Background
Methods
Sampling, isolation and identification of bacteria
Serovar | Poultry | Beef | (Poultry + beef) n (%) | ||||||
---|---|---|---|---|---|---|---|---|---|
Fresh meat (n = 30) | Frozen meat (n = 30) | Fresh organs (n = 30) | Total n (%) | Fresh meat (n = 30) | Frozen meat (n = 30) | Fresh organs (n = 30) | Total n (%) | ||
S. Typhimurium | 2 | 0 | 1 | 3 (3.3) | 3 | 1 | 2 | 6 (6.7) | 9 (60.0) |
S. Paratyphi A | 0 | 1 | 1 | 2 (6.7) | 0 | 0 | 1 | 1 (3.3) | 3 (20.0) |
S. Enteritidis | 1 | 0 | 0 | 1 (3.3) | 1 | 0 | 0 | 1 (3.3) | 2 (13.3) |
S. Kentucky | 1 | 0 | 0 | 1 (3.3) | 0 | 0 | 0 | 0 (0) | 1 (6.7) |
Total | 4 | 1 | 2 | 7 (7.8) | 4 | 1 | 3 | 8 (8.8) | 15 (100) |
Serogroup | Fresh poultry meat (n = 30) | Frozen poultry meat (n = 30) | Fresh poultry organs (n = 30) | Fresh beef meat (n = 30) | Frozen beef meat (n = 30) | Fresh beef organs (n = 30) | Total number of isolates | % of E. coli isolates |
---|---|---|---|---|---|---|---|---|
O1 | 1 | 1 | 0 | 0 | 0 | 0 | 2 | 9.5 |
O18 | 2 | 1 | 1 | 0 | 0 | 0 | 4 | 19 |
O20 | 1 | – | 1 | – | – | – | 2 | 9.5 |
O78 | 1 | 0 | 0 | 1 | 1 | 1 | 4 | 19 |
O103 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 4.8 |
O119 | 1 | – | – | 1 | – | – | 2 | 9.5 |
O126 | 0 | 0 | 1 | 0 | 1 | 0 | 2 | 9.5 |
O145 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 4.8 |
O146 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 4.8 |
O158 | 0 | 0 | 0 | 1 | 1 | 0 | 2 | 9.5 |
Total | 6 | 3 | 4 | 3 | 4 | 1 | 21 | 100 |
DNA extraction and purification
Molecular identification of S. enterica serovars and E. coli
Primer | Sequence (5′–3′) | Amplicon size (bp) | Amplification target | References |
---|---|---|---|---|
E. coli, Salmonella enterica and Salmonella serovar identification | ||||
ECO-f | GAC CTC GGT TTA GTT CAC AGA | 585 |
E. coli
| [67] |
ECO-r | CAC ACG CTG ACG CTG ACC A | |||
OMPCF | ATC GCT GAC TTA TGC AAT CG | 204 |
Salmonella genus | [68] |
OMPCR | CGG GTT GCG TTA TAG GTC TG | |||
ENTF | TGT GTT TTA TCT GAT GCA AGA GG | 304 |
S. Enteritidis | [68] |
ENTR | TGA ACT ACG TTC GTT CTT CTG G | |||
TYPHF | TTG TTC ACT TTT TAC CCC TGA A | 401 |
S. Typhimurium | [68] |
TYPHR | CCC TGA CAG CCG TTA GAT ATT | |||
β-Lactamases | ||||
TEM-F | ATA AAA TTC TTG AAG ACG AAA | 1080 |
bla
TEM
| [69] |
TEM-R | GAC AGT TAC CAA TGC TTA ATC | |||
OXA-F | TCA ACT TTC AAG ATC GCA | 591 |
bla
OXA
| [69] |
OXA-R | GTG TGT TTA GAA TGG TGA | |||
OXA-F-2 | ATT AAG CCC TTT ACC AAA CCA | Whole bla
OXA
| [69] | |
OXA-R-2 | AAG GGT TGG GCG ATT TTG CCA | |||
OXA-23-F | GAT CGG ATT GGA GAA CCA GA | 501 |
bla
OXA-23
| [25] |
OXA-23-R | ATT TCT GAC CGC ATT TCC AT | |||
CTX-M-F | CGC TTT GCG ATG TGC AG | 550 |
bla
CTX-M
| [69] |
CTX-M-R | ACC GCG ATA TCG TTG GT | |||
CTX-M-F2 | CCA GAA TAA GGA ATC CCA TG | Whole bla
CTX-M
| [69] | |
CTX-M-R2 | GCC GTC TAA GGC GAT AAA C | |||
CMY-F | GAC AGC CTC TTT CTC CAC A | 1007 |
bla
CMY
| [69] |
CMY-R | TGG AAC GAA GGC TAC GTA | |||
CMY-F2 | ACG GAA CTG ATT TCA TGA TG | Whole bla
CMY
| [69] | |
CMY-R2 | GAA AGG AGG CCC AAT ATC CT | |||
SHV-F | TTA TCT CCC TGT TAG CCA CC | 795 |
bla
SHV
| [69] |
SHV-R | GAT TTG CTG ATT TCG CTC GG | |||
Integrons | ||||
5′-CS | GGC ATC CAA GCA GCA AG | 152 | Class 1 integron | [69] |
3′-CS | AAG CAG ACT TGA CCT GA | |||
hep74 | CGG GAT CCC GGA CGG CAT GCA CGA TTT GTA | 491 | Class 2 integron | [69] |
hep51 | GAT GCC ATC GCA AGT ACG AG | |||
Plasmid-mediated quinolone resistance gene | ||||
qnrA-F | ATT TCT CAC GCC AGG ATT TG | 516 |
qnrA | [69] |
qnrA-R | GAT CGG CAA AGG TTA GGT CA | |||
qnrB-F | GAT CGT GAA AGC CAG AAA GG | 469 |
qnrB | [69] |
qnrB-R | ACG ATG CCT GGT AGT TGT CC | |||
qnrS-F | ACG ACA TTC GTC AAC TGC AA | 417 |
qnrS | [69] |
qnrS-R | TAA ATT GGC ACC CTG TAG GC | |||
Plasmid-mediated colistin resistance gene | ||||
CLR5-F | CGG TCA GTC CGT TTG TTC | 308 |
mcr-1 | [24] |
CLR5-R | CTT GGT CGG TCT GTA GGG |
Determination of antimicrobial susceptibility profiles
Antibiotic class | Antimicrobial agent | Conc. (μg) | Zone diameter (mm) |
S. Typhimurium (9) |
S. Enteritidis (2) |
S. Kentucky (1) |
S. Paratyphi A (3) | Total Salmonella (15) | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
R | I | S | R | I | S | R | I | S | R | I | S | R | I | S | R | I | S | |||
Penicillin | Amoxycillin–clavulanic acid | 20 | ≤ 13 | 14–17 | ≥ 18 | 5 | 4 | – | – | 2 | – | 1 | – | – | 2 | 1 | – | 8 (53%) | 7 (47%) | – |
Ampicillin | 10 | ≤ 13 | 14–16 | ≥ 17 | 7 | 2 | – | 2 | – | – | 1 | – | – | 3 | – | – | 13 (87%) | 2 (13%) | – | |
Cephalosporin | Cefotaxime | 30 | ≤ 22 | 23–25 | ≥ 26 | 7 | 1 | 1 | 2 | – | – | – | 1 | – | 3 | – | – | 12 (80%) | 2 (13%) | 1 (7%) |
Cefpodoxime | 20 | ≤ 17 | 18–20 | ≥ 21 | 7 | 2 | – | 1 | 1 | – | – | 1 | – | 1 | 2 | – | 9 (60%) | 6 (40%) | – | |
Ceftazidime | 30 | ≤ 17 | 18–20 | ≥ 21 | 3 | 5 | 1 | – | 2 | – | – | 1 | – | 1 | 2 | – | 4 (26%) | 10 (67%) | 1 (7%) | |
Ceftriaxone | 30 | ≤ 19 | 20–22 | ≥ 23 | 3 | 6 | – | – | 2 | – | – | 1 | – | 2 | 1 | – | 5 (33%) | 10 (67%) | – | |
Miscellaneous | Chloramphenicol | 30 | ≤ 12 | 13–17 | ≥ 18 | – | 5 | 4 | – | – | 2 | – | 1 | – | – | 1 | 2 | – | 7 (47%) | 8 (53%) |
Colistin | 10 | ≤ 11 | 12–13 | ≥ 14 | – | – | 9 | – | – | 2 | – | – | 1 | – | – | 3 | – | – | 15 (100%) | |
Fluoroquinolone | Ciprofloxacin | 5 | ≤ 15 | 16–20 | ≥ 21 | – | 3 | 6 | – | 1 | 1 | – | 1 | – | – | – | 3 | – | 5 (33%) | 10 (67%) |
Enrofloxacin | 5 | ≤ 17 | 18–20 | ≥ 21 | 1 | 7 | 1 | – | 1 | 1 | – | 1 | – | – | 2 | 1 | 1 (7%) | 11 (73%) | 3 (20%) | |
Nalidixic acid | 30 | ≤ 13 | 14–18 | ≥ 19 | 3 | 6 | – | 1 | 1 | – | 1 | – | – | – | 3 | – | 5 (33%) | 10 (67%) | – | |
Aminoglycoside | Streptomycin | 10 | ≤ 11 | 12–14 | ≥ 15 | 2 | 7 | – | 1 | 1 | – | 1 | – | – | – | 2 | 1 | 4 (26%) | 10 (67%) | 1 (7%) |
Tetracycline | Tetracycline | 30 | ≤ 11 | 12–14 | ≥ 15 | 3 | 6 | – | 2 | – | – | 1 | – | – | – | 3 | – | 6 (40%) | 9 (60%) | – |
Sulphonamide | Trimethoprim/sulphamethoxazole | 25 | ≤ 4 | 3 | ≥ 2 | 4 | 2 | 3 | 1 | 1 | – | 1 | – | – | 2 | 1 | – | 8 (53%) | 4 (26%) | 3 (20%) |
Antibiotic class | Antimicrobial agent | Conc. (μg) | Zone diameter (9) |
E. coli (21) | Resistance rate | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Poultry (15) | Beef (6) | Total (21) | Poultry | Beef | Total | ||||||||||||
R | I | S | R | I | S | R | I | S | R | I | S | % | % | % | |||
Penicillin | Amoxycillin–clavulanic acid | 20 | ≤ 13 | 14–17 | ≥ 18 | 10 | 4 | 1 | 3 | 3 | – | 13 | 7 | 1 | 66.7 | 50.0 | 61.9 |
Ampicillin | 10 | ≤ 13 | 14–16 | ≥ 17 | 12 | 2 | 1 | 3 | 3 | – | 15 | 5 | 1 | 80.0 | 50.0 | 71.4 | |
Cephalosporin | Cefotaxime | 30 | ≤ 22 | 23–25 | ≥ 26 | 6 | 7 | 2 | 1 | 3 | 2 | 7 | 10 | 4 | 40.0 | 16.7 | 33.3 |
Cefpodoxime | 20 | ≤ 17 | 18–20 | ≥ 21 | 3 | 12 | – | 2 | 4 | – | 5 | 16 | – | 20.0 | 33.3 | 23.8 | |
Ceftazidime | 30 | ≤ 17 | 18–20 | ≥ 21 | 5 | 9 | 1 | – | 6 | – | 5 | 15 | 1 | 33.3 | 0 | 23.8 | |
Ceftriaxone | 30 | ≤ 19 | 20–22 | ≥ 23 | 3 | 5 | 7 | 1 | 3 | 2 | 4 | 8 | 9 | 20.0 | 16.7 | 19.0 | |
Miscellaneous | Chloramphenicol | 30 | ≤ 12 | 13–17 | ≥ 18 | 3 | 9 | 3 | 1 | 1 | 4 | 4 | 10 | 7 | 20.0 | 16.7 | 19.0 |
Colistin | 10 | ≤ 11 | 12–13 | ≥ 14 | – | 1 | 14 | – | – | 6 | – | 1 | 20 | – | – | – | |
Fluoroquinolone | Ciprofloxacin | 5 | ≤ 15 | 16–20 | ≥ 21 | 4 | 3 | 8 | 2 | 2 | 2 | 6 | 5 | 10 | 26.7 | 33.3 | 28.6 |
Enrofloxacin | 5 | ≤ 17 | 18–20 | ≥ 21 | 2 | 5 | 8 | 1 | 4 | 1 | 3 | 9 | 9 | 13.3 | 16.7 | 14.3 | |
Nalidixic acid | 30 | ≤ 13 | 14–18 | ≥ 19 | 5 | 12 | 3 | 1 | 2 | 3 | 6 | 14 | 6 | 33.3 | 16.7 | 28.6 | |
Aminoglycoside | Streptomycin | 10 | ≤ 11 | 12–14 | ≥ 15 | 9 | 5 | 1 | 4 | 2 | – | 13 | 7 | 1 | 60.0 | 66.7 | 61.9 |
Tetracycline | Tetracycline | 30 | ≤ 11 | 12–14 | ≥ 15 | 12 | 3 | – | 5 | 1 | – | 17 | 4 | – | 80.0 | 83.3 | 81.0 |
Sulphonamide | Trimethoprim/sulphamethoxazole | 25 | ≤ 4 | 3 | ≥ 2 | 10 | 2 | 3 | 3 | – | 3 | 17 | 2 | 6 | 66.7 | 50.0 | 81.0 |
Molecular detection of antimicrobial resistance associated genes
Detection and sequencing of class 1 and class 2 integrons
Results
Serological characterization of S. enterica and E. coli
Molecular identification of Salmonella serovars
Determination of antimicrobial susceptibility profiles
Source | Serovar | Resistance phenotype | Resistance genes/class 1 integrons |
---|---|---|---|
Poultry | |||
Fresh poultry meat | Typhimurium | CPD, CTX, NAL |
bla
CTX-M-1, qnrA, qnrB, qnrS |
Fresh poultry meat | Typhimurium | AMC, AMP, CPD |
bla
TEM-1
|
Fresh poultry meat | Enteritidis | AMP, CTX, TET |
bla
CTX-M-1, bla
TEM-1
|
Fresh poultry meat | Kentucky | AMC, AMP, NAL, STR, TET, T/S |
bla
TEM-1, qnrA |
Fresh poultry organs (liver) | Typhimurium | AMC, AMP, CPD, CRO, CTX, NAL, T/S |
bla
CTX-M-1, bla
TEM-1, qnrA, dfrA15, dfrA17
|
Fresh poultry organs (liver) | Paratyphi A | AMC, AMP, CAZ CRO, CTX, T/S |
bla
CTX-M-14, bla
TEM-1
|
Frozen poultry meat | Paratyphi A | AMC, AMP, CRO, CTX, T/S |
bla
CTX-M-14, bla
TEM-1
|
Beef | |||
Fresh beef meat | Typhimurium | AMP, CAZ, CTX |
bla
CTX-M-1
|
Fresh beef meat | Typhimurium | AMP, CAZ, CRO, CTX |
bla
CTX-M-1
|
Fresh beef meat | Typhimurium | AMC, CPD, CTX, TET, T/S |
bla
CTX-M-3, bla
TEM-1
|
Fresh beef meat | Enteritidis | AMP, CPD, CTX, NAL, STR, TET, T/S |
bla
CMY-2, bla
CTX-M-1, bla
TEM-1, qnrA, qnrB |
Fresh beef organs (spleen) | Typhimurium | AMP, CPD, CTX, STR,TET, T/S |
bla
CTX-M-14, bla
TEM-1
|
Fresh beef organs (liver) | Typhimurium | AMC, AMP, CPD | – |
Fresh beef organs (liver) | Paratyphi A | AMP, CPD, CTX |
bla
TEM1
|
Frozen beef meat | Typhimurium | AMC, AMP, CAZ, CPD, CRO, CTX, ENR, NAL, STR, TET, T/S |
bla
CMY-2, bla
CTX-M-1, bla
TEM1, qnrA, qnrB, aadA2
|
Source |
E. coli serotype | Resistance phenotype | Resistance genes/class 1 integron |
---|---|---|---|
Poultry | |||
Fresh poultry meat | O1 | STR, TET | – |
Fresh poultry meat | O18 | AMP, CIP, CPD, NAL, STR, TET, T/S |
qnrA, qnrB |
Fresh poultry meat | O18 | AMC, AMP, CHL, CIP, CRO, CTX, NAL, STR, TET, T/S |
bla
CTX-M-1, bla
OXA-1, qnrA, qnrB |
Fresh poultry meat | O78 | AMC, AMP, CAZ, TET, T/S |
bla
CTX-M-1, bla
TEM-1
|
Fresh poultry meat | O119 | AMC, AMP, CAZ, CTX, STR, TET, T/S |
bla
CTX-M-14, bla
TEM-104
|
Fresh poultry meat | O20 | AMC, AMP, CPD, TET, T/S |
bla
CTX-M1, bla
OXA-1, bla
TEM-1
|
Fresh poultry organs (liver) | O18 | AMC, AMP, CAZ, CHL, CIP, CRO, CTX, ENR, NAL, STR, TET, T/S |
bla
CTX-M-1, bla
TEM-104,qnrA, qnrB, dfrA1-orf |
Fresh poultry organs (liver) | O126 | AMP, CTX, STR |
bla
CTX-M-1
|
Fresh poultry organs (gizzard) | O20 | AMC, AMP, STR, T/S |
bla
TEM-1
|
Fresh poultry organs (liver) | O145 | CAZ, TET | – |
Frozen poultry meat | O1 | CTX, NAL, TET |
qnrA, qnrB |
Frozen poultry meat | O18 | AMC, AMP, STR, TET, T/S |
bla
OXA-1, bla
TEM-1
|
Frozen poultry | 078 | AMC, AMP, CPD | |
Frozen poultry meat | O146 | AMC, AMP, TET, T/S |
bla
TEM-1
|
Frozen organ poultry | 078 | AMC, AMP, CAZ, CHL, CIP, CRO, CTX, ENR, NAL, STR, TET, T/S |
bla
CTX-M-14,bla
TEM-1, qnrA, qnrB, dfrA15 |
Beef | |||
Fresh beef meat | O119 | CPD | – |
Fresh beef meat | O158 | CPD, TET | – |
Fresh beef organs (liver) | O78 | AMC, AMP, CHL, CIP, CRO, CTX, ENR, NAL, STR, TET, T/S |
bla
CTX-M-1, bla
TEM-104, qnrA, qnrB, dfrA1, dfrA1-orf |
Frozen beef meat | O103 | STR, TET |
bla
CTX-M-1
|
Frozen beef meat | O126 | AMC, AMP, CIP, STR, TET, T/S |
bla
TEM-1, qnrA, qnrB |
Frozen beef meat | O158 | AMC, AMP, STR, TET, T/S |
bla
TEM-1
|