Identification of Bacteria Associated with Post-Operative Wounds of Patients with the Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Approach
Abstract
1. Introduction
2. Results and Discussion
2.1. Bacterial Identification
2.2. Mass Spectra Analysis
2.3. Identification of Proteins with Specific Peak to the Predicted Antibiotic Mechanism of Action and Bacterial Drug-Resistance
2.4. Selection of Sample Preparation Conditions to Determine Microorganisms Using the MALDI-TOF MS Technique
3. Experimental
3.1. Chemicals and Reagents
3.2. Patient and Sample Characterization
3.3. Isolation of Post-Operative Wound Bacteria
3.4. MALDI-TOF MS Analysis
3.5. Selection of Sample Preparation Conditions to Determine Microorganisms with MALDI-TOF MS
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Non-Selective Growth Media | |||||||||
BHI | MH | ||||||||
Sample Name | RAW | MSP | Sample Name | RAW | MSP | ||||
Best Match | Score Value | Best Match | Score Value | Best Match | Score Value | Best Match | Score Value | ||
B1a | Providencia stuartii DSM 4539T HAM | 2.10 | Providencia stuartii DSM 4539T HAM | 1.99 | B1a | Morganella morganii ssp morganii 15284_1 CHB | 2.04 | Morganella morganii ssp morganii 15284_1 CHB | 2.05 |
B1b | Enterobacter cloacae MB_5277_05 THL | 1.91 | Enterobacter cloacae MB_5277_05 THL | 1.78 | B1b | Morganella morganii 9544_1 CHB | 1.93 | Morganella morganii 9544_1 CHB | 1.93 |
B3a | Staphylococcus aureus ATCC 25923 THL | 2.35 | Staphylococcus aureus ATCC 33591 THL | 2.26 | B3aD | Enterobacter cloacae MB_5277_05 THL | 2.03 | Enterobacter cloacae MB11506_1 CHB | 1.99 |
B3b | Staphylococcus aureus ATCC 25923 THL | 2.19 | Staphylococcus aureus ATCC 29213 THL | 1.99 | B3aM | Staphylococcus aureus ATCC 33591 THL | 2.15 | Staphylococcus aureus ATCC 29213 THL | 2.12 |
B4a | Staphylococcus epidermidis 10547 CHB | 2.28 | - | 1.69 | B3b | Staphylococcus aureus ATCC 33591 THL | 2.23 | Staphylococcus aureus ATCC 29213 THL | 2.19 |
B4b | Staphylococcus epidermidis ATCC 14990T THL | 1.90 | - | 1.53 | B4a | Staphylococcus epidermidis 10547 CHB | 1.99 | - | 1.51 |
B7a | Enterococcus faecium 20218_1 CHB | 2.14 | Enterococcus faecium 20218_1 CHB | 2.15 | B4bD | Klebsiella pneumoniae ssp pneumoniae DSM 30104T HAM | 2.13 | Klebsiella pneumoniae ssp pneumoniae 9295_1 CHB | 1.90 |
B7b | Enterococcus faecium 11037 CHB | 2.09 | Enterococcus faecium 11037 CHB | 2.09 | B4bM | Staphylococcus epidermidis 0547 CHB | 1.98 | Staphylococcus epidermidis DSM 4851 DSM | 1.74 |
B8a | Enterococcus faecalis ATCC 7080 THL | 2.19 | Enterococcus faecalis ATCC 7080 THL | 2.14 | B7a | Enterococcus faecium 11037 CHB | 2.25 | Enterococcus faecium 11037 CHB | 2.25 |
B8b | Enterococcus faecalis DSM 2570 DSM | 2.18 | Enterococcus faecalis DSM 20409 DSM | 1.91 | B7b | Enterococcus faecium 11037 CHB | 2.35 | Enterococcus faecium 11037 CHB | 2.29 |
B9a | - | 1.45 | - | 1.39 | B8a | Enterococcus faecalis DSM 20409 DSM | 2.10 | Enterococcus faecalis DSM 20409 DSM | 2.03 |
B11a | Klebsiella pneumoniae ssp ozaenae DSM 16358T HAM | 2.15 | Klebsiella pneumoniae ssp rhinoscleromatis DSM 16231T HAM | 2.06 | B8b | Enterococcus faecalis ATCC 7080 THL | 2.12 | Enterococcus faecalis 20247_4 CHB | 1.91 |
B11b | Klebsiella pneumoniae ssp pneumoniae DSM 30104T HAM | 2.07 | Klebsiella pneumoniae ssp pneumoniae 9295_1 CHB | 2.04 | B11b | Klebsiella pneumoniae ssp pneumoniae 9295_1 CHB | 2.32 | Klebsiella pneumoniae ssp pneumoniae 9295_1 CHB | 2.32 |
B15a | - | 1.39 | - | 1.33 | B15a | Bacillus pumilus IAM 12469 PAH | 1.74 | - | 1.64 |
B15b | Enterococcus faecalis ATCC 7080 THL | 2.30 | Enterococcus faecalis DSM 20409 DSM | 2.26 | B15b | Enterococcus faecalis ATCC 7080 THL | 2.43 | Enterococcus faecalis DSM 20409 DSM | 2.35 |
B17a | Escherichia coli ATCC 25922 THL | 2.25 | Escherichia coli DH5alpha BRL | 2.18 | B17a | Escherichia coli MB11464_1 CHB | 1.99 | Escherichia coli MB11464_1 CHB | 1.88 |
B18a | Staphylococcus haemolyticus 10024 CHB | 2.23 | Staphylococcus haemolyticus 10024 CHB | 1.97 | B17b | Escherichia coli RV412_A1_2010_06a LBK | 1.95 | Escherichia coli RV412_A1_2010_06a LBK | 1.95 |
B18b | Staphylococcus epidermidis CCM 4505 CCM | 2.10 | - | 1.68 | B18a | Staphylococcus epidermidis DSM 1798 DSM | 1.85 | Staphylococcus epidermidis DSM 1798 DSM | 1.89 |
B21a | Bacillus pumilus IAM 12469 PAH | 1.77 | - | 1.68 | B18b | Staphylococcus haemolyticus Mb18803_2 CHB | 2.01 | Staphylococcus haemolyticus Mb18803_2 CHB | 1.93 |
B21a (white) | Lactobacillus pentosus DSM 20199 DSM | 1.82 | - | 1.21 | B21a | - | 1.68 | - | 1.59 |
Selective Growth Media | |||||||||
BCP | VRE | ||||||||
Sample Name | RAW | MSP | Sample name | RAW | MSP | ||||
Best Match | Score Value | Best Match | Score Value | Best Match | Score Value | Best Match | Score Value | ||
B1a | Escherichia coli ATCC 25922 THL | 2.22 | Escherichia coli ATCC 25922 THL | 2.09 | B1a | Morganella morgani 9544_1 CHB | 1.96 | Morganella morganii 9544_1 CHB | 1.80 |
B1b | Escherichia coli MB11464_1 CHB | 2.15 | Escherichia coli MB11464_1 CHB | 2.10 | B1b | Morganella morgani (E) 21086317 MLD | 1.99 | Morganella morganii (E) 21086317 MLD | 1.89 |
B3aD | Enterobacter kobei DSM 13645T DSM | 2.20 | Enterobacter cloacae 13159_1 CHB | 2.08 | B3a | Staphylococcus aureus ATCC 29213 THL | 2.47 | Staphylococcus aureus ATCC 29213 THL | 2.21 |
B3aM | Staphylococcus aureus ATCC 33591 THL | 2.03 | Staphylococcus aureus ATCC 33591 THL | 1.88 | B3b | Staphylococcus aureus ATCC 29213 THL | 2.47 | Staphylococcus aureus ATCC 33591 THL | 2.25 |
B3b | Staphylococcus aureus ATCC 33591 THL | 1.95 | Staphylococcus aureus ATCC 33591 THL | 1.84 | B4a | Enterococcus faecalis ATCC 7080 THL | 2.34 | Enterococcus faecalis DSM 20409 DSM | 2.33 |
B4a | Staphylococcus epidermidis ATCC 14990T THL | 2.04 | Staphylococcus epidermidis DSM 1798 DSM | 1.84 | B4b | Enterococcus faecalis ATCC 7080 THL | 2.06 | Enterococcus faecalis ATCC 7080 THL | 1.83 |
B4b | Staphylococcus epidermidis ATCC 14990T THL | 2.14 | Staphylococcus epidermidis DSM 1798 DSM | 1.84 | B7a | Enterococcus faecium 20218_1 CHB | 1.97 | Enterococcus faecium 20218_1 CHB | 1.95 |
B7a | Enterococcus faecium 11037 CHB | 2.19 | Enterococcus faecium 20218_1 CHB | 2.15 | B7b | Enterococcus faecium 20218_1 CHB | 1.95 | Enterococcus faecium 20218_1 CHB | 1.95 |
B7b | Enterococcus faecium 11037 CHB | 2.05 | Enterococcus faecium 20218_1 CHB | 2.09 | B8b | Enterococcus faecalis ATCC 7080 THL | 2.20 | Enterococcus faecalis ATCC 7080 THL | 2.15 |
B8a | Enterococcus faecalis DSM 20371 DSM | 1.98 | Enterococcus faecalis DSM 20371 DSM | 1.90 | B15a | Bacillus pumilus DSM 13835 DSM | 2.09 | Bacillus pumilus DSM 13835 DSM | 2.09 |
B8b | Enterococcus faecium 11037 CHB | 2.32 | Enterococcus faecium 11037 CHB | 2.38 | B15b | Enterococcus faecalis ATCC 29212 CHB | 2.20 | Enterococcus faecalis 20247_4 CHB | 2.11 |
B11a | Klebsiella pneumoniae ssp pneumoniae 9295_1 CHB | 2.22 | Klebsiella pneumoniae ssp pneumoniae 9295_1 CHB | 2.17 | B17a | Enterococcus avium 96 PIM | 1.87 | Enterococcus avium 96 PIM | 1.87 |
B11b | Klebsiella pneumoniae ssp pneumoniae 9295_1 CHB | 2.10 | Klebsiella pneumoniae ssp pneumoniae 9295_1 CHB | 2.10 | B18a | Staphylococcus epidermidis 10547 CHB | 2.16 | Staphylococcus epidermidis DSM 3269 DSM | 1.92 |
B15a | - | 1.61 | - | 1.61 | B18b | Staphylococcus epidermidis 10547 CHB | 1.92 | Staphylococcus epidermidis 4b_r ESL | 1.87 |
B15b | Enterococcus faecalis 20247_4 CHB | 2.29 | Enterococcus faecalis 20247_4 CHB | 2.18 | B21a | Bacillus altitudis CS 809_1 BRB | 1.72 | - | 1.57 |
B17a | Escherichia coli DH5alphaBRL | 2.19 | Escherichia coli DH5alpha BRL | 2.01 | |||||
B17b | Escherichia coli DH5alpha BRL | 2.08 | Escherichia coli MB11464_1 CHB | 1.84 | |||||
B18a | Staphylococcus epidermidis 10547 CHB | 2.08 | Staphylococcus epidermidis DSM 1798 DSM | 1.94 | |||||
B18b | Staphylococcus epidermidis DSM 1798 DSM | 2.00 | Staphylococcus epidermidis DSM 1798 DSM | 2.13 | |||||
B21a | Bacillus pumilus IAM 12469 PAH | 1.73 | - | 1.68 |
Bacteria from Biological Material | Reference Bacteria Strain |
---|---|
Bacillus | |
B. altitudis VRE.B21a | B. altitudis CS 809_1 BRB |
B. pumilus BCP.B21a | B. pumilus IAM 12469 PAH |
B. pumilus VRE.B15a | B. pumilus DSM 13835 DSM |
Enterobacter | |
E. cloacae BHI.B1b | E. cloacae MB_5277_05 THL |
E. kobei BCP.B3aD | E. kobei DSM 13645T DSM |
Enterococcus | |
E. avium VRE.B17a | E. avium 96 PIM |
E. faecalis BCP.B8a | E. faecalis DSM 20371 DSM |
E. faecalis BCP.B15b | E. faecalis 20247_4 CHB |
E. faecalis BHI.B15b | E. faecalis ATCC 7080 THL |
E. faecalis BHI.B8b | E. faecalis DSM 2570 DSM |
E. faecalis VRE.B15b | E. faecalis ATCC 29212 CHB |
E. faecalis MHA.B8a | E. faecalis DSM 20409 DSM |
E. faecium BHI.B7b | E. faecium 11037 CHB |
E. faecium BHI.B7a | E. faecium 20218_1 CHB |
Escherichia | |
E. coli BHI.B17a | E. coli ATCC 25922 THL |
E. coli BCP.B1b | E. coli MB11464_1 CHB |
E. coli BCP.B17a | E. coli DH5alpha BRL |
E. coli MHA.B17a | E. coli RV412_A1_2010_06a LBK |
Klebsiella | |
K. pneumoniae BCP.B11a | K. pneumoniae ssp pneumoniae 9295_1 CHB |
K. pneumoniae BHI.B11a | K. pneumoniae ssp ozaenae DSM 16358T HAM |
K.pneumoniae BHI.B11b | K.pneumoniae ssp pneumoniae DSM 30104T HAM |
Lactobacillus | |
L. pentosus BHI.B21a(white) | L. pentosus DSM 20199 DSM |
Morganella | |
M. morgani MHA.B1b | M. morgani 9544_1 CHB |
M. morgani VRE.B1b | M. morgani (E) 21086317 MLD |
M. morganii MHA.B1a | M. morganii ssp morganii 15284_1 CHB |
Providencia | |
P. stuartii BHI.B1a | P. stuartii DSM 4539T HAM |
Staphylococcus | |
S. aureus BCP.B3aM | S. aureus ATCC_33591_THL |
S. aureus BHI.B3b | S. aureus ATCC_25923_THL |
S. epidermidis BCP.B4a | S. epidermidis ATCC 14990T THL |
S. epidermidis BHI.B4a | S. epidermidis 10547 CHB |
S. epidermidis BCP.B18b | S. epidermidis DSM 1798 DSM |
S. epidermidis BHI. B18b | S. epidermidis CCM 4505 CCM |
S. haemolyticus BHI.B18a | S. haemolyticus 10024 CHB |
S. haemolyticus MHA.B18b | S. haemolyticus Mb18803_2 CHB |
S. aureus /ATCC 29213 THL/(Antibiotic: Clindamycin) | |||||
m/z | Control Sample | Test Sample | Protein | Putative Function | Ref. |
2656.8 | - | + | Phenol-soluble modulin PSM-alpha-3 | virulence factor | [46,47] |
3006.2 | - | + | TraR/DksA family transcriptional regulator | virulence, antibiotic resistance, pathogenesis | [48] |
4290.7 | + | - | 50S ribosomal protein L36 | builds ribosome, translation | [49] |
4365.1 | + | - | ABC transporter ATP-binding protein | [50,51] | |
4379.9 | + | - | Teichoic acid ABC transporter permease | [50,51] | |
4386.1 | + | - | Single-stranded DNA-binding protein | DNA replication | [52] |
4822.6 | + | - | Oligopeptide ABC superfamily ATP binding cassette transporter. binding protein (Protein CysJ) | [50,51] | |
5437.4 | + | - | 50S ribosomal protein L33. | builds ribosome, translation | [49] |
5872.7 | + | - | 50S ribosomal protein L33 | builds ribosome, translation | [49] |
6355.1 | - | + | Antibiotic transport-associated protein | [53] | |
6589.8 | - | + | Antibiotic resistance protein | [53] | |
6743.1 | - | + | Exported protein | [53] | |
7022.9 | + | - | ABC transporter ATP-binding protein | [50,51] | |
8111.7 | - | + | GCN5-related N-acetyltransferase (GNAT) | kanamycin and gentamycin resistance | [54] |
8908.0 | + | - | Compound ABC uptake transporter ATP-binding protein. | [50,51] | |
9898.5 | - | + | Immunoglobulin G binding protein A | binding protein A | [55] |
9909.4 | - | + | Protein A | pathogenic factor | [55] |
E. faecalis /ATCC 7080 THL/(antibiotics: piperacylin and tazobactam) | |||||
m/z | Control sample | Test sample | Protein | Putative Function | |
2443.3 | ‒ | + | Attenuator leader peptide | response to antibiotic | [56] |
6341.0 | + | ‒ | 50S ribosomal protein L30 | builds ribosome, translation | [57] |
E. cloacae /MB_5277_05 THL/(antibiotic: clindamycin) | |||||
m/z | Control sample | Test sample | Protein | Putative Function | |
3710.7 | ‒ | + | Agmatinase | catalysis of the reaction: agmatine + H2O = putrescine + urea | [58] |
5571.8 | + | ‒ | Isocitrate dehydrogenase (NADP(+)) | cofactor binding sites Mg2+, Mn2+; | [59] |
5598.8 | ‒ | + | Agmatinase | catalysis of the reaction: agmatine + H2O = putrescine + urea | [58] |
6417.9 | ‒ | + | Murein transglycosylase | break the glycosidic bond | [60] |
7620.8 | ‒ | + | Cellulose biosynthesis protein BcsF | necessary for biofilm formation | [61] |
9410.2 | ‒ | + | DNA polymerase V | part of the SOS response to DNA damage | [62] |
10,315.8 | + | ‒ | ParB partition protein | regulation of transcription | [63] |
10,883.9 | ‒ | + | Ribosome hibernation promoting factor HPF | primary metabolic process, regulation of translation | [64] |
Bacteria Species | Time | Standard Method | Sepsityper Method | ||
---|---|---|---|---|---|
Vials | TSB | Vials | TSB | ||
S. aureus | 4 h | 1.85–1.97 | 1.79–1.88 | 2.05–2.16 | |
6 h | 2.38–2.42 | 2.31–2.39 | 2.36–2.39 | 2.25–2.30 | |
24 h | 2.25–2.34 | 2.24–2.33 | 2.30–2.34 | 2.31–2.42 | |
E. coli | 4 h | - | 2.35–2.37 | - | 2.31–2.38 |
6 h | - | 2.31–2.33 | - | 2.20–2.28 | |
24 h | - | 2.27–2.32 | - | 2.10–2.17 | |
E. faecalis | 4 h | 2.33–2.35 | 2.26–2.36 | 2.28–2.32 | 2.06–2.18 |
6 h | 2.00–2.02 | 1.76–1.88 | 2.02–2.06 | ||
24 h | 2.39–2.42 | 2.45–2.47 | 2.19–2.24 |
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Szultka-Młyńska, M.; Janiszewska, D.; Pomastowski, P.; Złoch, M.; Kupczyk, W.; Buszewski, B. Identification of Bacteria Associated with Post-Operative Wounds of Patients with the Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Approach. Molecules 2021, 26, 5007. https://doi.org/10.3390/molecules26165007
Szultka-Młyńska M, Janiszewska D, Pomastowski P, Złoch M, Kupczyk W, Buszewski B. Identification of Bacteria Associated with Post-Operative Wounds of Patients with the Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Approach. Molecules. 2021; 26(16):5007. https://doi.org/10.3390/molecules26165007
Chicago/Turabian StyleSzultka-Młyńska, Małgorzata, Daria Janiszewska, Paweł Pomastowski, Michał Złoch, Wojciech Kupczyk, and Bogusław Buszewski. 2021. "Identification of Bacteria Associated with Post-Operative Wounds of Patients with the Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Approach" Molecules 26, no. 16: 5007. https://doi.org/10.3390/molecules26165007
APA StyleSzultka-Młyńska, M., Janiszewska, D., Pomastowski, P., Złoch, M., Kupczyk, W., & Buszewski, B. (2021). Identification of Bacteria Associated with Post-Operative Wounds of Patients with the Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Approach. Molecules, 26(16), 5007. https://doi.org/10.3390/molecules26165007