Multidrug-Resistance of Vibrio Species in Bivalve Mollusks from Southern Thailand: Isolation, Identification, Pathogenicity, and Their Sensitivity toward Chitooligosaccharide-Epigallocatechin-3-Gallate Conjugate
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemical and Microbial Media
2.2. Sample Collection and Vibrio Isolation
2.3. Classification and Identification of Vibrio
2.4. Pathogenicity Detection of Vibrio Isolates
2.5. Biofilm Formation
2.6. Detection of Virulence Genes
2.7. Antibiotic Susceptibility
2.8. Bacterial Identification
2.9. Effect of COS-EGCG Conjugate on Inhibition of MDR Vibrio Strains
2.9.1. Preparation of COS-EGCG Conjugate
2.9.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
2.9.3. Scanning Electron Microscopy (SEM)
2.10. Statistical Analysis
3. Results and Discussion
3.1. Morphological Identification of Vibrio spp. Colonies
3.2. Hemolysis
3.3. Presence of Virulence Gene
3.4. Biofilm Formation
3.5. Antibiotic Resistance of Selected Isolates
3.6. Molecular Identification Using 16s rRNA Gene Sequencing
3.7. Antimicrobial Activity of COS-EGCG Conjugate against Multidrug-Resistant (MDR) Vibrio Strains
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target Genes | Primers | Sequence (5’-3’) | Amplicon Size (bp) | References |
---|---|---|---|---|
tdh | tdh-F | GTAAAGGTCTCTGACTTTTGGAC | 269 | Siddique et al. (2021) [25] |
tdh-R | TGGAATAGAACCTTCATCTTCACC | |||
tlh | tlh-F | AAAGCGGATTATGCAGAAGCACTG | 450 | |
tlh-R | GCTACTTTCTAGCATTTTCTCTGC | |||
trh | trh-F | TTGGCTTCGATATTTTCAGTATCT | 500 | |
trh-R | CATAACAAACATATGCCCATTTCCG | |||
16s rRNA | 27F | AGAGTTTGATCCTGGCTCAG | 1400 | Palamae et al. (2022) [14] |
1429R | GGTTACCTTGTTACGACTT |
TCBS | HiCromeTM Vibrio | MALDI Biotyper® Analysis | BL | BB | M | Total of Each Character |
---|---|---|---|---|---|---|
Yellow | Creamy white | V. navarrensis | 82 | 44 | 12 | 138 |
Yellow | Indigo/Purple | V. fluvialis | 49 | 0 | 15 | 64 |
Yellow | Light green | V. alginolyticus | 123 | 80 | 7 | 210 |
Green | Light green | V. parahaemolyticus | 73 | 41 | 47 | 161 |
Green | Purple | V. vunificus | 14 | 22 | 0 | 36 |
Green | Creamy white | V. harveyi | 16 | 21 | 3 | 40 |
Total | 649 |
Vibrio Strains | Species |
---|---|
BL2, BL48, BL56, BL87, BL95, BL99, BB73, BB103, M69, BL133 | Vibrio alginolyticus |
BL105, M34, M68, M105 | Vibrio navarrensis |
BL186 | Vibrio fluvialis |
BL18, BL82, BL84, BL85, BL90, BL92 | Vibrio parahaemolyticus |
Antibiotics | Concentration (µg/mL) | Vibrio Strains | |||||||
---|---|---|---|---|---|---|---|---|---|
BL2, BL18, BL48, BL82, BL87, BL90, BL92, BL95, BL133, M68, M69 | BL56, BL84, BL85, BB103 | BB73 | BL99 | BL105 | BL186 | M34 | M105 | ||
AN | 8–32 | S | S | S | S | S | S | S | S |
AMC | 4/2–16/8 | S | S | S | S | I | R | I | I |
AM | 8–16 | NI | NI | NI | NI | NI | NI | NI | NI |
SAM | 4/2–16/8 | S | S | S | S | I | R | I | I |
FEP | 1–32 | S | S | S | S | S | S | I | S |
CTX | 1–32 | S | S | S | R | R | R | R | R |
FOX | 4–16 | NI | NI | NI | NI | NI | NI | NI | NI |
CAZ | 1–32 | S | S | S | S | S | S | S | S |
CRO | 0.5–32 | NI | NI | NI | NI | NI | NI | NI | NI |
CXM | 8–16 | S | I | NM | R | R | R | R | R |
CIP | 0.06–2 | S | S | S | S | S | S | S | S |
CL | 1–8 | NI | NI | NI | NI | NI | NI | NI | NI |
DOR | 0.5–16 | NI | NI | NI | NI | NI | NI | NI | NI |
ETP | 0.5–4 | NI | NI | NI | NI | NI | NI | NI | NI |
GM | 2–8 | S | S | S | S | S | S | S | S |
IPM | 0.5–16 | S | S | S | S | S | S | S | S |
LVX | 0.06–8 | S | S | S | S | S | S | S | S |
MEM | 0.5–16 | S | S | S | S | S | R | S | S |
NET | 8–16 | NI | NI | NI | NI | NI | NI | NI | NI |
TZP | 8/4–64/4 | S | S | S | S | S | S | S | S |
SXT | 1/19–4/76 | S | S | S | S | S | R | S | S |
Vibrio Strains | MIC (µg/mL) | MBC (µg/mL) | MBC/MIC Ratio |
---|---|---|---|
Vibrio neocaledonicus BL99 | 128 | 128 | 1 |
Vibrio cidicii BL105 | 64 | 128 | 2 |
Vibrio fluvialis BL186 | 64 | 64 | 1 |
Vibrio cidicii M34 | 128 | 128 | 1 |
Vibrio cidicii M105 | 64 | 64 | 1 |
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Sharma, M.H.; Palamae, S.; Yingkajorn, M.; Benjakul, S.; Singh, A.; Buatong, J. Multidrug-Resistance of Vibrio Species in Bivalve Mollusks from Southern Thailand: Isolation, Identification, Pathogenicity, and Their Sensitivity toward Chitooligosaccharide-Epigallocatechin-3-Gallate Conjugate. Foods 2024, 13, 2375. https://doi.org/10.3390/foods13152375
Sharma MH, Palamae S, Yingkajorn M, Benjakul S, Singh A, Buatong J. Multidrug-Resistance of Vibrio Species in Bivalve Mollusks from Southern Thailand: Isolation, Identification, Pathogenicity, and Their Sensitivity toward Chitooligosaccharide-Epigallocatechin-3-Gallate Conjugate. Foods. 2024; 13(15):2375. https://doi.org/10.3390/foods13152375
Chicago/Turabian StyleSharma, Mruganxi Harshad, Suriya Palamae, Mingkwan Yingkajorn, Soottawat Benjakul, Avtar Singh, and Jirayu Buatong. 2024. "Multidrug-Resistance of Vibrio Species in Bivalve Mollusks from Southern Thailand: Isolation, Identification, Pathogenicity, and Their Sensitivity toward Chitooligosaccharide-Epigallocatechin-3-Gallate Conjugate" Foods 13, no. 15: 2375. https://doi.org/10.3390/foods13152375