coli (ATCC 4157), (5) S. aureus (ATCC 33591), (6) E. coli (ATCC 51446), and (7) S. aureus (ATCC 6539). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 4 Advantages and limitations of common S. aureus detection methods. Method
Advantages
Limitations
Reference
Bacterial culture method
Cheap; able to detect and quantify S. aureus
[32]
Enzyme-linked immunosorbent assay (ELISA) Lateral flow immunoassays Polymerase chain reaction (PCR)
Specific; can be automated so that it is more time- and labor-efficient; can handle large numbers of samples Low cost; reliable; easy to operate; sensitive; specific High sensitivity; high specificity; automated; reliable results
Colorimetric optical biosensors
Cost-effective; simple; results can be read out by bare eyes and sometimes with the help of a strip reader; timely detection after reaction completion Feasible; sensitive; rapid; applicable for on-site detection; visual readout of the results
Time-consuming; labor-intensive; takes several days to provide conclusive results High cost; requires trained personnel; requires labelling of antibodies or antigens Require labelling of antibodies or antigens High cost; requires trained technicians; affected by PCR inhibitors; requires DNA purification Not suitable for multiple use Requires minimal detection-platform manipulation
Current
Agar-based biosensing platform
protease production. On the other hand, the negative control sample showed an intense green color. In situ pH-dependent detection of S. aureus culture was developed, to offer a point-of-care (POC) diagnostic assay. This colorimetric assay offers quick results, leading to a speedy decision concerning the treatment plan, reducing the emergency room time and number of hospital beds required, and ensuring the optimal use of professional time. It is worth mentioning that the current detection assay is simple and can be performed by untrained personnel anywhere in hospitals, clinics and physician offices, university and school medical centers, pharmaceutical clinical trial sites, and homes.
[33] [34,35] [36–38] [39–41]
2 h bacterial PBCs, including those of three S. aureus (ATCC 25932, ATCC 33591, and ATCC 6539) and four E. coli (ATCC 156, ATCC 51446, ATCC 4157, and ATCC 8739) species. These samples were labeled with numbers such that the bacteria identities were blinded to the person in charge of carrying out the test. Following this, the BTB dye reagent was added, and a green color for S. aureus strains and a yellow color for all the E. coli strains were observed, as shown in Fig. 6. 4. Conclusions In this research, a promising, breakthrough, simple, and low-cost colorimetric S. aureus detection assay was developed using S. aureus extracellular proteases as the organic biomarkers. Using this approach, the presence of S. aureus was qualitatively and semi-quantitatively detected within 15 min over a solid readout platform, with a lower LOD of
3.7. Specificity of S. aureus culture dye-based assay Validation of the assay specificity was carried out by blindly testing 37
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102 CFU mL−1. A further in situ specific detection of S. aureus culture was designed to spot S. aureus presence specifically among a number of blindly tested pathogens. This method was based on the use of the BTB dye reagent, which showed a change in color due to protease production during the 2 h incubation period. The LOD was 103 CFU mL−1. These assays are easy to perform and are applicable for patient POC testing without the need for many preparative steps and instruments.
(2012) 1–11. [17] O.G. Brakstad, K. Aasbakk, J.A. Maeland, Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene, J. Clin. Microbiol. 30 (1992) 1654–1660. [18] R.C. Kasana, R. Salwan, S.K. Yadav, Microbial proteases: detection, production, and genetic improvement, Crit. Rev. Microbiol. 37 (2011) 262–276. [19] G.A.R.Y. Suaifan, S. Alhogail, M. Zourob, Rapid and low-cost biosensor for the detection of Staphylococcus aureus, Biosens. Bioelectron. 90 (2017) 230–237. [20] S. Alhogail, G.A.R.Y. Suaifan, M. Zourob, Rapid colorimetric sensing platform for the detection of Listeria monocytogenes foodborne pathogen, Biosens. Bioelectron. 86 (2016) 1061–1066. [21] G.A.R.Y. Suaifan, S. Alhogail, M. Zourob, Paper-based magnetic nanoparticle-peptide probe for rapid and quantitative colorimetric detection of Escherichia coli O157:H7, Biosens. Bioelectron. 92 (2017) 702–708. [22] J. Gooch, B. Daniel, N. Frascione, Application of fluorescent substrates to the in situ detection of prostate specific antigen, Talanta 125 (2014) 210–214. [23] M. Rapala-Kozik, O. Bochenska, D. Zajac, J. Karkowska-Kuleta, M. Gogol, M. Zawrotniak, M. Kozik, Extracellular proteinases of Candida species pathogenic yeasts, Mol. Oral. Microbiol. 33 (2018) 113–124. [24] R. Ruchel, R. Tegeler, M. Trost, A comparison of secretory proteinases from different strains of Candida albicans, J. Med. Vet. Mycol. 20 (1982) 233–244. [25] P. Sacherer, G. Defago, D. Haas, Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0, FEMS Microbiol. Lett. 116 (1994) 155–160. [26] M.S. Lantz, P. Ciborowski, Zymographic techniques for detection and characterization of microbial proteases, Methods Enzymol. 235 (1994) 563–594. [27] S. Takeuchi, T. Kinoshita, T. Kaidoh, N. Hashizume, Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken, Vet. Microbiol. 67 (1999) 195–202. [28] C. Cupp-Enyard, Sigma's non-specific protease activity assay - casein as a substrate, J. Vis. Exp. 899 (2008) 1–3. [29] P. Vijayaraghavan, S.G.P. Vincent, A simple method for the detection of protease activity on agar plates using bomochresolgreen dye, J. Biochem. Tech. 4 (2013) 628–630. [30] N. Souissi, Y. Ellouz-Triki, A. Bougatef, M. Blibech, M. Nasri, Preparation and use of media for protease-producing bacterial strains based on by-products from Cuttlefish (Sepia officinalis) and wastewaters from marine-products processing factories, Microbiol. Res. 163 (2008) 473–480. [31] J.V. Moller, U. Kragh-Hansen, Indicator dyes as probes of electrostatic potential changes on macromolecular surfaces, Biochemistry 14 (1975) 2317–2323. [32] G.A. Suaifan, Colorimetric biosensors for bacterial detection, in: U.A. Minhaz, Z. Mohammed, T. Eiichi (Eds.), Food Biosensors. E-Royal Society of Chemistry Inc, United Kingdom, 2016, pp. 182–202. [33] S. Böcher, R. Smyth, G. Kahlmeter, J. Kerremans, M.C. Vos, R. Skov, Evaluation of four selective agars and two enrichment broths in screening for methicillin-resistant Staphylococcus aureus, J. Clin. Microbiol. 46 (2008) 3136–3138. [34] R.W. Bennett, Staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology, J. Food Prot. 68 (2005) 1264–1270. [35] C.Z. Li, K. Vandenberg, S. Prabhulkar, X. Zhu, L. Schneper, K. Methee, C.J. Rosser, E. Almeide, Paper based point-of-care testing disc for multiplex whole cell bacteria analysis, Biosens. Bioelectron. 26 (2011) 4342–4348. [36] C. Pöhlmann, I. Dieser, M. Sprinzl, A lateral flow assay for identification of Escherichia coli by ribosomal RNA hybridisation, Analyst 139 (2014) 1063–1071. [37] T. Trnčíková, V. Hrušková, K. Oravcová, D. Pangallo, E. Kaclíková, Rapid and sensitive detection of Staphylococcus aureus in food using selective enrichment and real-time PCR targeting a new gene marker, Food Anal. Methods 2 (2009) 241–250. [38] J.C. Cheng, C.L. Huang, C.C. Lin, C.C. Chen, Y.C. Chang, S.S. Chang, C.P. Tseng, Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR, Clin. Chem. 52 (2006) 1997–2004. [39] J.H. Yoon, S. Wei, D.H. Oh, A highly selective enrichment broth combined with real-time PCR for detection of Staphylococcus aureus in food samples, LWT 94 (2018) 103–110. [40] Y.J. Sung, H.J. Suk, H.Y. Sung, T. Li, H. Poo, M.G. Kim, Novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of Staphylococcus aureus in milk, Biosens. Bioelectron. 43 (2013) 432–439. [41] J. Yuan, S. Wu, N. Duan, X. Ma, Y. Xia, J. Chen, Z. Ding, Z. Wang, A sensitive gold nanoparticle-based colorimetric aptasensor for Staphylococcus aureus, Talanta 127 (2014) 163–168. [42] P. Liu, L. Han, F. Wang, V.A. Petrenko, A. Liu, Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus, Biosens. Bioelectron. 82 (2016) 195–203.
Acknowledgments Prof. Ghadeer Suaifan would like to acknowledge the funding from the Deanship of Scientific Research at The University of Jordan, Jordan (Grants number 1731, 2016) and the Ministry of Higher Education, Jordan (Scientific Research Support Fund number MPH/1/19/2015). Funding This work was funded by the Ministry of Higher Education, through the Scientific Research Support Fund [grant number MPH/1/19/2015], and The University of Jordan [Grant numbers 1731, 2016]. Declaration of Interest The authors declare no conflict of interest. References [1] G. Rachel, L. Franklin, Pathogenesis of methicillin-resistant Staphylococcus aureus infection, Clin. Infect. Dis. 46 (2008) S350–S359. [2] M. Aires de Sousa, H. Lencastre, Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones, FEMS Immunol. Med. Microbiol. 40 (2004) 101–111. [3] J. Bien, O. Sokolova, B. Bozko, Characterization of virulence factors of Staphylococcus aureus: novel function of known virulence factors that are implicated in activation of airway epithelial Proinflammatory response, J. Pathog. 2011 (2011) 1–13. [4] F.D. Lowy, Staphylococcus aureus infections, N. Engl. J. Med. 339 (1998) 520–532. [5] P.S. Loomba, J. Taneja, B. Mishra, Methicillin and vancomycin resistant S. aureus in hospitalized patients, J. Glob. Infect. Dis. 2 (2010) 275–283. [6] K. Plata, A.E. Rosato, G. Wegrzyn, Staphylococcus aureus as an infectious agent: overview of biochemistry and molecular genetics of its pathogenicity, Acta Biochim. Pol. 56 (2009) 597–612. [7] J.A. Hennekinne, M.L. De Buyser, S. Dragacci, Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation, FEMS Microbiol. 36 (2012) 815–836. [8] G.L. Archer, Staphylococcus aureus: a well-armed pathogen, Clin. Infect. Dis. 26 (1998) 1179–1181. [9] G.Y. Liu, Molecular pathogenesis of Staphylococcus aureus infection, Pediatr. Res. 65 (2009) 71R–77R. [10] S. Gröbner, M. Dion, M. Plante, V.A.J. Kempf, Evaluation of the BD GeneOhm StaphSR assay for the detection of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from spiked positive blood culture bottles, J. Clin. Microbiol. 47 (2009) 1689–1694. [11] T. Yen Tan, S. Cordan, R. Barnes, B. Cooksan, Rapid identification of methicillinresistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR, J. Clin. Microbiol. 39 (2001) 4529–4531. [12] P.K. Mandal, A.K. Biswas, K. Choi, U.K. Pal, Methods for rapid detection of food borne pathogens: an overview, Am. J. Food Technol. 6 (2011) 87–102. [13] X. Zhao, C.W. Lin, J. Wang, D.H. Oh, Advances in rapid detection methods for foodborne pathogens, J. Microbiol. Biotechnol. 24 (2014) 297–312. [14] M.J. Struelens, Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) and patient management, Clin. Microbiol. Infect. 12 (2006) 23–26. [15] O.G. Brakstad, J.F. Maeland, Comparison of tests designed to identify Staphylococcus aureus thermostable nuclease, Acta Pathol. Microbiol. Immunol. Scand. B 103 (1995) 219–224. [16] C.L. Pierce, J.C. Rees, F.M. Fernández, J.R. Barr, Viable Staphylococcus aureus quantitation using 15N metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow, Mol. Cell Proteom. 11
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Engineered colorimetric detection of Staphylococcus aureus extracellular proteases
T
Ghadeer A.R.Y. Suaifana, , Samah W.A. Al Nobania, Mayadah B. Shehadeha, Rula M. Darwishb ⁎
a b
Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan
ARTICLE INFO
ABSTRACT
Keywords: Colorimetric assay Staphylococcus aureus Extracellular proteases PH indicators Microorganism Proteolytic activity
This work describes the engineering of a feasible, sensitive, and specific colorimetric assay for the detection of Staphylococcus aureus (S. aureus) extracellular protease production, using a tailored agar-based readout platform. Firstly, S. aureus protease production was evaluated using different culture media. Maximum proteolytic activity was achieved after 24 h of incubation in brain heart infusion (BHI) broth at 37 °C, as confirmed using azo-casein and Sigma’s non-specific protease activity assays. Secondly, to enhance proteolysis detection, novel agar-based platform readouts for S. aureus proteases were developed. Clear proteolytic zones were observed after 48 h of incubation at 37 °C, using an agar-agar platform supplemented with 3% skim milk as a non-specific protease substrate. Thirdly, the colorimetric visualization of S. aureus proteolytic zones was evaluated using three different techniques, namely, the use of chromogenic media, dye flooding over the platform, and protease staining prior to testing. Colorimetric sensing of proteolysis was achieved by applying a Bromocresol Purple-colored protease solution on a skim milk plate count agar–PEG 4000 readout platform. Color change (yellow to burgundy) indicated a positive readout after 15 min of incubation. The lowest limit of S. aureus detection was 102 CFU mL−1. Moreover, direct detection of S. aureus BHI culture was achieved by sensing the pH change because of protease production, using bromothymol blue dye. A lightgreen color represented a positive readout. A direct relationship between S. aureus culture concentration and color change was observed, with a detection limit as low as 103 CFU mL−1. Examination of blind samples of Escherichia coli and S. aureus showed the potential of this assay to specifically detect S. aureus in situ. Therefore, the developed approach is anticipated to help detect S. aureus for biomedical applications.
1. Introduction Staphylococcus aureus (S. aureus) is a versatile pathogenic bacterium responsible for a significant number of acquired infections in communities and hospitals [1]. S. aureus causes broad-spectrum infections ranging from superficial lesions, such as wound infections and abscesses, to systemic life-threatening conditions such as bacteremia, endocarditis, meningitis, osteomyelitis, pneumonia, and brain abscesses [2,3]. The National Institutes of Health and Centers for Disease Control and Prevention proclaimed S. aureus as one of the major human pathogens, causing more than 500,000 infections in the United States of America (USA) annually, of which more than 90,000 cases are antibiotic-resistant [4]. Currently, less than 10% of S. aureus infections can be treated with penicillin [5]. Furthermore, S. aureus has been found to be responsible for toxinmediated diseases such as Staphylococcal foodborne diseases (SFDs) [6]. SFDs are considered the second most common reported cause of
⁎
food-borne illness, as 10% of the affected patients visited or were admitted to hospitals [7]. Hence, S. aureus infection control is of major importance. Hitherto, no vaccine is available against S. aureus infections in humans. Hyperimmune serums or monoclonal antibodies directed toward surface components like capsular polysaccharides or surface protein adhesions could theoretically prevent bacterial adherence, and initiate phagocytosis; however, they have not been applied yet [8,9]. Hence, there is a need for a rapid and reliable detection method, in order to start the appropriate therapy without delay, which is unacceptable in emergencies. Furthermore, early detection of S. aureus reduces mortality, the length of hospitalization, and therefore, elevated costs [10,11]. Definitive S. aureus detection methods include the conventional culturing technique [12]. However, the heterogeneous nature of S. aureus, especially methicillin-resistant S. aureus (MRSA), limits the method’s accuracy, sensitivity, and reliability [12,13]. In addition, the
Correspondence to: Department of Pharmaceutical Sciences, Faculty of Pharmacy, Amman 11942, Jordan. E-mail address: [email protected] (G.A.R.Y. Suaifan).
https://doi.org/10.1016/j.talanta.2019.01.067 Received 22 January 2019; Accepted 23 January 2019 Available online 23 January 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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technique is laborious and time-consuming [12–14]. Other selective in vitro detection methods include ELISA, oligonucleotide-embedded agar matrix application [15], PCR, and liquid chromatography–tandem mass spectrometry. However, these strategies require overnight culturing or sophisticated instrumentation [12,14,16,17]. All these methods have their own advantages and disadvantages. S. aureus produces a group of extracellular proteases that have been recognized to play an important role in its pathogenicity [18]. Proteolytic activity of S. aureus proteases was previously assessed on a solid agar medium. This method was a simple, inexpensive, and straightforward approach with great freedom for substrate selection. However, clear results necessitate incubation for at least 48–72 h, and sometimes up to a week, causing delayed result acquisition, which is by all means not acceptable in severe S. aureus infections. Recently, Suaifan et al. developed a diagnostic colorimetric biosensor for the specific detection of Escherichia coli (E. coli) and Listeria, by utilizing their extracellular proteases as biomarkers [19–21]. This biosensor was easy to operate and rapid, but expensive. In recent years, colorimetric analysis techniques have shown good development prospects due to their high specificity and sensitivity. Foremost among them is the use of a fluorogenic substrate [22]. Other strategies include zymographic techniques [23–26]. All these are promising breakthrough methods, but the need for sophisticated operation and complicated instruments limits their application. Hence, presently, researchers devote more time and energy to improve the simplicity, safety, and accuracy of colorimetric methods for implementation under a wide variety of settings in monitoring devices, following disinfection and sterilization processes in hospitals and nursing homes, as this notorious pathogen leads to serious infections. This study was aimed at the development of a simple, rapid, and specific colorimetric assay for the detection of S. aureus extracellular proteases. An agar-based platform supplemented with a peptide substrate was constructed to visually detect the proteolytic activity of stained S. aureus extracellular protease solutions. Upon substrate digestion, liberated tyrosine fragments alter the media ion concentration and result in a dye color change (Scheme 1).
sensitive S. aureus (MSSA) (ATCC 25923), methicillin-resistant S. aureus (MRSA) (ATCC 33591), S. aureus (ATCC 6539), E. coli O157:H7, E. coli (ATCC 51446), E. coli (ATCC 4157), and E. coli (ATCC 8739). The detection media used included brain heart infusion (BHI) agar and broth, Mueller-Hinton (MH) agar and broth, and malt extract (ME) agar, and were purchased from HiMedia (India). Todd Hewitt (TH) broth was obtained from Sigma Aldrich (India). Skim milk (SK) powder and polyethylene glycol (PEG) were purchased from Fluka–Sigma Aldrich (Germany). Agar-agar (AA), MacConkey (MAC) agar, and Sabouraud dextrose agar (SDA) agar were purchased from Oxoid (England). Nutrient agar (NA) was obtained from Scharlau (European Union). Gelatin from Biotech (USA) and skim milk plate count agar were obtained from Biolab (Hungary). Sterile syringe filters (0.22 µm) were purchased from Millipore (Amman, Jordan). 2.2. Chemicals L-cysteine, azo-casein, tris-buffered saline (pH 7.2), hydrochloric acid (HCl), trichloroacetic acid, potassium phosphate buffer (pH 7.5), casein from bovine milk, Folin & Ciocalteu’s phenol reagent, and anhydrous sodium acetate were purchased from Sigma-Aldrich (USA). Sodium hydroxide (NaOH) was obtained from Scharlau (Spain), and anhydrous sodium carbonate (Na2CO3) was purchased from SDFCL (India). L-tyrosine was purchased from Hopkin and Williams Ltd. (England). Methylene Blue (MB) was obtained from Gurr-BDH chemicals (England). Coomassie Brilliant Blue (CBB), naphthol blue black (NBB), bromothymol blue (BTB), and brilliant green (BG) were purchased from Fluka–Sigma Aldrich (Germany). Bromocresol green (BCG) and bromocresol purple (BCP) were purchased from Xilong (China). Neutral Red (NR) from Koch-Light (England) and pH-Fix test strips 0–14 were purchased from Macherey-Nagel (USA). 2.3. Bacterial counting and calibration curves Bacterial growth in BHI, MH, and TH broth media was determined by measuring the optical density using a microtiter plate reader spectrophotometer (Epoch-Biotec, California, USA). Cell growth was monitored using the viable plate count method. Blank samples (broth only) were included as a sterility control. Three replications were carried out on different days. The degree of correlation between S. aureus counts and optical densities (ODs) was investigated by linear regression of
2. Materials and methods 2.1. Bacterial strains and detection media The microorganisms used in this study included methicillin-
Scheme 1. Mechanism of colorimetric detection of S. aureus proteinase proteolytic activity using a skim milk plate count agar–PEG 4000 sensing platform. 31
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Table 1 Proteolytic activity of S. aureus culture supernatant proteases assayed using azo-casein assay and Sigma's non-specific protease activity assay. Proteolytic activity a
Azo-casein assay (U) Sigma's non-specific assay (U mL−1)b a b
ATCC 25932 BHI
ATCC 33591 BHI
ATCC 25932 MH
ATCC 33591 MH
ATCC 25932 TH
ATCC 33591 TH
25.5 0.227
26.7 0.234
23.8 0.157
24.1 0.123
24.8 0.168
25.1 0.170
One unit of activity was defined as an increase in optical density of 0.01 after 30 min of incubation at 37 °C. One unit of activity was defined as the amount in micromoles of tyrosine equivalents released from casein per minute.
continuous data. For the viable count, 100 µL of each S. aureus primary broth culture (PBC) was spread over the appropriate agar plate and incubated for 24 h at 37 °C.
Table 3 Detection of S. aureus culture supernatant protease proteolytic activity using agar-based platform supplemented with either skim milk or gelatin as substrates.
2.4. Optimization of S. aureus extracellular protease activity One colony each of S. aureus (ATCC 25923) and S. aureus (ATCC 33591) was isolated, inoculated onto BHI, MH, and TH broths, and incubated for 24 h at 37 °C. Each bacterial PBC was serially diluted to determine the bacterial colony forming unit (CFU) value of the original stock, using the viable count spread dilution method. Next, the S. aureus PBCs were centrifuged at 14,000 rpm for 10 min at RT to sediment the bacteria and filtered through a 0.22 µm sterile filter membrane to obtain the S. aureus extracellular protease solutions [19]. The pH value of the extracellular protease solutions was measured using pH-Fix test strips 0–14 (Table 1), and the solutions were stored at −80 °C for later use. The proteolytic activity of proteases from each bacterial culture supernatant was measured using the azo-casein assay [27] and Sigma's non-specific protease activity assay [28] (Table 2). The bacterial protease concentration was associated with the bacterial concentration and was referred to as a colony forming unit (CFU).
2.6.1. Chromogenic media Dye solutions of BCG (0.0015% w/v) and CBB (0.25% w/v) in methanol-acetic acid-water 5:1:4 (v/v/v) were used to stain 3% SK-AA media. Then, a 50 µL aliquot of S. aureus extracellular protease solution was injected into the well punched at the center of the solid media. Thereafter, the plates were incubated at 37 °C for 48 h [29]. The protease proteolytic activity was evaluated by observing the change in color of the zone around the well (Fig. 2I).
BHI BHI MH MH TH TH
6 5.5 7 8 7 7
25932 33591 25932 33591 25932 33591
-ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve
+vea +ve +ve +ve -ve +ve -ve +ve -ve -ve -ve +ve
Most clear proteolytic zone developed after incubation at 37 °C for 48 h.
2.7. Dye-based quantitative detection of S. aureus culture supernatant protease proteolytic activity To gain an insight into the applicability of the developed readout platform based on plate count skim milk supplemented with PEG 4000, different concentrations of BCP-colored S. aureus protease solutions prepared from a series of PBC concentrations (101 to 109 CFU mL−1) were tested, and the lower limit of detection (LOD) was determined (Fig. 4).
Table 2 pH values of S. aureus culture supernatant proteases prepared from different broth media.
ATCC ATCC ATCC ATCC ATCC ATCC
SK-AA SK-NA SK-MH SK-BHI SK-MEA SK-MAC G-AA G-NA G-MH G-BHI G-ME G -MAC
2.6.3. Protease solution dying S. aureus culture supernatant was spiked with a number of coloring agents. Then, 50 µL of the colored supernatant solution was injected into a well punched at the center of the biosensing platform (Fig. 2III). Proteolysis was evaluated by observing the color change of the proteolytic zone around the well. In this approach, combinations of pH indicators [BCP (0.6% w/v), BTB (0.4% w/v), BCG (0.1% w/v), NBB (0.1% w/v), BG (0.1% w/v), CBB (0.1% w/v), NR (0.1% w/v), MB (0.1% w/v), and TB (0.1% w/v)] and diffusivity agents [(PEG 400 (5%), PEG 4000 (5%), and PEG 6000 (5%)] were used (Fig. 2). Proteolysis was evaluated by visual observation of the change in color zone intensity versus the control (dyed broth only) (Fig. 3).
2.6. Optimization of dye-based qualitative visualization of S. aureus culture supernatant protease proteolytic activity
Protease solution pH value
48 h incubation period
2.6.2. Dye flooding technique S. aureus culture supernatant (50 µL) was injected into a well punched at the center of the biosensing platform. Thereafter, the plates were incubated at 37 °C for 48 h. Dye solutions BCG (0.0015% w/v) or CBB (0.25% w/v) were poured over the plates, which were re-incubated for 20–30 min at RT [29]. The protease activity was evaluated by visually observing the change in proteolytic zone color around the well (Fig. 2II).
To obtain an optimal agar-based readout platform, proteases from different S. aureus concentrations in CFU were screened using different platforms, including plate count skim milk agar, AA, NA, MH agar, BHI agar, MAC agar, and ME agar supplemented with one of the following non-specific protease substrates: gelatin (0.5% w/v) and SK (1%, 3%, 5%, and 7% w/v). Fifty microliters of S. aureus culture supernatant were added to the punch well (6 mm), and the plate was incubated at 37 °C for 24 and 48 h (Table 3, Fig. 1).
Culture broth medium
24 h incubation period
a
2.5. Optimization of agar-based readout platform
S. aureus
Substrate-agar platform
2.8. Qualitative and semi-quantitative assessment of S. aureus cultures In situ assessment of 2 h incubated S. aureus cultures (101 to 109 CFU mL−1) was performed using BTB (0.4% w/v). The change in color intensity as a function of pH change following protease production was 32
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Fig. 1. S. aureus protease agar–based biosensing platform supplemented with different SK percentages after incubation for 48 h at 37 °C. (A) 1% SK-AA; (B) 3% SK-AA (optimum percentage); (C) 5% SK-AA; and (D) 7% SK-AA.
observed and compared to the negative blank (dyed broth only) (Fig. 5).
3.2. Development of S. aureus extracellular protease agar–based biosensing platform Extracellular proteases of S. aureus (ATCC 25923 and ATCC 33591) cultivated in the optimized culture medium (BHI) preferentially hydrolyzed SK-containing agar platforms. Proteolytic zones were visually detected as a clear halo after inoculation on the SK-AA platform, followed by incubation for 48 h at 37 °C (Table 3). On the other hand, no clear zones were detected in the AA, MH agar, BHI agar, and ME agar platforms supplemented with gelatin. However, when proteases were inoculated on gelatin-NA and gelatin-MAC agar platforms, hazy proteolytic zones were observed. Accordingly, SK-AA is the favorable biosensing platform for the detection of S. aureus extracellular protease proteolytic activity. Visualization of S. aureus proteolysis using different percentages of SK-supplemented media (1%, 3%, 5%, and 7%) revealed a clear proteolytic zone in the 3% SK platform (Fig. 1I). Hence, the 3% SK-supplemented medium was designated as the optimal agar-based biosensing platform.
2.9. Preparation of blind control samples PCBs of different strains of S. aureus (ATCC 25932, ATCC 33591, and ATCC 6539) and E. coli (ATCC 156, ATCC 51446, ATCC 4157, and ATCC 8739) were prepared and labeled with a number to hide the identity of each bacterium for blind testing. Samples were incubated for 2 h at 37 °C, spiked with BTB (0.4% w/v), and assessed (Fig. 6). 3. Results and discussion 3.1. Effect of culture media composition on S. aureus extracellular protease activity The tested S. aureus cultures (ATCC 25923 and ATCC 33591) were cultivated in BHI (pH = 7.5), MH (pH = 7.5), and TH (pH = 8) broth media, and incubated at 37 °C for 48 h. BHI was the most appropriate culture medium for the production of active extracellular proteases, which were detected using the azo-casein assay (26.7 U) and Sigma's non-specific protease activity assay (0.234 U mL−1) [27,28]; the results are shown in Table 1. Additionally, it is to be mentioned that the S. aureus extracellular protease solutions prepared from BHI, MH, and TH broth media varied in pH, as indicated in Table 2. This variation is an indicative biomarker for the effect of media composition on the type of proteases produced, and consequently, the protease activity [30].
3.3. Dye-based qualitative assessment of S. aureus protease activity Two coloring agents (BCG and CBB) were used to visually assess the proteolytic zones of S. aureus proteinases, by dying the solid agar platform to produce a chromogenic medium (Fig. 2I), or by flooding the dye solution over the detecting platform (Fig. 2II). Unfortunately, the proteinase proteolytic zones were not clear. Therefore, a new approach using stained S. aureus protease solutions was adopted, wherein different coloring agents (BCG, NBB, BG, CBB, NR, MB, TB, and BCP) were 33
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Fig. 2. Visualization of S. aureus extracellular proteases cultivated in BHI broth using: (I) Chromogenic media dye incorporated into 3% SK-AA platform; (II) Dye flooding over the 3% SK-AA platform, followed by incubation for 20–30 min at RT. (III) In situ dying of S. aureus protease solution with different dyes (Coomassie Brilliant Blue (CBB), Bromocresol Green (BCG), Brilliant Green (BG), Naphthol Blue Black (NBB), Methylene Blue (MB), Thymol Blue (TB), Neutral Red (NR) (0.1% w/v) prior to application over the 3% SK-AA platform; and the chemical structures of the dyes. Positive visual readout for red-colored chemical structures. *C stands for control sample (no protease) and T stands for S. aureus culture supernatant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
added. Positive visual readout results with BCG (0.1% w/v), BG (0.1% w/v), and NBB (0.1% w/v) were observed, as shown in Fig. 2III. The color intensity difference between the stained test protease sample and the control (stained media only) could be correlated to the electrostatic interactions between dye sulfate (SOзˉ and HSO₄ˉ) groups and tyrosine amino groups [31]. Tyrosine liberated upon SK substrate digestion (proteolysis) may alter the chemical structure conjugation system and result in a color change. The low water solubility of the MB, TB, NR, and CBB dyes counteracted ionic interaction and resulted in negative readout results (Fig. 2III).
Interestingly, in situ staining of S. aureus extracellular protease solutions with the BCG, BG, and NBB visualizing agents shortened the time needed to detect proteinase activity to 18 h (Fig. 3I), compared with 48 h when unstained (Fig. 1B). To speed up the visual detection, simultaneous dying of the biosensing platform and S. aureus culture supernatant was conducted. Accordingly, PEG was incorporated into the 3% SK-AA platform to promote dye diffusion. Three different grades of PEG, 400, 4000, and 6000, were used. However, the presence of PEG in SK-AA prevented the formation of homogenous media after autoclaving. Therefore, other agar-based media were tested. Interestingly, 34
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Fig. 3. Qualitative detection of S. aureus extracellular proteases using different coloring agents and different biosensing platforms: (I) BG-colored protease over 3% skim milk agar-agar platform; (II) BG-colored protease over skim milk plate count agar–PEG 4000 platform at different time intervals. A 2 h; B 18 h; C 24 h; and D 48 h over black background. (III) A BCP pH color chart; B S. aureus and E. coli BCP-colored protease over skim milk plate count agar–PEG 4000 biosensing platform after 15 min of incubation. *C stands for control sample (no protease) and T stands for S. aureus culture supernatant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
homogeneity was achieved when PEG 4000 was incorporated into the skim milk plate count agar medium. Following the application of BGcolored S. aureus culture supernatant on the PEG 4000–skim milk plate count agar platform, visual detection of proteolysis was tracked at different time intervals (2–48 h) (Fig. 3II(A–D)). A difference in proteolytic zone color intensity was detected visually after 2 h of incubation at 37 °C (Fig. 3IIA). The inclusion of PEG 4000 in the medium improved dye diffusion through the solid agar platform and triggered water retention, which enabled protease proteolytic ability that manifested as a clear difference in proteolytic zone color intensity for the test sample when compared to that of the control (Fig. 3IIA). To optimize detection rapidity over the developed optical readout platform, the BCP dye reagent was used to track the change in pH following proteolysis, due to tyrosine amino acid liberation. The change in hydrogen ion concentration at the digested area resulted in a color shift from yellow to burgundy for S. aureus, compared to a purplish color for E. coli, following incubation at 37 °C for 15 min, as shown in
Fig. 3III. The pH of the negative control (BHI broth only) was adjusted using glacial acetic acid to match that for the tested protease at zero time. All the experiments were carried out in triplicates. 3.4. Quantitative detection of S. aureus culture supernatant proteolytic activity To gain an insight into the ability of the optical readout platform to semi-quantitatively detect S. aureus proteases, different concentrations of S. aureus (ATCC 25932) culture supernatants (109, 108, 107, 106, 105, 104, 103, 102, and 101 CFU mL−1) were prepared and examined. A direct relationship between different S. aureus proteinase culture densities and proteolytic zone burgundy chromophore intensities was observed, as shown in (Fig. 4I). At higher proteolytic activity (as measured using Sigma's non-specific protease activity assay (Fig. 4II(A and B)) more tyrosine residues could be released; thus, the media pH increased in association with an increase in chromophore generation. 35
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Fig. 4. Quantitative detection of proteolytic activity of different S. aureus ATCC 25932 culture supernatants prepared from different PBC concentrations: (I) Skim milk plate count agar–PEG 4000 biosensing platform under the effect of different BCP-colored S. aureus ATCC 25932 protease concentrations; (II) Average proteolytic activity of different S. aureus culture supernatants measured using Sigma's non-specific protease activity assay ± CV. The protease activity was determined in terms of Units, which are the amounts in micromoles of tyrosine equivalents released from casein per minute. *C stands for control sample (no protease) and T stands for S. aureus culture supernatant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A lower LOD of 100 CFU mL−1 was achieved without the use of any instrumentation and within 15 min of incubation (Fig. 4I). The LOD was based on visual detection of the lowest protease concentration incapable of developing a distinct proteolytic zone color change. It is to be mentioned that the solid agar biosensing platform showed an adequate long-term stability of up to three months if stored in a sterile container away from direct light at 2–8 °C.
3.6. Qualitative and semi-quantitative detection of S. aureus culture In accordance with our findings, the S. aureus PBC pH value varied with the incubation period at 37 °C. At zero time, the culture pH was > 7; however, after 24 h of incubation, the culture pH dropped to 5.5–6. Hence, the BTB coloring reagent (0.4% w/v) was used for in situ detection of S. aureus (ATCC 25932, ATCC 33591, and ATCC 6539) PBCs. This reagent has a yellow chromophore below pH 6.0 and a blue chromophore above pH 7.6. A light-green color indicated a positive visual readout result, whereas the negative control sample (BHI broth) produced a dark green–bluish color. Fig. 5 shows the semi-quantitative detection of different S. aureus culture concentrations (109, 108, 107, 106, 105, 104, 103, 102, and 101 CFU mL−1). A proportional decrease in culture media green chromophore intensity was observed with higher PBC concentrations. This may be attributed to the increase in media ion concentration with higher
3.5. Comparison with literature The colorimetric assay presented herein is simple and can be used hand-held by untrained personnel; it is cheap, with no need for any instrumentation to read out the results. At present, a number of detection methods have been applied [31–42]. A summary of their advantages and limitations is presented in Table 4. 36
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Fig. 5. Qualitative and semi-quantitative detection of S. aureus PBCs. Correlation between different S. aureus PBC concentrations (CFU mL−1) and BTB color intensity, illustrating a lower limit of detection of 103. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 6. Dye-based S. aureus culture assay specificity. Positive green color for S. aureus spp. and yellow color for E. coli spp.: (C) control, (1) E. coli (ATCC 8739), (2) S. aureus (ATCC 25932), (3) E. coli (ATCC 156), (4) E. coli (ATCC 4157), (5) S. aureus (ATCC 33591), (6) E. coli (ATCC 51446), and (7) S. aureus (ATCC 6539). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 4 Advantages and limitations of common S. aureus detection methods. Method
Advantages
Limitations
Reference
Bacterial culture method
Cheap; able to detect and quantify S. aureus
[32]
Enzyme-linked immunosorbent assay (ELISA) Lateral flow immunoassays Polymerase chain reaction (PCR)
Specific; can be automated so that it is more time- and labor-efficient; can handle large numbers of samples Low cost; reliable; easy to operate; sensitive; specific High sensitivity; high specificity; automated; reliable results
Colorimetric optical biosensors
Cost-effective; simple; results can be read out by bare eyes and sometimes with the help of a strip reader; timely detection after reaction completion Feasible; sensitive; rapid; applicable for on-site detection; visual readout of the results
Time-consuming; labor-intensive; takes several days to provide conclusive results High cost; requires trained personnel; requires labelling of antibodies or antigens Require labelling of antibodies or antigens High cost; requires trained technicians; affected by PCR inhibitors; requires DNA purification Not suitable for multiple use Requires minimal detection-platform manipulation
Current
Agar-based biosensing platform
protease production. On the other hand, the negative control sample showed an intense green color. In situ pH-dependent detection of S. aureus culture was developed, to offer a point-of-care (POC) diagnostic assay. This colorimetric assay offers quick results, leading to a speedy decision concerning the treatment plan, reducing the emergency room time and number of hospital beds required, and ensuring the optimal use of professional time. It is worth mentioning that the current detection assay is simple and can be performed by untrained personnel anywhere in hospitals, clinics and physician offices, university and school medical centers, pharmaceutical clinical trial sites, and homes.
[33] [34,35] [36–38] [39–41]
2 h bacterial PBCs, including those of three S. aureus (ATCC 25932, ATCC 33591, and ATCC 6539) and four E. coli (ATCC 156, ATCC 51446, ATCC 4157, and ATCC 8739) species. These samples were labeled with numbers such that the bacteria identities were blinded to the person in charge of carrying out the test. Following this, the BTB dye reagent was added, and a green color for S. aureus strains and a yellow color for all the E. coli strains were observed, as shown in Fig. 6. 4. Conclusions In this research, a promising, breakthrough, simple, and low-cost colorimetric S. aureus detection assay was developed using S. aureus extracellular proteases as the organic biomarkers. Using this approach, the presence of S. aureus was qualitatively and semi-quantitatively detected within 15 min over a solid readout platform, with a lower LOD of
3.7. Specificity of S. aureus culture dye-based assay Validation of the assay specificity was carried out by blindly testing 37
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102 CFU mL−1. A further in situ specific detection of S. aureus culture was designed to spot S. aureus presence specifically among a number of blindly tested pathogens. This method was based on the use of the BTB dye reagent, which showed a change in color due to protease production during the 2 h incubation period. The LOD was 103 CFU mL−1. These assays are easy to perform and are applicable for patient POC testing without the need for many preparative steps and instruments.
(2012) 1–11. [17] O.G. Brakstad, K. Aasbakk, J.A. Maeland, Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene, J. Clin. Microbiol. 30 (1992) 1654–1660. [18] R.C. Kasana, R. Salwan, S.K. Yadav, Microbial proteases: detection, production, and genetic improvement, Crit. Rev. Microbiol. 37 (2011) 262–276. [19] G.A.R.Y. Suaifan, S. Alhogail, M. Zourob, Rapid and low-cost biosensor for the detection of Staphylococcus aureus, Biosens. Bioelectron. 90 (2017) 230–237. [20] S. Alhogail, G.A.R.Y. Suaifan, M. Zourob, Rapid colorimetric sensing platform for the detection of Listeria monocytogenes foodborne pathogen, Biosens. Bioelectron. 86 (2016) 1061–1066. [21] G.A.R.Y. Suaifan, S. Alhogail, M. Zourob, Paper-based magnetic nanoparticle-peptide probe for rapid and quantitative colorimetric detection of Escherichia coli O157:H7, Biosens. Bioelectron. 92 (2017) 702–708. [22] J. Gooch, B. Daniel, N. Frascione, Application of fluorescent substrates to the in situ detection of prostate specific antigen, Talanta 125 (2014) 210–214. [23] M. Rapala-Kozik, O. Bochenska, D. Zajac, J. Karkowska-Kuleta, M. Gogol, M. Zawrotniak, M. Kozik, Extracellular proteinases of Candida species pathogenic yeasts, Mol. Oral. Microbiol. 33 (2018) 113–124. [24] R. Ruchel, R. Tegeler, M. Trost, A comparison of secretory proteinases from different strains of Candida albicans, J. Med. Vet. Mycol. 20 (1982) 233–244. [25] P. Sacherer, G. Defago, D. Haas, Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0, FEMS Microbiol. Lett. 116 (1994) 155–160. [26] M.S. Lantz, P. Ciborowski, Zymographic techniques for detection and characterization of microbial proteases, Methods Enzymol. 235 (1994) 563–594. [27] S. Takeuchi, T. Kinoshita, T. Kaidoh, N. Hashizume, Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken, Vet. Microbiol. 67 (1999) 195–202. [28] C. Cupp-Enyard, Sigma's non-specific protease activity assay - casein as a substrate, J. Vis. Exp. 899 (2008) 1–3. [29] P. Vijayaraghavan, S.G.P. Vincent, A simple method for the detection of protease activity on agar plates using bomochresolgreen dye, J. Biochem. Tech. 4 (2013) 628–630. [30] N. Souissi, Y. Ellouz-Triki, A. Bougatef, M. Blibech, M. Nasri, Preparation and use of media for protease-producing bacterial strains based on by-products from Cuttlefish (Sepia officinalis) and wastewaters from marine-products processing factories, Microbiol. Res. 163 (2008) 473–480. [31] J.V. Moller, U. Kragh-Hansen, Indicator dyes as probes of electrostatic potential changes on macromolecular surfaces, Biochemistry 14 (1975) 2317–2323. [32] G.A. Suaifan, Colorimetric biosensors for bacterial detection, in: U.A. Minhaz, Z. Mohammed, T. Eiichi (Eds.), Food Biosensors. E-Royal Society of Chemistry Inc, United Kingdom, 2016, pp. 182–202. [33] S. Böcher, R. Smyth, G. Kahlmeter, J. Kerremans, M.C. Vos, R. Skov, Evaluation of four selective agars and two enrichment broths in screening for methicillin-resistant Staphylococcus aureus, J. Clin. Microbiol. 46 (2008) 3136–3138. [34] R.W. Bennett, Staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology, J. Food Prot. 68 (2005) 1264–1270. [35] C.Z. Li, K. Vandenberg, S. Prabhulkar, X. Zhu, L. Schneper, K. Methee, C.J. Rosser, E. Almeide, Paper based point-of-care testing disc for multiplex whole cell bacteria analysis, Biosens. Bioelectron. 26 (2011) 4342–4348. [36] C. Pöhlmann, I. Dieser, M. Sprinzl, A lateral flow assay for identification of Escherichia coli by ribosomal RNA hybridisation, Analyst 139 (2014) 1063–1071. [37] T. Trnčíková, V. Hrušková, K. Oravcová, D. Pangallo, E. Kaclíková, Rapid and sensitive detection of Staphylococcus aureus in food using selective enrichment and real-time PCR targeting a new gene marker, Food Anal. Methods 2 (2009) 241–250. [38] J.C. Cheng, C.L. Huang, C.C. Lin, C.C. Chen, Y.C. Chang, S.S. Chang, C.P. Tseng, Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR, Clin. Chem. 52 (2006) 1997–2004. [39] J.H. Yoon, S. Wei, D.H. Oh, A highly selective enrichment broth combined with real-time PCR for detection of Staphylococcus aureus in food samples, LWT 94 (2018) 103–110. [40] Y.J. Sung, H.J. Suk, H.Y. Sung, T. Li, H. Poo, M.G. Kim, Novel antibody/gold nanoparticle/magnetic nanoparticle nanocomposites for immunomagnetic separation and rapid colorimetric detection of Staphylococcus aureus in milk, Biosens. Bioelectron. 43 (2013) 432–439. [41] J. Yuan, S. Wu, N. Duan, X. Ma, Y. Xia, J. Chen, Z. Ding, Z. Wang, A sensitive gold nanoparticle-based colorimetric aptasensor for Staphylococcus aureus, Talanta 127 (2014) 163–168. [42] P. Liu, L. Han, F. Wang, V.A. Petrenko, A. Liu, Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus, Biosens. Bioelectron. 82 (2016) 195–203.
Acknowledgments Prof. Ghadeer Suaifan would like to acknowledge the funding from the Deanship of Scientific Research at The University of Jordan, Jordan (Grants number 1731, 2016) and the Ministry of Higher Education, Jordan (Scientific Research Support Fund number MPH/1/19/2015). Funding This work was funded by the Ministry of Higher Education, through the Scientific Research Support Fund [grant number MPH/1/19/2015], and The University of Jordan [Grant numbers 1731, 2016]. Declaration of Interest The authors declare no conflict of interest. References [1] G. Rachel, L. Franklin, Pathogenesis of methicillin-resistant Staphylococcus aureus infection, Clin. Infect. Dis. 46 (2008) S350–S359. [2] M. Aires de Sousa, H. Lencastre, Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones, FEMS Immunol. Med. Microbiol. 40 (2004) 101–111. [3] J. Bien, O. Sokolova, B. Bozko, Characterization of virulence factors of Staphylococcus aureus: novel function of known virulence factors that are implicated in activation of airway epithelial Proinflammatory response, J. Pathog. 2011 (2011) 1–13. [4] F.D. Lowy, Staphylococcus aureus infections, N. Engl. J. Med. 339 (1998) 520–532. [5] P.S. Loomba, J. Taneja, B. Mishra, Methicillin and vancomycin resistant S. aureus in hospitalized patients, J. Glob. Infect. Dis. 2 (2010) 275–283. [6] K. Plata, A.E. Rosato, G. Wegrzyn, Staphylococcus aureus as an infectious agent: overview of biochemistry and molecular genetics of its pathogenicity, Acta Biochim. Pol. 56 (2009) 597–612. [7] J.A. Hennekinne, M.L. De Buyser, S. Dragacci, Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation, FEMS Microbiol. 36 (2012) 815–836. [8] G.L. Archer, Staphylococcus aureus: a well-armed pathogen, Clin. Infect. Dis. 26 (1998) 1179–1181. [9] G.Y. Liu, Molecular pathogenesis of Staphylococcus aureus infection, Pediatr. Res. 65 (2009) 71R–77R. [10] S. Gröbner, M. Dion, M. Plante, V.A.J. Kempf, Evaluation of the BD GeneOhm StaphSR assay for the detection of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from spiked positive blood culture bottles, J. Clin. Microbiol. 47 (2009) 1689–1694. [11] T. Yen Tan, S. Cordan, R. Barnes, B. Cooksan, Rapid identification of methicillinresistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR, J. Clin. Microbiol. 39 (2001) 4529–4531. [12] P.K. Mandal, A.K. Biswas, K. Choi, U.K. Pal, Methods for rapid detection of food borne pathogens: an overview, Am. J. Food Technol. 6 (2011) 87–102. [13] X. Zhao, C.W. Lin, J. Wang, D.H. Oh, Advances in rapid detection methods for foodborne pathogens, J. Microbiol. Biotechnol. 24 (2014) 297–312. [14] M.J. Struelens, Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) and patient management, Clin. Microbiol. Infect. 12 (2006) 23–26. [15] O.G. Brakstad, J.F. Maeland, Comparison of tests designed to identify Staphylococcus aureus thermostable nuclease, Acta Pathol. Microbiol. Immunol. Scand. B 103 (1995) 219–224. [16] C.L. Pierce, J.C. Rees, F.M. Fernández, J.R. Barr, Viable Staphylococcus aureus quantitation using 15N metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow, Mol. Cell Proteom. 11
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