Characterization of Clostridium Perfringens Isolates Collected from Three Agricultural Biogas Plants over a One-Year Period
Abstract
:1. Introduction
2. Materials and Methods
2.1. Biogas Plants and Sample Collection
2.2. Isolation of C. perfringens
2.3. DNA Extraction
2.4. Determination of the Toxin Genotype and Search for Antibiotic Resistance Genes
2.5. Testing Antimicrobial Susceptibility
2.6. Statistical Analysis
3. Results
3.1. Toxinotyping of C. perfringens Isolates
3.2. Tetracycline and Erythromycin Resistance Genes of C. perfringens Isolates
3.3. Antimicrobial Susceptibility of C. perfringens Isolates
4. Discussion
4.1. Toxinotyping of C. perfringens Isolates
4.2. Tetracycline and Erythromycin Resistance Genes of C. perfringens Isolates
4.3. Antimicrobial Susceptibility of C. perfringens Isolates
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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BGP1 | BGP2 | BGP3 | |||||
---|---|---|---|---|---|---|---|
Matrix | Manure | Digestate | Dairy Manure | Poultry Manure | Digestate | Manure | Digestate |
Nb isolates | 24 | 21 | 25 | 14 | 19 | 29 | 25 |
Gene | Primers | Sequences (5′-3′) | Amplicon Size (bp) | Reference |
---|---|---|---|---|
cpa | CPA5L | AGTCTACGCTTGGGATGGAA | 900 | [35] |
CPA5R | TTTCCTGGGTTGTCCATTTC | |||
cpb | CPBL | TCCTTTCTTGAGGGAGGATAAA | 611 | [35] |
CPBR | TGAACCTCCTATTTTGTATCCCA | |||
iap | CPIL | AAACGCATTAAAGCTCACACC | 293 | [35] |
CPIR | CTGCATAACCTGGAATGGCT | |||
etx | CPETXL | TGGGAACTTCGATACAAGCA | 396 | [35] |
CPEXTR | TTAACTCATCTCCCATAACTGCAC | |||
cpe | CPEL | GGGGAACCCTCAGTAGTTTCA | 506 | [35] |
CPER | ACCAGCTGGATTTGAGTTTAATG | |||
cpb2 | CPB2L | CAAGCAATTGGGGGAGTTTA | 200 | [35] |
CPB2R | GCAGAATCAGGATTTTGACCA | |||
netB | AKP78 | GCTGGTGCTGGAATAAATGC | 383 | [34] |
AKP79 | TCGCCATTGAGTAGTTTCCC | |||
erm(Q) | GE 350 | GAAGAGTTAAATYCACCAACTGA | 84 | This study |
GE 352 | ACTCTCTCTAGGTATTCCCA | |||
tetA(P) | GE 353 | TGTAGCACAGATTGTATGGGGA | 124 | This study |
GE 355 | CCCTGCTTGTGCTCCCTTTA | |||
tetB(P) | GE 359 | TTTTGGGCGACAGTAGGCTT | 90 | This study |
GE 361 | TGGCAATGACCCTACTGAAACA | |||
tet(M) | GE 362 | GCTATTGCCACAGAGAGAGAGA | 127 | This study |
GE 364 | CGGGTCACTGTCGGAGATTT |
Type (Gene) | BGP1 n (%) | BGP2 n (%) | BGP3 n (%) | Total n (%) | |||||
---|---|---|---|---|---|---|---|---|---|
Manure n = 23 | Digestate n = 20 | Dairy Manure n = 19 | Poultry Manure n = 14 | Digestate n = 14 | Manure n = 24 | Digestate n = 24 | Manure n = 80 | Digestate n = 58 | |
A (cpa) | 6 (26.1) | 3 (15) | 15 (78.9) | 12 (85.7) | 8 (57.1) | 6 (25) | 6 (25) | 39 (67.2) | 17(21.3) |
A (cpa + cpb2) | 13 (56.5) | 15 (75) | 2(10.5) | 2(14.3) | 5 (35.7) | 8 (33.3) | 7 (29.2) | 25 (43.1) | 27(33.8) |
G (netB) | 2 (8.7) | 1 (5) | 2(10.5) | - | - | 4 (16.7) | 1 (4.2) | 8 (13.8) | 2(2.5) |
G (netB + cpb2) | 2 (8.7) | 1 (5) | - | - | 1 (7.1) | 3 (12.5) | 6 (25) | 5 (8.6) | 8(10) |
C (cpb) | -1 | - | - | - | - | - | 1 (4.2) | - | 1 (1.3) |
C (cpb + cpb2) | - | - | - | - | - | 1 (4.2) | 3 (12.5) | 1 (1.7) | 3(3.8) |
D (etx) | - | - | - | - | - | 1 (4.2) | - | 1 (1.7) | - |
D (etx + cpb2) | - | - | - | - | - | 1 (4.2) | - | 1 (1.7) | - |
Gene | BGP1 n (%) | BGP2 n (%) | BGP3 n (%) | Total n (%) | |||||
---|---|---|---|---|---|---|---|---|---|
Manure n = 23 | Digestate n = 20 | Dairy Manure n = 19 | Poultry Manure n = 14 | Digestate n = 14 | Manure n = 24 | Digestate n = 24 | Manure n = 80 | Digestate n = 58 | |
tetA(P) | 12 (52.2) | 13 (65) | 2(10.5) | - | 2 (14.3) | 10 (41.7) | 5 (20.8) | 24 (30) | 20 (34.5) |
tetA(P) + tetB(P) | 9 (39.2) | 6 (30) | 10 (52.6) | - | 8 (57.1) | 5 (20.8) | 10 (41.7) | 24 (30) | 24 (41.4) |
tetA(P) + erm(Q) | 1 (4.3) | - | - | - | - | - | 1 (4.2) | 1 (1.25) | 1 (1.7) |
tetB(P) + erm(Q) | - 1 | - | - | - | - | 1 (4.2) | - | 1 (1.25) | - |
tetA(P) + tetB(P) + erm(Q) | 1 (4.3) | 1 (5) | - | - | - | - | 4 (16.7) | 1 (1.25) | 5 (8.6) |
tet(M) | - | - | - | - | - | - | - | - | - |
no ARG 2 | - | - | 7 (36.8) | 14 (100) | 4 (28.6) | 8 (33.3) | 4 (16.7) | 29 (36.3) | 8 (13.8) |
Antimicrobial Agents | Origin of Isolates | ||||||||
---|---|---|---|---|---|---|---|---|---|
Manure (n = 80) | Digestate (n = 58) | ||||||||
MIC50 | MIC90 | Min | Max | MIC50 | MIC90 | Min | Max | ||
β-lactams | Penicillin | ≤0.125 | 1 | ≤0.125 | >8 | ≤0.125 | 0.5 | ≤0.125 | 1 |
Ampicillin | ≤0.25 | ≤0.25 | ≤0.25 | >16 | ≤0.25 | ≤0.25 | ≤0.25 | 2 | |
Ceftiofur | ≤0.25 | 1 | ≤0.25 | 4 | ≤0.25 | 2 | ≤0.25 | 2 | |
Macrolides | Tartrate Tylosin | ≤0.5 | 4 | ≤0.5 | >32 | ≤0.5 | 8 | ≤0.5 | 16 |
Tilmicosin | ≤4 | 8 | ≤4 | >64 | ≤4 | ≤4 | ≤4 | >64 | |
Tulathromycin | 64 | >64 | 16 | >64 | 64 | >64 | 32 | >64 | |
Lincosamides | Clindamycin | 2 | 2 | ≤0.25 | >16 | 2 | 4 | ≤0.25 | >16 |
Fluoroquinolones | Enrofloxacin | 0.25 | >2 | 0.25 | >2 | 0.50 | >2 | ≤0.125 | >2 |
Danofloxacin | 1 | >1 | 0.25 | >1 | 1 | >1 | 0.5 | >1 | |
Pleuromutilins | Tiamulin | 4 | 8 | ≤0.5 | >32 | 4 | 16 | ≤0.5 | >32 |
Phenicols | Florfenicol | 1 | 2 | 0.5 | >8 | 1 | 1 | ≤0.25 | 2 |
Tetracyclines | Chlortetracycline | ≤0.5 | 8 | ≤0.5 | >8 | 1 | 4 | ≤0.5 | 8 |
Oxytetracycline | 2 | >8 | ≤0.5 | >8 | 8 | >8 | ≤0.5 | >8 |
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Derongs, L.; Druilhe, C.; Ziebal, C.; Le Maréchal, C.; Pourcher, A.-M. Characterization of Clostridium Perfringens Isolates Collected from Three Agricultural Biogas Plants over a One-Year Period. Int. J. Environ. Res. Public Health 2020, 17, 5450. https://doi.org/10.3390/ijerph17155450
Derongs L, Druilhe C, Ziebal C, Le Maréchal C, Pourcher A-M. Characterization of Clostridium Perfringens Isolates Collected from Three Agricultural Biogas Plants over a One-Year Period. International Journal of Environmental Research and Public Health. 2020; 17(15):5450. https://doi.org/10.3390/ijerph17155450
Chicago/Turabian StyleDerongs, Lorine, Céline Druilhe, Christine Ziebal, Caroline Le Maréchal, and Anne-Marie Pourcher. 2020. "Characterization of Clostridium Perfringens Isolates Collected from Three Agricultural Biogas Plants over a One-Year Period" International Journal of Environmental Research and Public Health 17, no. 15: 5450. https://doi.org/10.3390/ijerph17155450