Phenotypic and Genotypic Characterization with MALDI-TOF-MS Based Identification of Staphylococcus spp. Isolated from Mobile Phones with their Antibiotic Susceptibility, Biofilm Formation, and Adhesion Properties
Abstract
:1. Introduction
2. Results
2.1. Staphylococcus spp. Morphotypes on CSA
2.2. Species Identification with MALDI-TOF-MS and Hydrolytic Enzymes Production
2.3. Susceptibility to Antibiotics and Detection of mecA Gene
2.4. Adhesive Properties
2.5. Detection of Biofilm, Exoenzymes, and Haemolysin Related Genes
2.6. Statistical Analysis
3. Discussion
4. Materials and Methods
4.1. Sample Collection and Bacteriological Analysis
4.2. Susceptibility to Antibiotics
4.3. Adhesive Properties of the Identified Bacteria
4.4. Detection of Methicillin Resistance, Protease (sspA, sspB), Lipase (geh), α-Hemolysin (hla), and Adhesion Genes (icaA, icaD, cna, fnbA) in Staphylococcus spp. Strains
4.5. Data Analysis
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Organism (No. Tested) | Color of Isolated Colonies |
---|---|
Methicillin susceptible (1) | Mauve |
Methicillin resistant (18) | Mauve |
S. haemolyticus (2) | Light blue |
S. warneri (3) | White |
Strains | MALDI-TOF-MS | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
---|---|---|---|---|---|---|---|---|---|---|
ATCC 43300 | S. aureus | + | + | + | + | − | + | + | + | β |
12C | S. aureus | + | + | + | + | − | + | − | + | β |
26M | S. aureus | + | + | + | + | + | + | + | + | β |
32T | S. aureus | + | + | + | + | + | − | + | − | α |
34B | S. aureus | + | + | + | + | − | − | + | + | α |
37C | S. aureus | + | + | + | + | − | − | + | − | α |
40B | S. aureus | + | + | + | − | + | + | + | + | α |
47C | S. aureus | + | + | + | − | + | − | + | − | β |
56M | S. aureus | + | + | + | + | − | − | + | − | α |
57 M | S. aureus | + | + | + | + | − | − | + | − | α |
59B | S. aureus | + | + | + | + | + | − | + | − | σ |
65C | S. aureus | + | + | + | + | + | − | − | − | σ |
66B | S. aureus | + | + | + | − | − | − | + | − | α |
67M | S. aureus | + | + | + | − | − | + | + | + | β |
68T | S. aureus | + | + | + | − | − | + | + | − | β |
74M | S. aureus | + | + | + | + | + | + | + | − | α |
76C | S. aureus | + | + | + | + | − | − | + | + | β |
78T | S. aureus | + | + | + | + | + | − | + | − | σ |
83M | S. aureus | + | + | + | + | + | + | + | + | β |
96M | S. aureus | + | + | + | + | + | + | + | − | σ |
7T | S. warneri | − | + | + | + | − | − | − | − | α |
31C | S. warneri | − | + | + | + | − | + | − | − | α |
47B | S. warneri | − | + | + | − | − | + | + | − | σ |
30C | S. haemolyticus | − | + | + | + | + | + | + | − | α |
39B | S. haemolyticus | − | + | + | − | − | − | + | − | α |
% positivity | 80 | 100 | 100 | 70.83 | 45.83 | 45.83 | 83.33 | 29.16 | 29.16 (β) |
Strains | Antibiotics | MARI | mecA | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | |||
ATCC 43300 | R | S | R | R | R | R | R | S | S | R | S | S | R | R | S | S | R | R | 0.611 | mecA+ |
12C | R | S | S | R | R | S | R | S | R | R | R | S | R | R | S | R | R | R | 0.666 | mecA− |
26M | R | S | S | S | R | S | R | S | R | R | S | S | R | S | S | S | R | R | 0.444 | mecA+ |
32T | R | S | R | S | R | S | R | S | R | R | R | S | R | R | S | S | R | R | 0.611 | mecA− |
34B | R | S | R | S | R | S | S | S | R | S | R | S | R | S | S | S | R | S | 0.388 | mecA− |
37C | R | S | R | S | R | S | R | S | R | R | R | S | R | S | S | S | R | R | 0.555 | mecA− |
40B | R | S | R | S | R | S | S | S | S | S | R | S | R | R | S | S | R | R | 0.444 | mecA− |
47C | R | S | R | S | R | S | S | S | R | R | R | S | R | R | S | S | R | R | 0.555 | mecA− |
56M | S | S | R | R | R | S | R | S | R | R | S | S | R | R | S | S | R | R | 0.555 | mecA− |
57M | R | S | R | R | R | R | S | S | R | R | R | S | R | S | S | R | R | R | 0.666 | mecA− |
59B | R | S | R | R | R | R | R | R | R | R | R | S | R | S | S | R | R | R | 0.777 | mecA+ |
65C | R | S | S | S | R | S | S | S | S | R | S | S | R | R | S | S | R | R | 0.388 | mecA+ |
67M | R | S | R | R | R | S | R | S | R | R | R | S | R | R | S | S | R | R | 0.666 | mecA− |
66B | R | S | S | S | R | S | S | R | R | R | R | S | R | S | S | R | R | R | 0.555 | mecA+ |
68T | R | R | R | R | - | S | - | S | R | R | R | R | R | S | R | R | R | R | 0.812 | mecA+ |
74M | R | S | R | S | R | R | R | S | S | R | S | R | R | R | S | S | R | R | 0.611 | mecA+ |
76C | R | S | S | S | R | S | R | S | R | S | R | S | R | R | S | S | R | R | 0.500 | mecA− |
78T | R | S | R | S | R | S | R | S | S | R | R | S | R | R | S | S | R | R | 0.555 | mecA+ |
83M | R | S | S | S | R | S | R | S | S | R | R | S | R | R | R | S | R | R | 0.555 | mecA− |
96M | R | S | R | R | R | R | R | S | R | R | R | S | R | R | S | S | R | R | 0.722 | mecA− |
7T | R | R | S | R | R | S | R | S | R | R | R | S | R | R | S | R | R | R | 0.722 | mecA+ |
31C | R | S | R | R | R | R | R | R | R | R | R | S | R | S | S | R | R | R | 0.777 | mecA− |
47B | R | S | S | S | R | R | - | S | R | R | S | S | R | R | S | S | R | R | 0.529 | mecA− |
39B | R | S | R | R | R | S | R | S | R | R | R | R | R | R | S | S | R | R | 0.722 | mecA+ |
30C | R | S | S | R | R | S | R | S | R | R | R | S | R | R | S | R | R | R | 0.666 | mecA− |
Strains | Safranin Assay | Slime Phenotype (CRA) | Biofilm on Polystyrene | Biofilm on Glass | |||
---|---|---|---|---|---|---|---|
(OD570) ± SD | Interpretation | (OD570) ± SD | Interpretation | ||||
ATCC 43300 | +++ | Black | S+ | 1.89 ± 0.13 | H | 1.48 ± 0.15 | H |
12C | +++ | Black | S+ | 1.34 ± 0.18 | H | 1.09 ± 0.05 | H |
26M | +++ | Red | S− | 1.31 ± 0.15 | H | 0.57 ± 0.1 | M |
32T | + | Bordeaux | S− | 0.57 ± 0.4 | M | 0.55 ± 0.04 | M |
34B | +++ | Red | S− | 0.64 ± 0 | M | 0.65 ± 0.09 | M |
37C | ++ | Red | S− | 0.16 ± 0.35 | M | 0.99 ± 0.01 | M |
40B | +++ | Bordeaux | S− | 0.28 ± 0 | M | 1.10 ± 0.16 | H |
47C | ++ | Red | S− | 0.64 ± 0 | M | 0.57 ± 0.06 | M |
56M | ++ | Red with black center | S+ | 0.21 ± 0 | M | 1.11 ± 0.08 | H |
57 M | +++ | Red | S− | 0.74 ± 0 | M | 1.68 ± 0.2 | H |
59B | +++ | Pink | S− | 0.16 ± 0 | M | 0.717 ± 0.12 | M |
65C | ++ | Pink | S− | 0.15 ± 0 | M | 0.88 ± 0.05 | M |
66B | ++ | Red | S− | 2.73 ± 0.56 | H | 0.75 ± 0.2 | M |
67M | +++ | Orange | S− | 1.27 ± 0.17 | H | 1.28 ± 0.1 | H |
68T | +++ | Red | S− | 2.14 ± 0.64 | H | 0.45 ± 0.09 | M |
74M | +++ | Pink | S− | 0.40 ± 0 | M | 0.50 ± 0.09 | M |
76C | +++ | Black | S+ | 1.63 ± 0.57 | H | 0.38 ± 0.04 | M |
78T | + | Red | S− | 0.47 ± 0 | M | 0.90 ± 0.15 | M |
83M | +++ | Red with black center | S+ | 0.85 ± 0.13 | M | 0.32 ± 0.04 | M |
96M | +++ | Red | S− | 0.36 ± 0.3 | M | 0.30 ± 0.05 | M |
7T | +++ | Red | S− | 0.33 ± 0 | M | 0.87 ± 0.13 | M |
31C | +++ | Pink | S− | 0.59 ± 0 | M | 0.46 ± 0.08 | M |
47B | ++ | Pink | S− | 0.66 ± 0.12 | M | 0.56 ± 0.03 | M |
30C | + | Red | S− | 0.17 ± 0 | M | 0.98 ± 0.07 | M |
39B | ++ | Pink | S− | 1.07 ± 0.19 | H | 0.84 ± 0.11 | M |
Strains | Adhesion | Haemolysins | Exoenymes | |||||
---|---|---|---|---|---|---|---|---|
icaA | icaD | cna | fnbA | Hla | geh | sspA | sspB | |
ATCC 43300 | + | + | + | + | + | + | + | + |
12C | − | − | − | + | − | + | − | + |
26M | − | − | + | + | + | + | − | + |
32T | + | + | − | + | + | + | − | − |
34B | + | + | + | + | − | + | − | + |
37C | + | + | + | + | − | − | − | + |
40B | − | − | − | − | − | − | − | + |
47C | − | + | + | − | + | − | + | + |
56M | + | + | + | + | + | + | − | + |
57M | + | + | − | + | − | + | − | + |
59B | − | + | + | − | + | − | + | + |
65C | − | − | + | + | + | − | − | + |
66B | + | − | − | + | − | − | − | + |
67M | + | + | + | − | + | − | − | − |
68T | + | + | + | + | + | + | − | + |
74M | + | + | + | + | − | − | − | − |
76C | + | + | + | + | − | − | − | + |
78T | + | + | + | + | − | − | − | + |
83M | + | − | − | + | − | − | − | − |
96M | + | − | − | + | + | − | − | − |
7T | − | + | − | − | − | − | − | − |
31C | − | + | − | − | − | − | − | + |
47B | − | + | − | + | − | − | − | − |
30C | + | + | − | + | − | − | + | − |
39B | + | − | + | + | − | + | + | + |
% positivity | 62.5 | 66.66 | 50.00 | 75.00 | 37.50 | 33.33 | 16.66 | 66.66 |
p-Value (Univariate Analysis) | Adhesion Related Genes | Haemolysin Gene | Exoenymes Genes | |||||
---|---|---|---|---|---|---|---|---|
icaA | icaD | cna | fnbA | Hla | geh | sspA | sspB | |
Biofilm on glass | 0.489 | 0.783 | 0.783 | 0.489 | 0.581 | 0.945 | 0.836 | 0.581 |
Biofilm on polystyrene | 0.489 | 0.783 | 0.783 | 0.489 | 0.581 | 0.945 | 0.836 | 0.581 |
Primer | Primer Sequence | PCR Conditions | Product Size (pb) | Reference |
---|---|---|---|---|
mecA-F | 5′-GTA GAA ATG ACT GAA CGT CCG ATAA-3′ | 94 °C for 5 min 30 Cycles (1 min at 94 °C, 1 min at 55 °C, 2 min at 72 °C) 72 °C for 10 min | 310 | [78] |
mecA-R | 5′-CCAATT CCA CAT TGT TTC GGT CTAA-3′ | |||
sspA-F | 5′-GAC AAC AGC GAC ACT TGT GA-3′ | 94 °C for 5 min 30 Cycles (30 s at 94 °C, 30 s min at 45 °C, 45 s at 72 °C) 72 °C for 10 min | 292 | [79] |
sspA-R | 5′-AGT ATC TTT ACC TAC AAC TAC A-3′ | |||
sspB-F | 5′-TGA AGA AGA TGG CAA AGT TAG-3′ | 94 °C for 5 min 30 Cycles (30 s at 94 °C, 30 s min at 47 °C, 45 s at 72 °C) 72 °C for 10 min | 493 | |
sspB-R | 5′-TTG AGA TAC ACT TTG TGC AAG-3′ | |||
geh-F | 5′-GCACAAGCCTCGG-3′ | 94 °C for 5 min 30 Cycles (30 s at 94 °C, 30 s min at 40 °C, 45 s at 72 °C) 72 °C for 10 min | 473 | [20] |
geh-R | 5′-GACGGGGGTGTAG-3′ | |||
icaA-F | 5′-ACACTTGCTGGCGCAGTCAA-3′ | 94 °C for 5 min 30 Cycles (30 s at 94 °C, 30 s min at 45 °C, 45 s at 72 °C) 72 °C for 10 min | 188 | [80] |
icaA-R | 5′-TCTGGAACCAACATCCAACA-3′ | |||
icaD-F | 5′-ACACTTGCTGGCGCAGTCAA-3′ | 94 °C for 5 min 30 Cycles (30 s at 94 °C, 30 s min at 55 °C, 45 s at 72 °C) 72 °C for 10 min | 198 | |
icaD-R | 5′-TCTGGAACCAACATCCAACA-3′ | |||
cna-F | 5′-AAAGCGTTGCCTAGTGGAGA-3′ | 94 °C for 5 min 30 Cycles (30 s at 94 °C, 30 s min at 62 °C, 45 s at 72 °C) 72 °C for 10 min | 192 | [57] |
cna-R | 5′-AGTGCCTTCCCAAACCTTTT-3′ | |||
fnbA-F | 5′-GATACAAACCCAGGTGGTGG-3′ | 191 | ||
fnbA-R | 5′-TGTGCTTGACCATGCTCTTC-3′ | |||
hla-F | 5′CAACTGATAAAAAAGTAGGCTGGAAAGTGAT-3′ | 94 °C for 5 min 35 Cycles (30 s at 94 °C, 60 s min at 59 °C, 60 s at 72 °C) 72 °C for 10 min | 201 | [81] |
hla-R | 5′-CTGGTGAAAACCCTGAAGATAATAGAG-3′ |
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Noumi, E.; Merghni, A.; Alreshidi, M.; Del Campo, R.; Adnan, M.; Haddad, O.; De Feo, V.; Snoussi, M. Phenotypic and Genotypic Characterization with MALDI-TOF-MS Based Identification of Staphylococcus spp. Isolated from Mobile Phones with their Antibiotic Susceptibility, Biofilm Formation, and Adhesion Properties. Int. J. Environ. Res. Public Health 2020, 17, 3761. https://doi.org/10.3390/ijerph17113761
Noumi E, Merghni A, Alreshidi M, Del Campo R, Adnan M, Haddad O, De Feo V, Snoussi M. Phenotypic and Genotypic Characterization with MALDI-TOF-MS Based Identification of Staphylococcus spp. Isolated from Mobile Phones with their Antibiotic Susceptibility, Biofilm Formation, and Adhesion Properties. International Journal of Environmental Research and Public Health. 2020; 17(11):3761. https://doi.org/10.3390/ijerph17113761
Chicago/Turabian StyleNoumi, Emira, Abderrahmen Merghni, Mousa Alreshidi, Rosa Del Campo, Mohd Adnan, Ons Haddad, Vincenzo De Feo, and Mejdi Snoussi. 2020. "Phenotypic and Genotypic Characterization with MALDI-TOF-MS Based Identification of Staphylococcus spp. Isolated from Mobile Phones with their Antibiotic Susceptibility, Biofilm Formation, and Adhesion Properties" International Journal of Environmental Research and Public Health 17, no. 11: 3761. https://doi.org/10.3390/ijerph17113761