Antimicrobial Resistance of Lactic Acid Bacteria from Nono, a Naturally Fermented Milk Product
Abstract
:1. Introduction
2. Results
2.1. MIC Determination
2.2. Determination of Resistance Genes
2.3. In Vitro Conjugation Experiments for the Transfer of tet(S), tet(M), and aad(E) Genes
2.4. Determination of the Presence of the Resistance Genes in the Potential Transconjugants
3. Discussion
4. Material and Methods
4.1. Bacterial Isolates
4.2. Determination of Minimal Inhibitory Concentration (MIC) of the Isolates
4.3. Detection of Resistance Genes for Specific Antimicrobials
4.4. Screening Transferability of Resistance Genes (In Vitro Conjugation)
4.5. Confirmation of the Transconjugants Using MIC Determination and PCR
4.6. Determination of the Presence and Location of the Tetracycline and Streptomycin Genes in the Potential Transconjugants
4.7. Determination of the Presence of Transposons
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Antimicrobial | Isolate/MIC (µg/mL) a | ||||||
---|---|---|---|---|---|---|---|
Ent. thailandicus | S. infantarius | Lent. senioris | L. fermentum | Lact. delbrueckii subsp. indicus | Leuc. pseudomesenteriodes | S. thermophilus | |
Ampicillin | ≤0.12 s | ≤0.12 s | 0.5 s | 0.25 s | ≤0.12 s | 0.25 s | ≤0.12 s |
Ceftriaxone | <8 s | <8 s | <8 s | <8 s | <8 s | <8 s | <8 s |
Clindamycin | >2 r | ≤0.12 s | 2 r | ≤0.12 s | 2 r | ≤0.12 s | 2 s |
Ciprofloxacin | 4 s | 8 r | ≥16 r | ≥16 r | ≥16 r | 8 r | 2 s |
Daptomycin | >8 r | 8 r | >8 r | >8 r | >8 r | >8 r | 1 s |
Erythomycin | >4 r | 4 r | >4 r | >4 r | 1 s | 1 s | ≤0.25 s |
Gatifloxacin | 2 s | 2 s | 8 r | >8 r | 8 r | ≤2 s | ≤1 s |
Gentamicin | 64 r | 16 s | 32 r | 32 r | 64 r | 8 s | 16 s |
Levofloxacin | 8 r | 8 r | >8 r | >8 r | >8 r | 8 r | 2 s |
Linezolid | 4 s | 4 s | 4 s | 8 r | 2 s | 2 s | 2 s |
Oxacillin + 2% NaCl | 2 s | 2 s | 2 s | 1 s | ≤0.25 s | 0.5 s | ≤0.25 s |
Penicillin | ≤0.06 s | ≤0.06 s | 0.5 s | 0.12 s | ≤0.06 s | ≤0.06 s | >8 r |
Quinupristin/Dalfopristin | 4 s | ≤0.12 s | 4 s | 1 s | 1 s | 0.5 s | 2 s |
Rifampin | 2 s | ≤0.5 s | ≤0.5 s | ≤0.5 s | ≤0.5 s | ≤0.5 s | ≤0.5 s |
Streptomycin | 512 r | 64 s | 256 r | 256 r | 32 r | 64 s | >32 s |
Tetracycline | 64 r | 32 r | 16 r | 16 r | ≤2 s | ≤2 s | ≤2 s |
Trimethoprim/Sulfamethoxazole | >4/76 s | 2/28 s | >4/76 s | >4/76 s | >4/76 s | >4/76 s | >4/76 s |
Vancomycin | 2 s | ≤1 s | >128 r | >128 r | ≤1 s | >128 r | 16 r |
Antimicrobial | Isolate/Resistance Genes | ||||||
---|---|---|---|---|---|---|---|
Ent. thailandicus | S. infantarius | Lent. senioris | L. fermentum | Lact. delbrueckii subsp. indicus | Leuc. pseudomesenteriodes | S. thermophilus | |
Chloramphenicol | - | - | - | - | - | - | - |
Erythromycin | - | - | - | - | - | - | - |
Gentamicin | - | - | - | - | - | - | - |
Kanamycin | - | - | - | - | - | - | - |
Penicillin | - | - | - | - | - | - | - |
Streptomycin | aad(E) | - | - | - | - | - | - |
Tetracycline | tet(S)/tet(M) | tet(S)/tet(M) | - | - | - | - | - |
Vancomycin | - | - | - | - | - | - | - |
Isolate | Antimicrobial/MIC µg/mL | ||
---|---|---|---|
Tetracycline | Streptomycin | ||
Donor | Ent. thailandicus 52 | 64 r | 512 r |
Recipient | Ent. faecalis JH2-2 | <1 s | 512 r |
Transconjugants | T1 | 8 r | 128 r |
T2 | 8 r | 128 r | |
T3 | 8 r | 128 r | |
T4 | 8 r | 256 r | |
T5 | 32 r | 256 r | |
T6 | 32 r | 128 r | |
T7 | 16 r | 128 r | |
T8 | 8 r | 128 r |
Antimicrobial | Resistance Genes | Sequence (5′–3′) | Annealing Temperature (°C) |
---|---|---|---|
Tetracycline | tet(M) | 5′-GTTAAATAGTGTTCTTGGAG-3′ 5′-CTAAGATATGGCTCTAACAA-3′ | 45 °C |
tet(L) | 5′-GTTGCGCGCTATATTCCAAA-3′ 5′-TTAAGCAAACTCATTCCAGC-3′ | 54 °C | |
tet(S) | 5′-TGGAACGCCAGAGAGGTATT-3′ 5′-ACATAGACAAGCCGTTGACC-3′ | 55 °C | |
tet(Q) | 5′-ATGTTCAATATCGGTATCAATGA-3′ 5′-GCGGATATCACCTTGCTTC-3′ | 55 °C | |
tet(K) | 5′-TTAGGTGAAGGGTTAGGTCC-3′ 5′-GCAAACTCATTCCAGAAGCA-3′ | 55 °C | |
tet(O) | 5′-GATGGCATACAGGCACAGAC-3′ 5′-CAATATCACCAGAGCAGGCT-3′ | 55 °C | |
tet(W) | 5′-GCCATCTTGGTGATCTCC-3′ 5′-TGGTCCCCTAATACATCGTT-3′ | 55 °C | |
Kanamycin | aph(3″)-I | 5′-AACGTCTTGCTCGAGGCCGCG-3′ 5′-GGCAAGATCCTGGTATCGGTCTGCG-3′ | 68 °C |
aph(3″)-III | 5′-GCCGATGTGGATTGCGAAAA-3′ 5′-GCTTGATCCCCAGTAAGTCA-3′ | 52 °C | |
Gentamicin | ant(2″)-I | 5′-GGGCGCGTCATGGAGGAGTT-3′ 5′-TATCGCGACCTGAAAGCGGC-3′ | 67 °C |
aac(6′)aph(2″) | 5′-CCAAGAGCAATAAGGGCATA-3′ 5′-CACTATCATAACCACTACCG-3′ | 48 °C | |
aac(3″)IV | 5′-GTGTGCTGCTGGTCCACAGC-3′ 5′-AGTTGACCCAGGGCTGTCGC-3′ | 63 °C | |
Streptomycin | str(A) | 5′-CTTGGTGATAACGGCAATTC-3′ 5′-CCAATCGCAGATAGAAGGC-3′ | 55 °C |
str(B) | 5′-ATCGTCAAGGGATTGAAACC-3′ 5′-GGATCGTAGAACATATTGGC-3′ | 56 °C | |
Streptomycin | aad(A) | 5′-ATCCTTCGGCGCGATTTTG-3′ 5′-GCAGCGCAATGACATTCTTG-3′ | 56 °C |
aad(E) | 5′-ATGGAATTATTCCCACCTGA-3′ 5′-TCAAAACCCCTATTAAAGCC-3′ | 50 °C | |
Erythromycin | erm(A) | 5′-AAGCGGTAAAACCCCTCTGAG-3′ 5′-TCAAAGCCTGTCGGAATTGG-3′ | 55 °C |
erm(B) | 5′-CATTTAACGACGAAACTGGC-3′ 5′-GGAACATCTGTGGTATGGCG-3′ | 52 °C | |
erm(C) | 5′-CAAACCCGTATTCCACGATT-3′ 5′-ATCTTTGAAATCGGCTCAGG-3′ | 48 °C | |
Vancomycin | vanA | 5′-AACAACTTACGCGGCACT-3′ 5′-AAAGTGCGAAAAACCTTG-3′ | 55 °C |
vanB | 5′-GATATTCAAAGCTCCGCAGC-3′ 5′-TGATGGATGCGGAAG ATACC-3′ | 55 °C | |
vanX | 5′-TGCGATTTTGCGCTTCATTG-3′ 5′-ACTTGGGATAATTTCACCGG-3′ | 55 °C | |
Chloramphenicol | cmlA | 5′-TACTCGGATCCATGCTGGCC-3′ 5′-TCCTCGAAGAGCGCCATTGG-3′ | 65 °C |
cat501 | 5′-GGATATGAAATTTATCCCTC-3′ 5′-CAATCATACCCTATGAAT-3′ | 47 °C | |
catA1 | 5′-CGCCTGATGAATGCTCATCCG-3′ 5′-CCTGCCACTCATCGCAGTAC-3′ | 60 °C | |
Penicillin | blaZ | 5′-CAGTTCACATGCCAAAGAG-3′ 5′-TACACTCTTGGCGGTTTC-3′ | 54 °C |
Transposon | Tn916-1545 | 5′-GCGTGATTGTATCTCACT-3′ 5′-GACGCTCCTGTTGCTTCT-3′ | 50 °C |
Tn916 | 5′-GGCTGTCGCTGTAGGATAGAG-3′ 5′-GGGTACTTTTAGGGCTTAGT-3′ | 50 °C |
Bacteria * | Related Genes |
---|---|
Salmonella Rissen 7522486-1 | aph(3″)-I |
Enterococcus faecalis pEF418 | aad(E) |
Salmonella enterica #74 | aad(A) |
Staphylococcus aureus RN422 | erm(C) |
Enterococcus faecalis JH2-2 Tn1545 | erm(B) |
Staphylococcus aureus 1206 Tn554 | erm(A) |
Staphylococcus aureus pSTS9-like | tet(L) |
Staphylococcus aureus pT181-like | tet(K) |
Staphylococcus intermedius 2567 | tet(M) |
Escherichia coli pBT-1 | tet(Q) |
Listeria monocytogenes BM4210 pIP811 | tet(S) |
Escherichia coli K2 | ant(2″)-I |
Enterococcus faecalis JH2-1-5 | aph(3″)-III |
Escherichia coli TetW | tet(W) |
Enterococcus faecium BM4147 | vanA |
Enterococcus faecalis V583 | vanB |
Enterococcus faecium UW6605 | vanX |
Enterococcus faecium JH2-2 cat pip 501 | cat501 |
Escherichia coli K13 aac(3)-IV | |
No positive control | tet(O), str(A), str(B), aac(6′)aph(2″), cmlA, and catA1 |
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Obioha, P.I.; Anyogu, A.; Awamaria, B.; Ghoddusi, H.B.; Ouoba, L.I.I. Antimicrobial Resistance of Lactic Acid Bacteria from Nono, a Naturally Fermented Milk Product. Antibiotics 2023, 12, 843. https://doi.org/10.3390/antibiotics12050843
Obioha PI, Anyogu A, Awamaria B, Ghoddusi HB, Ouoba LII. Antimicrobial Resistance of Lactic Acid Bacteria from Nono, a Naturally Fermented Milk Product. Antibiotics. 2023; 12(5):843. https://doi.org/10.3390/antibiotics12050843
Chicago/Turabian StyleObioha, Promiselynda I., Amarachukwu Anyogu, Brigitte Awamaria, Hamid B. Ghoddusi, and Labia Irene I. Ouoba. 2023. "Antimicrobial Resistance of Lactic Acid Bacteria from Nono, a Naturally Fermented Milk Product" Antibiotics 12, no. 5: 843. https://doi.org/10.3390/antibiotics12050843