Identification of Risk Factors Associated with Resistant Escherichia coli Isolates from Poultry Farms in the East Coast of Peninsular Malaysia: A Cross Sectional Study
Abstract
:1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. Study Area
4.2. Study Design, Definitions, and Data Sources
- Intensive management system is defined as mainly concentrated and often mechanized operations that use controlled-environment systems to provide the ideal thermal environment for the poultry.
- Semi-intensive system is that which relies on natural airflow though the shed for ventilation.
- Extensive system is mainly pasture-based and land-based where birds in the household flock are typically housed overnight in the shelter and are let out in the morning to forage during the day.
- The criteria of the farm size included large-scale commercial farms that has more than ≥10,000 birds, medium-scale commercial farms that has more 5000–10,000, and small-scale farms where birds are often kept in single-age groups of >1000.
- A poor sewage system is defined as that which retains high volumes of wastewater with low flow rate, blackish appearance, and sewage smell odour as a result of composing agricultural waste—probably as leakage from nearby irrigated effluent which is used for agricultural land application along with the presence of food waste, green waste, plastic, and heavy materials.
- A good sewage system is that which has good drainage with no agricultural waste and relatively low heavy materials.
- Excellent swage system is that which has significant drainage, no agriculture, and heavy materials.
4.3. Microbiological Testing
4.4. Antimicrobial Susceptibility Testing
4.5. PCR
4.5.1. DNA Extraction of Escherichia Coli Isolates
4.5.2. Primers and PCR Assay for Specific Genes
4.6. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Risk Factors | Samples Tested | Affected (%) | p-Value |
---|---|---|---|
Age | 0.511 | ||
Young | 187 | 123 (65.8%) | |
Adult | 184 | 115 (62.5%) | |
Management system | 0.541 | ||
Intensive | 187 | 115 (61.5%) | |
Semi-intensive | 158 | 105 (66.5%) | |
Mixed | 26 | 18 (69.2%) | |
Production system | 0.278 | ||
Broiler | 212 | 129 (60.8%) | |
Layer | 53 | 35 (66%) | |
Mixed | 106 | 74 (69.8%) | |
State | 0.012 | ||
Kelantan | 158 | 115 (72.8%) | |
Terengganu | 80 | 46 (57.5%) | |
Pahang | 133 | 77 (57.9%) | |
Districts | 0.001 | ||
Bachok | 52 | 36 (69.2%) | |
Kota Bharu | 26 | 18 (69.2%) | |
Machang | 28 | 24 (85.7%) | |
Pasir Mas | 26 | 14 (53.8%) | |
Jeli | 26 | 23 (88.5%) | |
Kuantan | 79 | 49 (62%) | |
Pekan | 54 | 28 (51.9%) | |
kuala terengganu | 26 | 20 (76.9%) | |
Marang | 54 | 26 (48.1%) | |
Sample source | 0.001 | ||
Cloaca swab | 259 | 172 (66.4%) | |
Faecal Sample | 84 | 58 (69%) | |
Sewage | 14 | 5 (35.7%) | |
Tape Water | 14 | 3 (21.4%) | |
Farm size | 0.013 | ||
Small | 104 | 77 (74%) | |
Medium | 188 | 119 (63.2%) | |
Large | 79 | 42 (53.2%) | |
Origin of the poultry | 0.005 | ||
Local | 26 | 18 (69.2%) | |
Imported | 133 | 71 (53.4%) | |
Both | 212 | 149 (70.3%) |
Risk Factors | No Antimicrobial Resistance n = 137 | Resistance to at least One Antimicrobial n = 234 | p-Value |
---|---|---|---|
Age | 0.44 | ||
Young | 65 (47.4%) | 122 (52.1%) | |
Adult | 72 (52.6%) | 112 (47.9%) | |
Origin of the poultry | 0.01 | ||
Local | 10 (7.3%) | 16 (6.8%) | |
Imported | 63 (46%) | 70 (29.9%) | |
Both | 64 (46.7%) | 148 (63.2%) | |
Management system | 0.18 | ||
Intensive | 80 (58.4%) | 115 (49.1%) | |
Semi-intensive | 47 (34.3%) | 103 (44%) | |
Mixed | 10 (7.3%) | 16 (6.8%) | |
Production system | 0.21 | ||
Broiler | 86 (62.8%) | 125 (53.4%) | |
Layer | 18 (13.1%) | 37 (15.8%) | |
Mixed | 33 (24.1%) | 72 (30.8%) | |
Farm size | 0.02 | ||
Small | 29 (21.2%) | 75 (32.1%) | |
Medium | 70 (51.1%) | 117 (50%) | |
Large | 38 (27.7%) | 42 (17.9%) | |
Source of sample | <0.001 | ||
Cloacal swab | 89 (65%) | 170 (72.6%) | |
Faecal sample | 26 (19%) | 58 (24.8%) | |
sewage | 9 (6.6%) | 5 (2.1%) | |
Tap water | 13 (9.5%) | 1 (0.4%) | |
Water source | 0.02 | ||
Surface water | 37 (27%) | 69 (29.5%) | |
Bond water | 61 (44.5%) | 72 (30.8%) | |
Pump water | 39 (28.5%) | 93 (39.7%) | |
Sewage system | 0.60 | ||
Excellent | 38 (27.7%) | 71 (30.3%) | |
Good | 82 (59.9%) | 128 (54.7%) | |
Poor | 17 (12.4%) | 35 (15%) | |
Feed source | 0.53 | ||
Endogenous | 50 (36.5%) | 82 (35%) | |
Exogenous | 75 (54.7%) | 138 (59%) | |
Other | 12 (8.8%) | 14 (6%) |
Antimicrobials | Cloacal n = 259 | Faecal n = 84 | Sewage n = 14 | Tape Water n = 14 | p-Value |
---|---|---|---|---|---|
No identified resistance | <0.001 | ||||
No antimicrobial resistance | 89 (34.4%) | 26 (31%) | 9 (64.3%) | 13 (92.9%) | |
Resistance to at least one antimicrobial | 170 (65.6%) | 58 (69%) | 5 (35.7%) | 1 (7.1%) | |
Antimicrobial class resistance | 0.003 | ||||
No antimicrobial resistance | 89 (34.4%) | 26 (31%) | 9 (64.3%) | 13 (92.9%) | |
Resistant to 1 class | 4 (1.5%) | 1 (1.2%) | 0 (0%) | 0 (0%) | |
Resistant to 2 classes | 13 (5%) | 3 (3.6%) | 2 (14.3%) | 0 (0%) | |
Resistant to 3 classes | 46 (17.8%) | 19 (22.6%) | 2 (14.3%) | 1 (7.1%) | |
Resistant to 4 classes | 74 (28.6%) | 19 (22.6%) | 1 (7.1%) | 0 (0%) | |
Resistant to 5 or more classes | 33 (12.7%) | 16 (19%) | 0 (0%) | 0 (0%) | |
Source of antimicrobials | 1 | ||||
Drug supplier | 112 (43.2%) | 36 (42.9%) | 6 (42.9%) | 6 (42.9%) | |
Feed store | 147 (56.8%) | 48 (57.1%) | 8 (57.1%) | 8 (57.1%) | |
Tetracyclines | <0.001 | ||||
Not resistant | 93 (35.9%) | 26 (31%) | 9 (64.3%) | 13 (92.9%) | |
Resistant | 166 (64.1%) | 58 (69%) | 5 (35.7%) | 1 (7.1%) | |
Penicillins | 0.048 | ||||
Not resistant | 151 (58.3%) | 47 (56%) | 10 (71.4%) | 13 (92.9%) | |
Resistant | 108 (41.7%) | 37 (44%) | 4 (28.6%) | 1 (7.1%) | |
Aminoglycosides | 0.246 | ||||
Not resistant | 219 (84.6%) | 68 (81%) | 13 (92.9%) | 14 (100%) | |
Resistant | 40 (15.4%) | 16 (19%) | 1 (7.1%) | 0 (0%) | |
Quinolones | 0.002 | ||||
Not resistant | 142 (54.8%) | 49 (58.3%) | 13 (92.9%) | 13 (92.9%) | |
Resistant | 117 (45.2%) | 35 (41.7%) | 1 (7.1%) | 1 (7.1%) | |
Sulfonamides | <0.001 | ||||
Not resistant | 104 (40.2%) | 29 (34.5%) | 11 (78.6%) | 14 (100%) | |
Resistant | 155 (59.8%) | 55 (65.5%) | 3 (21.4%) | 0 (0%) | |
Cephelosporins | 0.645 | ||||
Not resistant | 246 (95%) | 79 (94%) | 14 (100%) | 14 (100%) | |
Resistant | 13 (5%) | 5 (6%) | 0 (0%) | 0 (0%) | |
Other classes | 0.025 | ||||
Not resistant | 224 (86.5%) | 65 (77.4%) | 14 (100%) | 14 (100%) | |
Resistant | 35 (13.5%) | 19 (22.6%) | 0 (0%) | 0 (0%) |
Variables | OR | 2.5% | 97.5% | Pr (>|z|) | |
---|---|---|---|---|---|
Farms | |||||
Farm 1 | 10.20 | 2.95 | 42.89 | <0.001 | *** |
Farm 2 | 2.72 | 0.91 | 8.53 | 0.07 | . |
Farm 3 | 13.03 | 3.46 | 65.56 | <0.001 | *** |
Farm 4 | 1.98 | 0.66 | 6.09 | 0.22 | |
Farm 5 | 2.31 | 0.78 | 7.18 | 0.134 | |
Farm 6 | 7.14 | 2.16 | 27.16 | 0.002 | ** |
Farm 7 | 2.89 | 0.97 | 9.02 | 0.05 | . |
Farm 8 | Ref | Ref | Ref | Ref | |
Farm 9 | 4.61 | 1.48 | 15.63 | 0.01 | * |
Farm 10 | 3.82 | 1.25 | 12.52 | 0.02 | * |
Farm 11 | 1.16 | 0.38 | 3.53 | 0.78 | |
Farm 12 | 1.06 | 0.34 | 3.25 | 0.91 | |
Farm 13 | 5.66 | 1.78 | 20.13 | 0.004 | ** |
Farm 14 | 2.26 | 0.77 | 6.87 | 0.13 | |
Sample source | |||||
Cloaca swab | 24.83 | 4.82 | 454.74 | 0.002 | ** |
Faecal sample | 29.0 | 5.35 | 540.73 | 0.001 | ** |
Sewage | 7.22 | 0.95 | 151.21 | 0.09 | . |
Tap water | Ref | Ref | Ref | Ref | |
Age | |||||
Young | 1.21 | 0.79 | 1.84 | 0.38 | |
Adult | Ref | Ref | Ref | Ref | |
Poultry origin | |||||
Local | 1.44 | 0.61 | 3.50 | 0.41 | |
Both | 2.08 | 1.32 | 3.26 | 0.001 | ** |
Imported | Ref | Ref | Ref | Ref | |
Management system | |||||
Semi-intensive | 1.52 | 0.97 | 2.39 | 0.06 | . |
Mixed | 1.11 | 0.48 | 2.65 | 0.80 | |
Intensive | Ref | Ref | Ref | Ref | |
Production system | |||||
Layer | 1.41 | 0.76 | 2.69 | 0.27 | |
Broiler | Ref | Ref | Ref | 0.11 | |
Mixed | 1.50 | 0.91 | 2.48 | Ref | |
Farm size | |||||
Small | 2.33 | 1.27 | 4.35 | 0.001 | ** |
Medium | 1.51 | 0.88 | 2.57 | 0.125 | |
Large | Ref | Ref | Ref | Ref | |
Water source | |||||
Surface water | 1.57 | 0.93 | 2.68 | 0.08 | . |
Pump water | 2.02 | 1.22 | 3.36 | 0.01 | ** |
Bond water | |||||
Sewage system | |||||
Excellent | 0.91 | 0.44 | 1.81 | 0.786 | |
Good | 0.75 | 0.39 | 1.42 | 0.398 | |
Poor | Ref | Ref | Ref | Ref |
OR | 2.5% | 97.5% | Pr (>|z|) | ||
---|---|---|---|---|---|
Cloaca swab | 26.50 | 5.08 | 487.69 | 0.001 | ** |
Feacal sample | 30.92 | 5.63 | 579.63 | 0.001 | ** |
Sewage | 7.43 | 0.96 | 156.87 | 0.09 | . |
Farm size Small | 2.50 | 1.33 | 4.77 | 0.004 | ** |
Farm size medium | 1.55 | 0.89 | 2.67 | 0.114 |
Antimicrobial Class/Agent | Resistance Gene | % Isolates | Total # Tested |
---|---|---|---|
Gentamicin | aac(3)-IV | 12 (85.7%) | 14 |
Tetracyclines | tet(A), tet(B) | 9 (64.2%) | 14 |
Chloramphenicol | catA1 | 2 (14.2%) | 14 |
Sulfonamides | sul1 | 14 (100%) | 14 |
β-Lactams | blaSHV | 6 (42.8%) | 14 |
Trimethoprim | dhfrI | 4 (28.5%) | 14 |
Genes | Primer Sequence(5′ to 3′) | PCR Condition | Product Size | References |
---|---|---|---|---|
β-Lactams | F- CTATCGCCAGCAGGATCTGG R- ATTTGCTGATTTCGCTCGGC | 3 min at 95 °C; 35 cycles of 1 min at 94 °C, 90 s at 55 °C and 1 min at 72 °C; 10 min at 72 °C | 543 | [16] |
Gentamicin aac(3)-IV | F-CTTCAGGATGGCAAGTTGGT R-TCATCTCGTTCTCCGCTCAT | 3 min at 95 °C; 35 cycles of 1 min at 94 °C, 90 s at 55 °C and 1 min at 72 °C; 10 min at 72 °C | 286 | [17] |
Sulfonamide sul1 | F- ACTGCAGGCTGGTGGTTATG R- ACCGAGACCAATAGCGGAAG | 3 min at 95 C; 35 cycles of 1 min at 94 C, 90 s at 55 °C and 1 min at 72 °C; 10 min at 72 °C | 271 | [8] |
Tetracycline tet(A) | F-CCTCAATTTCCTGACGGGCT R-GGCAGAGCAGGGAAAGGAAT | 3 min at 95 °C; 35 cycles of 1 min at 94 C, 90 s at 55 °C and 1 min at 72 °C; 10 min at 72 °C | 712 | [18] |
Tetracycline tet(B) | F-ACCACCTCAGCTTCTCAACG R-GTAAAGCGATCCCACCACCA | 3 min at 95 °C; 35 cycles of 1 min at 94 C, 90 s at 55 °C and 1 min at 72 °C; 10 min at 72 °C | 586 | [18] |
Chloramphenicol catA1 | F- GAAAGACGGTGAGCTGGTGA R- TAGCACCAGGCGTTTAAGGG | 3 min at 95 °C; 35 cycles of 1 min at 94 °C, 90 s at 55 °C and 1 min at 72 °C; 10 min at 72 °C | 473 | [8] |
Trimethoprim dhfrI | F-AAGAATGGAGTTATCGGGAATG R-GGGTAAAAACTGGCCTAAAATTG | 15 min at 95 °C; 30 cycles of 30 s at 94 °C; 30 s at 58 °C; 1 min at 72 °C; 10 min 72 °C. | 391 | [8] |
Ampicillin CITM | F-TGGCCAGAACTGACAGGCAAA R-TTTCTCCTGAACGTGGCTGGC | 15 min at 95 °C; 30 cycles of 30 s at 94 °C; 30 s at 58 °C; 1 min at 72 °C; 10 min 72 °C. | 462 | [8] |
E.coli | F-TGACGTTACCCGCAGAAGAA R- CTCCAATCCGGACTACGACG | 3 min at 95 °C; 35 cycles of 15s at 95 °C, 90 s at 55 °C and 15s at 72 °C; 10 min at 72 °C | 832 | [19] |
O157 | F-GTGTCCATTTATACGGACATCCATG R-CCTATAACGTCATGCCAATATTGCC | 2 min at 94 °C; 35 cycles of 30s at 94 °C, 30 s at 55 °C and 30s at 72 °C; 5 min at 72 °C | 292 | [20] |
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Elmi, S.A.; Simons, D.; Elton, L.; Haider, N.; Abdel Hamid, M.M.; Shuaib, Y.A.; Khan, M.A.; Othman, I.; Kock, R.; Osman, A.Y. Identification of Risk Factors Associated with Resistant Escherichia coli Isolates from Poultry Farms in the East Coast of Peninsular Malaysia: A Cross Sectional Study. Antibiotics 2021, 10, 117. https://doi.org/10.3390/antibiotics10020117
Elmi SA, Simons D, Elton L, Haider N, Abdel Hamid MM, Shuaib YA, Khan MA, Othman I, Kock R, Osman AY. Identification of Risk Factors Associated with Resistant Escherichia coli Isolates from Poultry Farms in the East Coast of Peninsular Malaysia: A Cross Sectional Study. Antibiotics. 2021; 10(2):117. https://doi.org/10.3390/antibiotics10020117
Chicago/Turabian StyleElmi, Sharifo Ali, David Simons, Linzy Elton, Najmul Haider, Muzamil Mahdi Abdel Hamid, Yassir Adam Shuaib, Mohd Azam Khan, Iekhsan Othman, Richard Kock, and Abdinasir Yusuf Osman. 2021. "Identification of Risk Factors Associated with Resistant Escherichia coli Isolates from Poultry Farms in the East Coast of Peninsular Malaysia: A Cross Sectional Study" Antibiotics 10, no. 2: 117. https://doi.org/10.3390/antibiotics10020117