Background
Methods
Bacterial isolates
Determination of minimal inhibitory concentration
Antimicrobial substance | Group of antimicrobial agent | Diluent to prepare stock solution | Range of concentration (µg/ml) |
---|---|---|---|
Ampicilin | β-Lactams | BR IIa
| 0.125–64 |
Tetracycline | Tetracyclines | 0.1 M HCl | 1–512 |
Doxycycline | 0.5–256 | ||
Erythromycin | Macrolides | Methanol 99.9% | 0.125–64 |
Tylosin | H2O | 0.125–64 | |
Lincomycin | Lincosamides | 2–1024 | |
Streptomycin | Aminoglicosides | H2O | 2–1024 |
Gentamicin | BR IIa
| 1–512 | |
Neomycin | BR IIa
| 1–512 | |
Chloramphenicol | Chloramphenicol | Methanol 50% | 0.5–256 |
Tiamulin | Semisynthetic derivative of pleuromutilin | – | 0.5–256 |
Enrofloxacin | Fluoroquinolones | – | 0.5–512 |
Flumequine | 0.1 M NH4OH | 2–1024 |
Detection of resistance genes
Determining resistance to | Target gene | Primer sequence (5′–3′) | Amplicon size (bp) | Annealing temperature (°C) | Reference |
---|---|---|---|---|---|
Tetracyclines |
tetM
| GTG GAC AAA GGT ACA ACG AG CGG TAA AGT TCG TCA CAC AC | 406 | 60 | [14] |
tetK
| GAT CAA TTG TAG CTT TAG GTG AAG G TTT TGT TGA TTT ACC AGG TAC CAT T | 155 | 60 | ||
tetL
| TGG TGG AAT GAT AGC CCA TT CAG GAA TGA CAG CAC GCT AA | 229 | 60 | ||
tetO
| AAC TTA GGC ATT CTG GCT CAC TCC CAC TGT TCC ATA TCG TCA | 515 | 60 | ||
tetQ
| TTA TAC TTC CTC CGG CAT CG ATC GGT TCG AGA ATG TCC AC | 904 | 55 | [64] | |
tetW
| GAG AGC CTG CTA TAT GCC AGC GGG CGT ATC CAC AAT GTT AAC | 168 | 64 | [65] | |
Macrolides |
ermA
| CCC GAA AAA TAC GCA AAA TTT CAT CCC TGT TTA CCC ATT TAT AAA CG | 590 | 60 | [14] |
ermB
| TGG TAT TCC AAA TGC GTA ATG CTG TGG TAT GGC GGG TAA GT | 745 | 60 | ||
ermC
| AAT CGT CAA TTC CTG CAT GT TAATCGTGGAATACGGGTTTG | 299 | 58 | [31] | |
ermT
| TAT TAT TGA GAT TGG TTC AGG G GGA TGA AAG TAT TCT CTA GGG ATT T | 395 | 55 | [33] | |
msrA/B
| GCA AAT GGT GTA GGT AAG ACA ACT ATC ATG TGA TGT AAA CAA AAT | 399 | 54 | [66] | |
msrC
| AAG GAA TCC TTC TCT CTC CG GTA AAC AAA ATC GTT CCC G | 343 | 55 | [67] | |
mefA/E
| CAA TAT GGG CAG GGC AAG AAG CTG TTC CAA TGC TAC GC | 317 | 60 | [14] | |
lnuA
| GGT GGC TGG GGG GTA GAT GTA TTA ACT GG GCT TCT TTT GAA ATA CAT GGT ATT TTT CGA TC | 323 | 61 | [32] | |
Aminoglicosides |
aac(6’)-Ie-aph(2″)-Ia
| CAG AGC CTT GGG AAG ATG AAG CCT CGT GTA ATT CAT GTT CTG GC | 348 | 57 | [15] |
aph3IIIa
| GGC TAA AAT GAG AAT ATC ACC GG CTT TAA AAA ATC ATA CAG CTC GCG | 523 | |||
ant(4’)-Ia
| CAA ACT GCT AAA TCG GTA GAA GCC GGA AAG TTG ACC AGA CAT TAC GAA CT | 294 | |||
aph(2″)-Ic
| CCA CAA TGA TAA TGA CTC AGT TCC C CCA CAG CTT CCG ATA GCA AGA G | 444 | |||
aph(2″)-Id
| GTG GTT TTT ACA GGA ATG CCA TC CCC TCT TCA TAC CAA TCC ATA TAA CC | 641 | |||
ant(6)-Ia
| CGG GAG AAT GGG AGA CTT TG CTG TGG CTC CAC AAT CTG AT | 563 | 56 | [54] | |
aac(6’)-Ii
| TGGCCGGAAGAATATGGAGA GCATTTGGTAAGACACCTACG | 410 | 55 | ||
aadA
| ATC CTT CGG CGC GAT TTT G GCA GCG CAA TGA CAT TCT TG | 282 | 55 | [47] | |
aadE
| ATG GAA TTA TTC CCA CCT GA TCA AAA CCC CTA TTA AAG CC | 1100 | 51 | [51] | |
Chloramphenicol |
cat
| TAA GGT TAT TGG GAT AAG TTA GCA TGR TAA CCA TCA CAW AC | ~300 | 54 | [18] |
Tiamulin |
cfr
| TGA AGT ATA AAG CAG GTT GGG AGT CA ACC ATA TAA TTG ACC ACA AGC AGC | 746 | 58 | [68] |
lsaE
| TGT CAA ATG GTG AGC AAA CG TGT AAA ACG GCT TCC TGA TG | 496 | 54 | [69] | |
lsaC
| GGC TAT GTA AAA CCT GTA TTT G ACT GAC AAT TTT TCT TCC GT | 429 | 50 | [70] | |
vgaA
| AGT GGT GGT GAA GTA ACA CG CTC TTG TTC TAA TTC TTC CG | 1287 | 53 | [70] | |
vgaAv
| CTC CGT GTT GAA GAT GTT TCG GGA TTC AAA CGC CTC TAT AGC C | 459 | 56 | [71] | |
Penicillins |
blaZ
| ACT TCA ACA CCT GCT GCT TTC TAG GTT CAG ATT GGC CCT TAG | 240 | 60 | [72] |
mecA
| AGT TCT GCA GTA CCG GAT TTG C AAA ATC GAT GGT AAA GGT TGG C | 533 | 55 | [73] | |
pbp5
| AAC AAA ATG ACA AAC GGG TAT CCT TGG TTA TCA GGG | 779 | 52 | [74] | |
Many antibiotics |
acrA
| CTC TCA GGC AGC TTA GCC CTA A TGC AGA GGT TCA GTT TTG ACT GTT | 107 | 58 | [58] |
mdeA
| CTT TCA GGT TAC CTT GTT GAA TAT TTA AAC ATC AAT AGG TAC TTT AAT TGT AGT TCC AAC | 180 | 56 | [75] | |
norA
| TTT GTT TTC AGT GTC AGA ATT TAT GTT TG GGC TTG GTG AAA TAT CAG CTA TTA AAC | 140 | 56 | ||
mepA
| ATG GTA TAG GTT TCT TGT TTA CTG GTA TG AAT GAT AAT TGC ACC TTG TAA AAT GGC | 150 | 57 |
Nitrocefin test
Results
Antimicrobial susceptibility testing
Number of strains | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Do not showing any resistance | Susceptible to one drug only | With multidrug resistancea
| Displayed resistance against | |||||||||||
Ampicillin (MIC ≥2 or 4 or 8 μg/ml) | Tetracyclines (MIC ≥8 or 16 or 32 μg/ml) | Macrolides (MIC ≥2 μg/ml) | Lincomycin (MIC ≥16 μg/ml) | Streptomycin (MIC ≥32 or 128 μg/ml) | Gentamicin (MIC ≥16 or 32 μg/ml) | Chloramphenicol (MIC ≥8 μg/ml) | Tiamulin (MIC ≥8 μg/ml) | Enrofloxacin (MIC ≥64 μg/ml) | Macrolides and lincomycin | Streptomycin and gentamicin | ||||
L. salivarius (n = 31) | 0 (0%) | 2 (6%) | 27 (87%) | 4 (13%) | 23 (74%) | 18 (58%) | 22 (71%) | 16 (52%) | 6 (19%) | 16 (52%) | 29 (93.5%) | 18 (58%) | 18 (58%) | 4 (13%) |
L. agilis (n = 3) | 0 (0%) | 2 (67%) | 1 (33%) | 0 (0%) | 1 (33%) | 0 (0%) | 1 (33%) | 1 (33%) | 1 (33%) | 0 (0%) | 2 (67%) | 1 (33%) | 0 (0%) | 1 (33%) |
L. johnsoni (n = 21) | 0 (0%) | 3 (14%) | 16 (76%) | 6 (28%) | 14 (67%) | 10 (48%) | 14 (67%) | 3 (14%) | 0 (0%) | 2 (9.5%) | 21 (100%) | 10 (48%) | 10 (48%) | 0 (0%) |
L. crispatus (n = 12) | 0 (0%) | 0 (0%) | 10 (83%) | 8 (67%) | 9 (75%) | 3 (25%) | 8 (67%) | 2 (17%) | 2 (17%) | 1 (8%) | 12 (100%) | 11 (92%) | 3 (25%) | 1 (8.3%) |
L. reuteri (n = 10) | 0 (0%) | 0 (0%) | 9 (90%) | 5 (50%) | 9 (90%) | 4 (40%) | 10 (100%) | 2 (20%) | 0 (0%) | 1 (10%) | 7 (70%) | 1 (10%) | 4 (40%) | 0 (0%) |
L. ingluviei (n = 8) | 2 (25%) | 0 (0%) | 5 (62.5%) | 0 (0%) | 7 (87%) | 1 (12%) | 5 (62%) | 0 (0%) | 0 (0%) | 0 (0%) | 5 (62.5%) | 0 (0%) | 1 (12%) | 0 (0%) |
L. saerimneri (n = 3) | 0 (0%) | 0 (0%) | 2 (67%) | 0 (0%) | 2 (67%) | 1 (33%) | 2 (67%) | 3 (100%) | 2 (67%) | 0 (0%) | 3 (100%) | 1 (33%) | 1 (33%) | 2 (67%) |
Total: 88 | 2 (2%) | 7 (8%) | 70 (79.5%) | 23 (26%) | 65 (74%) | 37 (42%) | 62 (70%) | 27 (31%) | 11 (12.5%) | 20 (23%) | 79 (90%) | 42 (48%) | 37 (42%) | 8 (9%) |
Detection of antibiotic resistance genes
Resistant gene |
tetL
|
tetM
|
tetW
|
ermB
|
ermC
|
LnuA
|
tetL + tetM
|
tetL + tetM + ermB
|
tetW + ermB
|
ermB + ermC
|
ermB + lnuA
|
cat
|
ant(6)-Ia
|
aadE
|
ant(6)-Ia + aadE
|
aac(6’)-Ie-aph(2’)-Ia
|
aph(2″)-Ic
|
lsaE
|
lsaE + ant(6)-Ia
|
acrA
|
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
L. salivarius (n = 31) | 19 (61%) | 21 (68%) | 0 | 16 (52%) | 6 (19%) | 15 (48%) | 18 (58%) | 14 (45%) | 0 | 2 (1%) | 12 (39%) | 8 (26%) | 8 (26%) | 4 (13%) | 4 (13%) | 6 (19%) | 4 (13%) | 8 (26%) | 7 (22%) | 31 (100%) |
L. johnsonii (n = 21) | 0 | 0 | 13 (62%) | 10 | 0 | 6 (3%) | 0 | 0 | 7 (3%) | 0 | 4 (2%) | 11 (48%) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 21 (100%) |
L. crispatus (n = 12) | 1 (8%) | 1 (8%) | 7 (58%) | 2 (17%) | 1 (8%) | 1 (8%) | 1 (8%) | 1 (8%) | 1 (8%) | 0 | 0 | 8 (7%) | 0 | 0 | 0 | 1 (8%) | 0 | 0 | 0 | 12 (100%) |
L. reuteri (n = 10) | 0 | 0 | 9 (90%) | 4 (40%) | 2 (20%) | 7 (70%) | 0 | 0 | 4 (40%) | 1 (1%) | 3 (3%) | 5 (50%) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 10 (100%) |
L. ingluviei (n = 8) | 0 | 0 | 6 (75%) | 1 (12%) | 0 | 5 (62%) | 0 | 0 | 1 (12%) | 0 | 1 (12%) | 1 (12%) | 1 (12%) | 0 | 0 | 0 | 0 | 2 (25%) | 1 (12%) | 8 (100%) |
L. agilis (n = 3) | 1 (33%) | 1 (33%) | 0 | 0 | 1 (33%) | 0 | 1 (33%) | 0 | 0 | 0 |
–
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 (100%) |
L. saerimneri (n = 3) | 0 | 0 | 2 (67%) | 1 (33%) | 1 (33%) | 0 |
0
| 0 | 1 (33%) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (33%) | 0 | 0 | 3 (100%) |
Total: 88 | 21 (24%) | 23 (26%) | 37 (42%) | 34 (39%) | 11 (12%) | 34 (39%) | 20 (23%) | 15 (17%) | 14 (16%) | 3 (3%) | 20 (23%) | 33 (37.5%) | 9 (10%) | 4 (4.5%) | 4 (4.5%) | 7 (8%) | 5 (6%) | 10 (11%) | 8 (9%) | 88 (100%) |
Nitrocefin test
Distribution of resistant isolates on farms
Farm Age of birds/no. of Lactobacillus isolates | Broiler chickens | Green-legged Partridge hens | ||||||
---|---|---|---|---|---|---|---|---|
I 45 days/11 (%) | II 49 days/13 (%) | III 17 days/17 (%) | IV 42 days/7 (%) | V 40 days/8 (%) | VI 48 days/12 (%) | VII 2 days/7 (%) | VIII 49 days/13 (%) | |
Ampicillin | 9 | 15 | 29 | 43 | 62.5 | 25 | 28.5 | 15 |
Tetracyclines | 100 | 92 | 94 | 86 | 100 | 58 | 14 | 31 |
Lincomycin | 91 | 100 | 100 | 86 | 62.5 | 50 | 57 | 8 |
Macrolides | 54.5 | 92 | 70.5 | 43 | 25 | 8 | 0 | 8 |
Streptomycin | 45 | 54 | 29 | 0 | 0 | 50 | 28.5 | 15 |
Gentamicin | 9 | 23 | 23.5 | 0 | 0 | 17 | 14 | 0 |
Chloramphenicol | 9 | 31 | 53 | 0 | 12.5 | 25 | 0 | 15 |
Tiamulin | 82 | 92 | 88 | 100 | 75 | 100 | 86 | 92 |
Enrofloxacin | 27 | 61.5 | 82 | 57 | 25 | 42 | 28.5 | 31 |
Isolates without any resistance | 0 | 0 | 0 | 0 | 12.5 | 0 | 0 | 8 |
Resistance to one drug only | 0 | 0 | 0 | 0 | 0 | 0 | 43 | 31 |
Multidrug resistant isolates | 100 | 100 | 100 | 100 | 75 | 67 | 57 | 31 |