Assessment of the Antibiotic Resistance Profile, Genetic Heterogeneity and Biofilm Production of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from The Italian Swine Production Chain
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sampling Design
2.2. Isolation of Suspected MRSA
2.3. MRSA Confirmation
2.4. Antibiotic Resistance Profiling of Confirmed MRSA Isolates
2.4.1. Disk Diffusion Assays
2.4.2. Minimum Inhibitory Concentration Assays
2.4.3. Antimicrobial Resistance Genes Determination
- Multiplex 1 (aacA-aphD, tetL, tetM, dfrA, dfrD): 95 °C (5 min), followed by 32 cycles of 95 °C (30 s), 45 °C (45 s), 72 °C (1 min) and a final extension at 72 °C (10 min).
- Multiplex 2 (blaZ, dfrK, vgaE): 95 °C (5 min), followed by 32 cycles of 95 °C (30 s), 47 °C (45 s), 72 °C (50 s) and a final extension at 72 °C (10 min).
- Multiplex 3 (ermT, norA, vgaA): 95 °C (5 min), followed by 31 cycles of 95 °C (30 s), 49 °C (40 s), 72 °C (1 min) and a final extension at 72 °C (10 min).
- Multiplex 4 (aadD, cfr): 95 °C (5 min), followed by 31 cycles of 95 °C (30 s), 52 °C (40 s), 72 °C (50 s) and a final extension at 72 °C (10 min).
- PCR 5 (fexA): 95 °C (5 min), followed by 31 cycles of 95 °C (30 s), 56 °C (40 s), 72 °C (1 min) and a final extension at 72 °C (10 min).
2.5. Enterotoxin Gene Determination
2.6. MRSA Molecular Subtyping and Phylogenetic Analysis
2.7. Quantification of Biofilm Production
2.8. Statistical Analysis
3. Results
3.1. MRSA Detection
3.2. Antibiotic Resistance Profiles of MRSA Isolates
3.2.1. Phenotypic Resistance
3.2.2. Minimum Inhibitory Concentration (MIC) Assays
3.2.3. Presence of Antibiotic Resistance Genes
3.3. Presence of Enterotoxin Genes
3.4. Molecular Subtyping of MRSA Isolates
3.5. Biofilm Production Assays
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Antibiotic Resistance Pattern | Genotypes (No. Isolates) |
---|---|
P-AMC-OX-AMP-TET-T | ST398/t011 (5); ST398/t899 (1); ST398/t4474 (1) |
P-AMC-OX-AMP-FUR-TET-T | ST398/t011 (5) |
P-AMC-OX-AMP-FUR-TET-ENR | ST398/t899 (8); ST97/t4795 (1) |
P-AMC-OX-AMP-FUR-TET-DXT-T | ST398/t011 (6) |
P-AMC-OX-AMP-FUR-KF-ENR-CN | ST1/t127 (3) |
P-AMC-OX-AMP-FUR-TET-DXT-ENR | ST5422/t1730 (1); ST398/t1939 (1) |
P-AMC-OX-AMP-KF-ENR-CN | ST1/t127 (2) |
P-AMC-OX-AMP-FUR-TET-DXT-C-T | ST398/t011 (2) |
P-AMC-OX-AMP-FUR-TET-C-FFC-CN-SxT | ST398/t899 (2) |
P-AMC-OX-AMP-FUR-TET-DXT-C-FFC-SxT | ST398/t899 (2) |
P-AMC-OX-AMP-FUR-TET-DXT-C-FFC-T-SxT | ST398/t899 (2) |
Isolate ID | Source | ST | spa-Type | Antibiotic Resistance Genes | Enterotoxin Genes | Biofilm Production |
---|---|---|---|---|---|---|
SA1 | Animal | 398 | t011 | blaZ, tetL, tetM, norA, aadD, dfrA, dfrK | n.d. | Moderate |
SA2 | Environment | 398 | t899 | blaZ, tetL, tetM, norA, fexA, aacAaphD, aadD, vgaA, dfrA | seb, sed, see | Strong |
SA3 | Environment | 398 | t899 | blaZ, tetL, tetM, norA, fexA, aadD, vgaA, dfrA | sea, seb, see | Weak |
SA4 | Environment | 398 | t899 | tetL, aadD, vgaA | seb, see | Weak |
SA5 | Animal | 97 | t4795 | blaZ, tetL, tetM, norA, aadD, vgaA, dfrA | seb, see | Moderate |
SA6 | Animal | 97 | t4795 | n.d. | n.d. | Moderate |
SA7 | Animal | 5422 | t1730 | blaZ, tetM, norA, dfrA, | sea, seb, sed | Strong |
SA8 | Animal | 398 | t899 | tetM, vgaA, dfrA, dfrK | n.d. | Strong |
SA9 | Animal | 398 | t011 | blaZ, tetM, vgaA, dfrA, dfrD | seb | Moderate |
SA10 | Animal | 97 | t1730 | blaZ, tetL, tetM, norA, cfr, dfrA, dfrD | sea | Strong |
SA11 | Animal | 398 | t899 | blaZ, tetL, tetM, fexA, aadD, vgaA, dfrA | sea, seb | Weak |
SA12 | Animal | 398 | t011 | blaZ, tetL, tetM | sea, seb | Moderate |
SA13 | Environment | 398 | t1939 | blaZ, tetM, norA, dfrA | sec | Weak |
SA14 | Environment | 398 | t011 | tetL | sea, seb | Non-producer |
SA15 | Environment | 30 | t318 | blaZ, tetL, tetM, fexA, cfr, aadD, dfrA | sea, seb | Strong |
SA16 | Animal | 398 | t899 | blaZ, dfrA | see | Moderate |
SA17 | Environment | 398 | t1939 | blaZ, tetM, norA, fexA | n.d. | Non-producing |
SA18 | Environment | 398 | t034 | blaZ, tetL, tetM, norA, fexA, cfr, aadD, dfrA | seb, see | Strong |
SA19 | Environment | 398 | t18494 | blaZ, tetM, norA, fexA, dfrA | sea, seb, see | Weak |
SA20 | Environment | 398 | t1200 | tetL, fexA, cfr, aadD, dfrK | n.d | Moderate |
SA21 | Animal | 398 | t1200 | blaZ, tetM, norA, vgaE, dfrA, dfrK | seb, sec | Weak |
SA22 | Animal | 398 | t18494 | blaZ, tetL, tetM, vgaE, dfrA, dfrK | n.d. | Weak |
SA23 | Animal | 398 | t011 | tetL, tetM, aadD, vgaE, dfrA, dfrK | sea, seb | Weak |
SA24 | Animal | 4894 | t18494 | cfr | sea, sec, see | Weak |
SA25 | Animal | 398 | t18494 | blaZ, tetL, tetM, cfr, dfrD | sea, seb, sec | Weak |
SA26 | Animal | 398 | t899 | blaZ, tetM, norA, vgaE, dfrA, dfrK | see | Weak |
SA27 | Animal | 398 | t899 | blaZ, tetM, norA, vgaE, dfrA, dfrK | sec, see | Weak |
SA28 | Animal | 398 | t899 | blaZ, tetM, norA, vgaE, dfrA, dfrK | sec | Weak |
SA29 | Animal | 398 | t899 | blaZ, tetM, norA, vgaE, dfrA, dfrK | n.d. | Moderate |
SA30 | Animal | 398 | t899 | blaZ, tetM, norA, aadD, vgaE, dfrA, dfrK | n.d. | Weak |
SA31 | Animal | 398 | t899 | tetM, aadD, dfrA | see | Non-producer |
SA32 | Animal | 97 | t1730 | blaZ, tetL, tetM, norA, aacAaphD, aadD, vgaA, vgaE, dfrA, dfrK, ermT | n.d. | Weak |
SA33 | Animal | 398 | t011 | blaZ, tetM, norA, vgaE, dfrA, dfrK | n.d. | Weak |
SA34 | Animal | 398 | t011 | blaZ, tetM, norA, aadD, dfrA, dfrK | sec, see | Weak |
SA35 | Animal | 398 | t899 | blaZ, tetM, norA, vgaA, vgaE, dfrA | sec, see | Weak |
SA36 | Animal | 398 | t899 | blaZ, tetM, norA, aadD, vgaE, dfrA, dfrD, dfrK | sea, seb, see | Non-producer |
SA37 | Animal | 398 | t011 | blaZ, tetM, norA, aadD, vgaE, dfrA, dfrD, dfrK | sea, seb | Weak |
SA38 | Animal | 398 | t011 | blaZ, tetL, tetM, norA, aadD, dfrA | n.d. | Non-producer |
SA39 | Animal | 398 | t011 | blaZ, tetM, norA, aadD, vgaA, vgaE, dfrA, dfrK | sea, seb, sec, sed | Weak |
SA40 | Animal | 398 | t011 | tetL, tetM, norA, aadD, vgaE, dfrK | seb | Weak |
SA41 | Animal | 1 | t127 | blaZ, norA, aacAaphD, aadD, vgaE, dfrA, dfrK, ermT | sec | Weak |
SA42 | Animal | 1 | t127 | blaZ, norA, aacAaphD, aadD, vgaE, dfrA, ermT | seb, sed | Moderate |
SA43 | Animal | 1 | t127 | blaZ, norA, aacAaphD, aadD, vgaE, dfrA, ermT | seb, sec | Weak |
SA44 | Animal | 1 | t127 | norA, aacAaphD, vgaA, vgaE, dfrA, dfrK, ermT | sec | Weak |
SA45 | Animal | 1 | t127 | norA, aacAaphD, vgaA, vgaE, dfrA | seb, sec, see | Non-producer |
SA46 | Animal | 398 | t899 | blaZ, tetM, norA, vgaE, dfrA, dfrK | seb | Non-producer |
SA47 | Animal | 398 | t1939 | blaZ, tetM, fexA, cfr, aadD, vgaA, vgaE, dfrA, dfrK | seb, sec, see | Non-producer |
SA48 | Animal | 398 | t899 | blaZ, fexA, aadD, vgaA, vgaE, dfrA, dfrK | n.d. | Non-producer |
SA49 | Animal | 398 | t899 | blaZ, tetL, tetM, norA, aacAaphD, aadD, vgaA, vgaE, dfrA, dfrK | sec, see | Non-producer |
SA50 | Animal | 398 | t011 | blaZ, tetM, norA, aadD, vgaE, dfrA | seb, see | Non-producer |
SA51 | Animal | 398 | t899 | blaZ, norA, aadD, vgaE, dfrA, dfrD, dfrK | sea, seb | Moderate |
SA52 | Animal | 398 | t899 | tetM, norA, aadD, vgaE, dfrA, dfrK | sec, see | Non-producer |
SA53 | Animal | 398 | t899 | blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK | seb, sed, see | Non-producer |
SA54 | Animal | 398 | t011 | blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK | see | Non-producer |
SA55 | Animal | 398 | t011 | blaZ, tetM, norA, vgaA, vgaE | see | Non-producer |
SA56 | Animal | 398 | t011 | blaZ, tetM, norA, vgaA, vgaE, dfrK | n.d. | Non-producer |
SA57 | Animal | 398 | t011 | blaZ, tetM, norA, aadD, vgaE, ermT | seb | Non-producer |
SA58 | Animal | 398 | t4474 | blaZ, tetM, norA, vgaA, vgaE, dfrA | sec, see | Non-producer |
SA59 | Animal | 398 | t4474 | blaZ, tetM, norA, vgaE, dfrA | n.d. | Weak |
SA60 | Animal | 398 | t011 | blaZ, tetM, norA, vgaA, vgaE, dfrA | see | Non-producer |
SA61 | Animal | 398 | t011 | blaZ, tetM, norA, vgaA, dfrA, dfrK | seb, sec, see | Weak |
SA62 | Animal | 398 | t899 | blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK | n.d. | Weak |
SA63 | Animal | 398 | t899 | blaZ, tetL, tetM, aacAaphD, aadD, vgaA, vgaE, dfrA, dfrK, ermT | sec | Moderate |
SA64 | Animal | 398 | t899 | blaZ, tetM, norA, dfrA, dfrK | seb, see | Moderate |
SA65 | Animal | 398 | t899 | blaZ, tetL, tetM, aacAaphD, aadD, vgaA, vgaE, dfrA, dfrK, ermT | n.d. | Weak |
SA66 | Animal | 398 | t4558 | blaZ, tetM, norA, fexA, aacAaphD, vgaA, vgaE, dfrA, dfrK | sea, sec, sed, see | Weak |
SA67 | Animal | 398 | t899 | blaZ, tetM, norA, vgaE, dfrA, dfrK | sec | Weak |
SA68 | Animal | 398 | t899 | blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK | n.d. | Weak |
SA69 | Animal | 398 | t899 | blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK | sec | Weak |
SA70 | Animal | 1 | t127 | blaZ, norA, aacAaphD, vgaA, vgaE, dfrA, dfrK, ermT | n.d. | Weak |
SA71 | Animal | 398 | t011 | blaZ, tetM, norA, aadD, vgaA, vgaE, dfrA, dfrD | sea | Weak |
SA72 | Animal | 398 | t011 | tetL, tetM, norA, aadD, vgaA, vgaE, dfrA, dfrK | sea | Non-producer |
SA73 | Animal | 398 | t011 | tetM, norA, aadD, vgaA, vgaE, dfrD, dfrK | sea | Weak |
SA74 | Animal | 398 | t011 | tetM, norA, aadD, vgaA, vgaE, dfrA, dfrD, dfrK | seb, see | Non-producer |
SA75 | Animal | 398 | t011 | tetL, tetM, norA, aadD, vgaA, vgaE, dfrA, dfrK | sea | Weak |
SA76 | Animal | 398 | t011 | tetM, norA, aadD, vgaE, dfrA, dfrD, dfrK | n.d. | Non-producer |
SA77 | Animal | 1 | t127 | blaZ, tetL, norA, aacAaphD, aadD, vgaA, vgaE, dfrA, ermT | sea | Weak |
SA78 | Animal | 1 | t127 | blaZ, tetL, norA, aacAaphD, aadD, vgaA, dfrA, dfrK, ermT | sea | Weak |
SA79 | Animal | 398 | t899 | tetM, norA, vgaE, dfrA | sea | Non-producer |
SA80 | Animal | 398 | t899 | blaZ, tetM, norA, vgaE, dfr | sea | Non-producer |
SA81 | Animal | 398 | t899 | blaZ, tetM, norA, vgaE, dfr | sea, seb | Non-producer |
SA82 | Animal | 398 | t899 | tetM, norA, vgaE, dfrA | sea, see | Non-producer |
SA83 | Animal | 398 | t011 | blaZ, tetM, norA, aadD, vgaE, dfrA, dfrD | see | Non-producer |
SA84 | Animal | 398 | t899 | blaZ, tetM, norA, vgaA, vgaE | see | Non-producer |
SA85 | Animal | 398 | t899 | blaZ, tetM, aadD, vgaA, vgaE, dfrD | seb | Weak |
SA86 | Animal | 398 | t011 | blaZ, tetM, norA, dfrA | sea | Non-producer |
SA87 | Animal | 398 | t011 | blaZ, tetM, norA, vgaE, dfrA | n.d. | Non-producer |
Genotype | Number of Isolates (%) |
---|---|
ST398/t899 | 33 (37.93%) |
ST398/t011 | 27 (31.03%) |
ST398/t1939 | 3 (3.45%) |
ST398/t034 | 1 (1.15%) |
ST398/t18494 | 3 (3.45%) |
ST398/t1200 | 2 (2.30%) |
ST398/t4474 | 2 (2.30%) |
ST398/t4558 | 1 (1.15%) |
ST1/t127 | 8 (9.20%) |
ST97/t1730 | 2 (2.30%) |
ST97/t4795 | 2 (2.30%) |
ST4894/t18494 | 1 (1.15%) |
ST30/t318 | 1 (1.15%) |
ST5422/t1730 | 1 (1.15%) |
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Rodríguez-López, P.; Filipello, V.; Di Ciccio, P.A.; Pitozzi, A.; Ghidini, S.; Scali, F.; Ianieri, A.; Zanardi, E.; Losio, M.N.; Simon, A.C.; et al. Assessment of the Antibiotic Resistance Profile, Genetic Heterogeneity and Biofilm Production of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from The Italian Swine Production Chain. Foods 2020, 9, 1141. https://doi.org/10.3390/foods9091141
Rodríguez-López P, Filipello V, Di Ciccio PA, Pitozzi A, Ghidini S, Scali F, Ianieri A, Zanardi E, Losio MN, Simon AC, et al. Assessment of the Antibiotic Resistance Profile, Genetic Heterogeneity and Biofilm Production of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from The Italian Swine Production Chain. Foods. 2020; 9(9):1141. https://doi.org/10.3390/foods9091141
Chicago/Turabian StyleRodríguez-López, Pedro, Virginia Filipello, Pierluigi Aldo Di Ciccio, Alessandra Pitozzi, Sergio Ghidini, Federico Scali, Adriana Ianieri, Emanuela Zanardi, Marina Nadia Losio, Ancuta Cezara Simon, and et al. 2020. "Assessment of the Antibiotic Resistance Profile, Genetic Heterogeneity and Biofilm Production of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from The Italian Swine Production Chain" Foods 9, no. 9: 1141. https://doi.org/10.3390/foods9091141