Supporting Information Wiley-VCH 2014 69451 Weinheim, Germany
Identification of Bacteria in Water by a Fluorescent Array** Wenwen Chen, Qizhai Li, Wenshu Zheng, Fang Hu, Guanxin Zhang, Zhuo Wang,* Deqing Zhang,* and Xingyu Jiang* anie_201407606_sm_miscellaneous_information.pdf
Supporting Information Table of Contents
Page
Experimental procedure for the whole process of bacteria identification.
2
Synthetic route and characterization of probe A1
3
Optimize the working concentration of probe A1-A5
3
Supplementary explanation for Figure 1 in the manuscript
4
Figure S1. Confocal microscopy fluorescent images of four kinds of bacteria in the presence of A1-A5 in F-array. 7 Figure S2. The normalized fluorescence intensity of eight kinds of bacteria stained with A3, A4 and A5 probes. 8 Interaction research of probe A1 and E. coli
9
Firgue S3. Confocal microscopy fluorescent images of lysozyme and trypsin treated E. coli incubated with probe A1 9 Figure S4. The cross validation results of eight kinds of bacteria.
10
Figure S5. Confocal microscopy bright field and fluorescent images of S. aureus incubated with probe A1 in PBS and urine 11 Table S1. Time Comparison of manual methods, automatic instruments and F-array 12 Table S2. Zeta potential of eight kinds of bacteria.
12
Table S3. Identification of twelve unknown bacterial samples with QDA using F-array. 12
1
Experimental Procedure for the whole process of bacteria identification The structures of molecules (named A1, A2, A3, A4, A5) we applied in the experiment are shown in figure 1. We used eight kinds of bacteria including four gram negative bacteria (Escherichia coli, Salmonella choleraesuis, Klebsiella pneumonia,Multi-drug resistant, MDR, Escherichia coli), four gram positive bacteria (Staphyloccocus aureus, Staphyloccocus epidermidis, Bacillus subtilis, methicillin-resistant Staphyloccocus aureus (MRSA) ). Standard strains of E. coli (ATCC 11775), S. choleraesuis (ATCC 14028), K. pneumonia (ATCC 13883), S. aureus(ATCC 6538P), S. epidermidis (ATCC 12228) and B. subtilis(ATCC 6633) were purchased from China General Microbiological Culture Collection Center. Two clinical isolates, MDR E. coli and MRSA were from local hospitals in China. We cultured the bacterial cells in Luria broth agar medium (2 mL) at 37oC to an optical density of 1.0 at 600 nm. The cultures were centrifuged (8000 rpm, 3 min) and washed with phosphate buffer saline (10 mM, pH 7.2) once, resuspended in phosphate buffer, and diluted to an absorbance of 2×10-3 at 600 nm. For the five fluorescent probes, we dissolved them in dimethyl sulfoxide (DMSO) with the concentration of 0.1 M and diluted them to the concentration of 100 μM using phosphate buffer. We mixed the bacteria (OD600= 2×10-3) with probe solution (v/v = 1:1) and put the mixture at room temperature for half an hour. We carried out six repeated experiments for each bacteria incubated with every probe. After the incubation, we used confocal microscopy to record images of each kind of bacteria incubated by different probes. We applied flow cytometry to characterize the fluorescence data of different bacteria. For each sample, in the flow cytometry characterization, we used three different excitation wavelengths ( 488 nm, 561 nm and 405 nm) to get various fluorescence emission in 11 different channels (FITC, Percp-Cy5.5, PE, PE-Texas Red and PE-Cy5, Pacific Blue, Amcyan, Qdot 605, Qdot 655, Qdot 705, Qdot 800). 11 different channels indicate the emission wavelengths at 530±15 nm, 695±20 nm, 582±7.5 nm, 610±10 nm, 670±15 nm, 450±25 nm, 525±25 nm, 610±10 nm, 670±15 nm, 710±25 nm and 780±30 nm respectively. So we could obtain 11 fluorescence data for each bacterium with a fluorescent probe. 2
We recorded all the fluorescence data and introduced them in mathematical analysis to receive bacteria identification results.
Synthetic route and Characterization of Probe A1 Scheme 1. Synthetic route to probe A1a
a
Reagents and conditions: i) 1,3-dibromopropane, K2CO3, DMF, 50 °C; ii) Me3N,
THF, 25 °C. Compound 1 was synthesized according to reported procedures.[1] Synthesis of Probe A1. The mixture of compound 1 (0.12 g, 0.23 mmol), 1,3-dibromopropane (0.18 g, 0.92 mmol) and K2CO3 (95 mg, 0.69 mmol) in 8 mL DMF was heated at 50 oC overnight. Then the mixutre was poured into water and extracted with ethyl acetate. The organic layer was washed with brine and evaporated to dryness. The residue was subjected to column chromatography to afford compound 2 (0.14 g, 0.19 mmol) as red oil in 82.1% yield. Compound 2 was mixed with Me3N (0.11 g, 1.9 mmol) in anhydrous THF to stir at 25 °C for 2 days. Then, the mixture was filtered and the residue was washed by excessive THF to afford probe A1 (69.9 mg, 0.08 mmol) as red solid in 42.1% yield. 1H NMR (400 MHz, CD3OD, δ): 7.71 (d, J = 7.2 Hz, 1H), 7.64 – 7.55 (m, 1H), 7.54 – 7.49 (m, 2H), 7.48 – 7.41 (m, 1H), 7.40 – 7.28 (m, 1H), 7.26 – 7.09 (m, 4H), 7.08 – 6.98 (m, 3H), 6.98 – 6.82 (m, 5H), 6.79 – 6.65 (m, 4H), 4.04 (t, J = 4.8 Hz, 4H), 3.56 (t, J = 8.0 Hz, 4H), 3.17 (s, 18H), 2.31 – 2.17 (m, 4H); HR-MS (ESI, positive) m/z: [M-2Br]2+/2 calcd. for [C48H52N4O2]2+/2, 358.20396; found, 358.20413. Optimize the working concentration of probe A1-A5 We screened the effects of different concentrations including 100 μM, 50 μM and 10 μM for bacteria identification. With the probes at 10 μM, the fluorescence of several bacteria was too weak to detect. The fluorescence data collected with probes at 3
concentration of 50 μM can be used to do mathematical treatment and get good (reproducible) results for bacteria identification. 100 μM of probes also worked well, while considering saving reagents, we chose the final concentration of 50 μM in the experiments.
Supplementary explanation for Figure 1 in the manuscript: We are very sorry for the breezing of Figure 1. We have tried our best to increase the quality of the figure. Two factors affect the quality of fluorescent images: 1) the size of bacteria is very small from 1 to several micrometers, such as the size of MRSA and S. aureus is even less than 1 μm, and we apply a confocal microscope (Zeiss LSM710, 63×objective lens) to record the bacteria that are live in aqueous solution, which means the bacteria can move and cause the problem of focusing; 2)the fluorescence of probes is not very bright, which can cause the breezing of images in some degree. Probe A1 and A4 are brighter, so the images are clear. Probe A3 and A5 are weaker, and the corresponding images are blurring. Additionally, we supply the bright field images of Figure 1 side by side here, and the bright images are easier to observe the bacterial morphology than fluorescent ones. The bright field images can not overlap fluorescent images exactly because of the quick moving of bacteria in the confocal dish. We hope the referee can see bacteria clearly, and we can add these bright field images in the supporting information based on the comments from referees in the follow-up revision. Figure 1 wants to certify that different bacteria incubated with different probes have diverse fluorescence. The specific fluorescence information is characterized and recorded by flow cytometry. In the following mathematical treatment, we used the data received from flow cytometry. We hope our explanation and supplementary figures are helpful to understand the work.
4
Incubated by A1 MRSA
S.aureus
MDR E. coli
E. coli
Incubated by A2 MRSA
S.aureus
MDR E. coli
E. coli
Incubated by A3 MRSA
S.aureus
MDR E. coli
E. coli
5
Incubated by A4 MRSA
S.aureus
MDR E. coli
E. coli
Incubated by A5 MRSA
S.aureus
MDR E. coli
E. coli
6
Figure S1. Confocal microscopy fluorescent images of four kinds of bacteria in the presence of A1-A5 in F-array. The different color representation (red, orange, green) is consistent with the maximum value of fluorescence recorded in flow cytometry.
7
Figure S2. The normalized fluorescence intensity of eight kinds of bacteria stained with A3, A4 and A5 probes. Each value is an average of 6 repeated measurements. Every bacteria incubated by one probe has eleven fluorescence received from 11 channels using flow cytometer. The excitation wavelengths are 488 nm, 561 nm and 405 nm, the eleven channels are Pacific Blue, Amcyan, Qdot 605, Qdot 655, Qdot 705, Qdot 800, Percp-Cy5.5, FITC, PE, PE-Texas Red and PE-Cy5 respectively and we listed emission wavelengths for each channel in the figure. 8
Interaction research of probe A1 and E. coli To examine the interaction modes between probes and bacteria, we used E. coli and probe A1 to study. We try to detect whether probes binds to the cell walls or protoplasm of the bacteria. We adopted lysozyme (1 mg/mL) to digest cell walls on ice for 1 hour, then centrifuged (8000 rpm, 3 min) and used probe A1 (50 μM, PBS solution) to resuspend and incubate bacteria for half an hour. We recorded the confocal microscopy image. Without the cell wall, E. coli became smaller and some of them are round-shaped. And after incubated with probe A1, the fluorescent still existed. We can speculate that the protoplasm rather than the cell wall plays an important role in the interaction of probe and the bacteria. Furthermore, E. coli was treated with 1% trypsin for 24 h at 37 oC to strip the surface proteins of bacteria.[2] And then we centrifuged (8000 rpm, 3 min) and used probe A1 (50 μM, PBS solution) to resuspend and incubate bacteria for half an hour. E. coli was fluorescent even without membrane protein. So we deduce the fluorescence of bacteria comes from the protoplasm.
Figure S3. Confocal microscopy fluorescent images of lysozyme (a) and trypsin (b) treated E.coli incubated with probe A1. Left column is the bright field images. Scale bar is 20 μm. 9
Firgue S4. The cross validation results of eight kinds of bacteria. Five subjects in each bacteria group are used to fabricate the discrimination model, and the remained one subject is used to evaluate the effect of identification. (Each kind of bacteria has six replicates that are the six subjects.) The dots having different colors are the remaining one subject in every bacteria group.
The fluorecent images of probes and S.aureus in PBS and artiifical urine To see whether the complex solution, like urine, influence the probes stained bacteria, we take probe A1 stained S. aureus as an example. We cultured the S. aureus in Luria broth agar medium (2 mL) at 37oC to an optical density of 1.0 at 600 nm (logarithmic phase). The cultures were centrifuged (8000 rpm, 3 min) and washed with phosphate buffer saline (10 mM, pH 7.2) once, resuspended in phosphate buffer and artificial urine respectively. We mixed the bacteria with probe solution (v/v = 1:1) and put the mixture at room temperature for half an hour. After the incubation, we used confocal microscopy to record images of S. aureus incubated with probe A1.
10
Figure S5. Confocal microscopy bright field and fluorescent images of S.aureus incubated with probe A1 in PBS and urine samples. The different color representation (orange, green) is consistent with the maximum value of fluorescence recorded in flow cytometry. Scale bar is 20 μm.
11
Table S1. Time Comparison of manual method, automatic instruments and F-array.[2]
Method Standard Methods of Identification and Susceptibility test Direct Identification and Susceptibility test
Instrument (eg.) Manuel and instruments aid
Identification time 24-46 hours
Compact biochemical analysis (eg.VITEK 2 Compact) Strain Typing (Diversilab) MALDI-TOF F-array
6-10 hours
4 hours 2.5-3 hours 1-2 hours
Table S2. Zeta potential of eight kinds of bacteria.
Bacteria MRSA S. aureus S. epidermidis B. subtilis MDR E. coli E. coli K. pneumonia S. choleraesuis
Zeta potential (mV) -8.30±0.69 -7.84±0.48 -10.21±0.51 -0.092±0.090 -15.7667±0.72 -11.7667±1.25 -8.33±0.51 -0.77667±0.092
Table S3. Identification of twelve unknown bacterial samples with QDA using F-array.
No 1 2 3 4 5 6 7 8 9 10 11 12
QDA Identification Bacteria E. coli E. coli S. aureus K. pneumonia K. pneumonia S. choleraesuis B. subtilis B. subtilis S. epidermidis MDR E. coli MRSA MRSA
Correct Identification Yes/No Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes
12
References [1] Hu, F.; Huang, Y.; Zhang, G.; Zhao, R.; Yang, H.; Zhang, D. Anal. Chem. 2014, , 86, 7987−7995. [2] M.-P. Romero-Gómez, R. Gómez -Gil, J. R. Paño-Pardo, J. Mingorance, J. Infection 2012, 65, 513-520
13
Identification of Bacteria in Water by a Fluorescent Array** Wenwen Chen, Qizhai Li, Wenshu Zheng, Fang Hu, Guanxin Zhang, Zhuo Wang,* Deqing Zhang,* and Xingyu Jiang* anie_201407606_sm_miscellaneous_information.pdf
Supporting Information Table of Contents
Page
Experimental procedure for the whole process of bacteria identification.
2
Synthetic route and characterization of probe A1
3
Optimize the working concentration of probe A1-A5
3
Supplementary explanation for Figure 1 in the manuscript
4
Figure S1. Confocal microscopy fluorescent images of four kinds of bacteria in the presence of A1-A5 in F-array. 7 Figure S2. The normalized fluorescence intensity of eight kinds of bacteria stained with A3, A4 and A5 probes. 8 Interaction research of probe A1 and E. coli
9
Firgue S3. Confocal microscopy fluorescent images of lysozyme and trypsin treated E. coli incubated with probe A1 9 Figure S4. The cross validation results of eight kinds of bacteria.
10
Figure S5. Confocal microscopy bright field and fluorescent images of S. aureus incubated with probe A1 in PBS and urine 11 Table S1. Time Comparison of manual methods, automatic instruments and F-array 12 Table S2. Zeta potential of eight kinds of bacteria.
12
Table S3. Identification of twelve unknown bacterial samples with QDA using F-array. 12
1
Experimental Procedure for the whole process of bacteria identification The structures of molecules (named A1, A2, A3, A4, A5) we applied in the experiment are shown in figure 1. We used eight kinds of bacteria including four gram negative bacteria (Escherichia coli, Salmonella choleraesuis, Klebsiella pneumonia,Multi-drug resistant, MDR, Escherichia coli), four gram positive bacteria (Staphyloccocus aureus, Staphyloccocus epidermidis, Bacillus subtilis, methicillin-resistant Staphyloccocus aureus (MRSA) ). Standard strains of E. coli (ATCC 11775), S. choleraesuis (ATCC 14028), K. pneumonia (ATCC 13883), S. aureus(ATCC 6538P), S. epidermidis (ATCC 12228) and B. subtilis(ATCC 6633) were purchased from China General Microbiological Culture Collection Center. Two clinical isolates, MDR E. coli and MRSA were from local hospitals in China. We cultured the bacterial cells in Luria broth agar medium (2 mL) at 37oC to an optical density of 1.0 at 600 nm. The cultures were centrifuged (8000 rpm, 3 min) and washed with phosphate buffer saline (10 mM, pH 7.2) once, resuspended in phosphate buffer, and diluted to an absorbance of 2×10-3 at 600 nm. For the five fluorescent probes, we dissolved them in dimethyl sulfoxide (DMSO) with the concentration of 0.1 M and diluted them to the concentration of 100 μM using phosphate buffer. We mixed the bacteria (OD600= 2×10-3) with probe solution (v/v = 1:1) and put the mixture at room temperature for half an hour. We carried out six repeated experiments for each bacteria incubated with every probe. After the incubation, we used confocal microscopy to record images of each kind of bacteria incubated by different probes. We applied flow cytometry to characterize the fluorescence data of different bacteria. For each sample, in the flow cytometry characterization, we used three different excitation wavelengths ( 488 nm, 561 nm and 405 nm) to get various fluorescence emission in 11 different channels (FITC, Percp-Cy5.5, PE, PE-Texas Red and PE-Cy5, Pacific Blue, Amcyan, Qdot 605, Qdot 655, Qdot 705, Qdot 800). 11 different channels indicate the emission wavelengths at 530±15 nm, 695±20 nm, 582±7.5 nm, 610±10 nm, 670±15 nm, 450±25 nm, 525±25 nm, 610±10 nm, 670±15 nm, 710±25 nm and 780±30 nm respectively. So we could obtain 11 fluorescence data for each bacterium with a fluorescent probe. 2
We recorded all the fluorescence data and introduced them in mathematical analysis to receive bacteria identification results.
Synthetic route and Characterization of Probe A1 Scheme 1. Synthetic route to probe A1a
a
Reagents and conditions: i) 1,3-dibromopropane, K2CO3, DMF, 50 °C; ii) Me3N,
THF, 25 °C. Compound 1 was synthesized according to reported procedures.[1] Synthesis of Probe A1. The mixture of compound 1 (0.12 g, 0.23 mmol), 1,3-dibromopropane (0.18 g, 0.92 mmol) and K2CO3 (95 mg, 0.69 mmol) in 8 mL DMF was heated at 50 oC overnight. Then the mixutre was poured into water and extracted with ethyl acetate. The organic layer was washed with brine and evaporated to dryness. The residue was subjected to column chromatography to afford compound 2 (0.14 g, 0.19 mmol) as red oil in 82.1% yield. Compound 2 was mixed with Me3N (0.11 g, 1.9 mmol) in anhydrous THF to stir at 25 °C for 2 days. Then, the mixture was filtered and the residue was washed by excessive THF to afford probe A1 (69.9 mg, 0.08 mmol) as red solid in 42.1% yield. 1H NMR (400 MHz, CD3OD, δ): 7.71 (d, J = 7.2 Hz, 1H), 7.64 – 7.55 (m, 1H), 7.54 – 7.49 (m, 2H), 7.48 – 7.41 (m, 1H), 7.40 – 7.28 (m, 1H), 7.26 – 7.09 (m, 4H), 7.08 – 6.98 (m, 3H), 6.98 – 6.82 (m, 5H), 6.79 – 6.65 (m, 4H), 4.04 (t, J = 4.8 Hz, 4H), 3.56 (t, J = 8.0 Hz, 4H), 3.17 (s, 18H), 2.31 – 2.17 (m, 4H); HR-MS (ESI, positive) m/z: [M-2Br]2+/2 calcd. for [C48H52N4O2]2+/2, 358.20396; found, 358.20413. Optimize the working concentration of probe A1-A5 We screened the effects of different concentrations including 100 μM, 50 μM and 10 μM for bacteria identification. With the probes at 10 μM, the fluorescence of several bacteria was too weak to detect. The fluorescence data collected with probes at 3
concentration of 50 μM can be used to do mathematical treatment and get good (reproducible) results for bacteria identification. 100 μM of probes also worked well, while considering saving reagents, we chose the final concentration of 50 μM in the experiments.
Supplementary explanation for Figure 1 in the manuscript: We are very sorry for the breezing of Figure 1. We have tried our best to increase the quality of the figure. Two factors affect the quality of fluorescent images: 1) the size of bacteria is very small from 1 to several micrometers, such as the size of MRSA and S. aureus is even less than 1 μm, and we apply a confocal microscope (Zeiss LSM710, 63×objective lens) to record the bacteria that are live in aqueous solution, which means the bacteria can move and cause the problem of focusing; 2)the fluorescence of probes is not very bright, which can cause the breezing of images in some degree. Probe A1 and A4 are brighter, so the images are clear. Probe A3 and A5 are weaker, and the corresponding images are blurring. Additionally, we supply the bright field images of Figure 1 side by side here, and the bright images are easier to observe the bacterial morphology than fluorescent ones. The bright field images can not overlap fluorescent images exactly because of the quick moving of bacteria in the confocal dish. We hope the referee can see bacteria clearly, and we can add these bright field images in the supporting information based on the comments from referees in the follow-up revision. Figure 1 wants to certify that different bacteria incubated with different probes have diverse fluorescence. The specific fluorescence information is characterized and recorded by flow cytometry. In the following mathematical treatment, we used the data received from flow cytometry. We hope our explanation and supplementary figures are helpful to understand the work.
4
Incubated by A1 MRSA
S.aureus
MDR E. coli
E. coli
Incubated by A2 MRSA
S.aureus
MDR E. coli
E. coli
Incubated by A3 MRSA
S.aureus
MDR E. coli
E. coli
5
Incubated by A4 MRSA
S.aureus
MDR E. coli
E. coli
Incubated by A5 MRSA
S.aureus
MDR E. coli
E. coli
6
Figure S1. Confocal microscopy fluorescent images of four kinds of bacteria in the presence of A1-A5 in F-array. The different color representation (red, orange, green) is consistent with the maximum value of fluorescence recorded in flow cytometry.
7
Figure S2. The normalized fluorescence intensity of eight kinds of bacteria stained with A3, A4 and A5 probes. Each value is an average of 6 repeated measurements. Every bacteria incubated by one probe has eleven fluorescence received from 11 channels using flow cytometer. The excitation wavelengths are 488 nm, 561 nm and 405 nm, the eleven channels are Pacific Blue, Amcyan, Qdot 605, Qdot 655, Qdot 705, Qdot 800, Percp-Cy5.5, FITC, PE, PE-Texas Red and PE-Cy5 respectively and we listed emission wavelengths for each channel in the figure. 8
Interaction research of probe A1 and E. coli To examine the interaction modes between probes and bacteria, we used E. coli and probe A1 to study. We try to detect whether probes binds to the cell walls or protoplasm of the bacteria. We adopted lysozyme (1 mg/mL) to digest cell walls on ice for 1 hour, then centrifuged (8000 rpm, 3 min) and used probe A1 (50 μM, PBS solution) to resuspend and incubate bacteria for half an hour. We recorded the confocal microscopy image. Without the cell wall, E. coli became smaller and some of them are round-shaped. And after incubated with probe A1, the fluorescent still existed. We can speculate that the protoplasm rather than the cell wall plays an important role in the interaction of probe and the bacteria. Furthermore, E. coli was treated with 1% trypsin for 24 h at 37 oC to strip the surface proteins of bacteria.[2] And then we centrifuged (8000 rpm, 3 min) and used probe A1 (50 μM, PBS solution) to resuspend and incubate bacteria for half an hour. E. coli was fluorescent even without membrane protein. So we deduce the fluorescence of bacteria comes from the protoplasm.
Figure S3. Confocal microscopy fluorescent images of lysozyme (a) and trypsin (b) treated E.coli incubated with probe A1. Left column is the bright field images. Scale bar is 20 μm. 9
Firgue S4. The cross validation results of eight kinds of bacteria. Five subjects in each bacteria group are used to fabricate the discrimination model, and the remained one subject is used to evaluate the effect of identification. (Each kind of bacteria has six replicates that are the six subjects.) The dots having different colors are the remaining one subject in every bacteria group.
The fluorecent images of probes and S.aureus in PBS and artiifical urine To see whether the complex solution, like urine, influence the probes stained bacteria, we take probe A1 stained S. aureus as an example. We cultured the S. aureus in Luria broth agar medium (2 mL) at 37oC to an optical density of 1.0 at 600 nm (logarithmic phase). The cultures were centrifuged (8000 rpm, 3 min) and washed with phosphate buffer saline (10 mM, pH 7.2) once, resuspended in phosphate buffer and artificial urine respectively. We mixed the bacteria with probe solution (v/v = 1:1) and put the mixture at room temperature for half an hour. After the incubation, we used confocal microscopy to record images of S. aureus incubated with probe A1.
10
Figure S5. Confocal microscopy bright field and fluorescent images of S.aureus incubated with probe A1 in PBS and urine samples. The different color representation (orange, green) is consistent with the maximum value of fluorescence recorded in flow cytometry. Scale bar is 20 μm.
11
Table S1. Time Comparison of manual method, automatic instruments and F-array.[2]
Method Standard Methods of Identification and Susceptibility test Direct Identification and Susceptibility test
Instrument (eg.) Manuel and instruments aid
Identification time 24-46 hours
Compact biochemical analysis (eg.VITEK 2 Compact) Strain Typing (Diversilab) MALDI-TOF F-array
6-10 hours
4 hours 2.5-3 hours 1-2 hours
Table S2. Zeta potential of eight kinds of bacteria.
Bacteria MRSA S. aureus S. epidermidis B. subtilis MDR E. coli E. coli K. pneumonia S. choleraesuis
Zeta potential (mV) -8.30±0.69 -7.84±0.48 -10.21±0.51 -0.092±0.090 -15.7667±0.72 -11.7667±1.25 -8.33±0.51 -0.77667±0.092
Table S3. Identification of twelve unknown bacterial samples with QDA using F-array.
No 1 2 3 4 5 6 7 8 9 10 11 12
QDA Identification Bacteria E. coli E. coli S. aureus K. pneumonia K. pneumonia S. choleraesuis B. subtilis B. subtilis S. epidermidis MDR E. coli MRSA MRSA
Correct Identification Yes/No Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes
12
References [1] Hu, F.; Huang, Y.; Zhang, G.; Zhao, R.; Yang, H.; Zhang, D. Anal. Chem. 2014, , 86, 7987−7995. [2] M.-P. Romero-Gómez, R. Gómez -Gil, J. R. Paño-Pardo, J. Mingorance, J. Infection 2012, 65, 513-520
13
