Antimicrobial susceptibility profile of E. coli isolates
A total of 110 E. coli strains were isolated from 122 human stool samples obtained from poultry workers on farms and LBMs; 111 faecal samples obtained from chickens on farms and LBMs; and 196 poultry litter and water samples obtained from farm and LBM environments. Of the 110 E. coli strains 42.7% (n = 47) were recovered from humans; 37.7% (n = 36) from chickens and 24.5% (n = 27) from poultry environment. High resistance rates were observed for tetracycline (n = 101; 91.8%), trimethoprim/ sulfamethoxazole (n = 94; 85.5%), streptomycin (n = 87; 79.1%) ampicillin (n = 87; 79.1%), nalidixic acid (n = 72; 65.5%) and gentamicin (n = 63; 57.3). Low frequencies of resistance were, however, observed for colistin (n = 13; 11.8%), imipenem (n = 12; 10.9%), ceftazidime (n = 11; 10%), amoxicillin/clavulanic acid (n=10; 9.1%), cefuroxime (n=7; 6.4%), cefotaxime (n=6; 5.5%) and ceftriaxone (n=5; 4.6%) as shown in Table 1.
Analysis of resistance profiles of the 110 isolates showed that a single isolate (0.9%) from a poultry farmer was susceptible to all antimicrobial drugs tested; 4 (3.6%) were resistant to only one antimicrobial drug, 4 (3.6%) were resistant to two antimicrobial drugs and interestingly 101 (91.8%) were MDR (resistant to three or more classes of antimicrobial drugs).
Table 1: Antimicrobial resistance profiles of E. coli isolates from humans, chickens and farm/market environments in Abuja - Nigeria, 2019
Drug Class
|
Drug
|
Resistance break point µg/mL
|
Human
n = 47
(%)
|
Chicken
n = 36
(%)
|
Environment
n = 27
(%)
|
Total
n = 110
(%)
|
Tetracyclines
|
Tetracycline
|
< 11
|
39 (83.0)
|
35 (97.2)
|
27 (100.0)
|
101 (91.8)
|
Folate Pathway antagonists
|
Sulfamethoxazole/Trimethoprim
|
< 10
|
39 (83.0)
|
31 (86.1)
|
24 (88.9)
|
94 (85.5)
|
Penicillins
|
Ampicillin
|
< 13
|
36 (76.6)
|
31 (86.1)
|
20 (74.1)
|
87 (79.1)
|
Quinolones
|
Nalidixic acid
|
< 13
|
26 (55.3)
|
27 (75.0)
|
19 (70.4)
|
72 (65.5)
|
Aminoglycosides
|
Streptomycin
|
< 11
|
35 (74.5)
|
30 (83.3)
|
22 (81.5)
|
87 (79.1)
|
Gentamicin
|
< 12
|
20 (42.5)
|
27 (75.0)
|
16 (59.3)
|
63 (57.3)
|
Phenicols
|
Chloramphenicol
|
< 12
|
15 (31.9)
|
17 (47.2)
|
7 (25.9)
|
39 (35.5)
|
1st Generation
Cephalosporins
|
Cephalothin
|
< 14
|
13 (27.7)
|
15 (41.7)
|
5 (18.5)
|
33 (30.0)
|
Nitrofurans
|
Nitrofurantoin
|
< 14
|
5 (10.6)
|
13 (36.1)
|
8 (29.6)
|
26 (23.6)
|
Carbapenems
|
Imipenem
|
< 19
|
3 (6.4)
|
6 (16.7)
|
3 (11.1)
|
12 (10.9)
|
B-lactam inhibitors
|
Amoxicillin-clavulanate
|
< 13
|
2 (4.3)
|
5 (13.9)
|
3 (11.1)
|
10 (9.1)
|
3rd and 4th Generation
Cephalosporins
|
Ceftriaxone
|
< 19
|
3 (6.4)
|
1 (2.8)
|
1 (3.7)
|
5 (4.6)
|
Cefuroxime
|
< 14
|
4 (8.5)
|
3 (8.3)
|
0 (0)
|
7 (6.4)
|
Cefotaxime
|
< 22
|
4 (8.5)
|
1 (2.8)
|
1 (3.7)
|
6 (5.5)
|
Ceftazidime
|
< 17
|
4 (8.5)
|
2 (5.6)
|
5 (18.5)
|
11 (10.0)
|
Polymyxin
|
Colistin
|
< 11
|
7 (14.9)
|
3 (8.3)
|
3 (11.1)
|
13 (11.8)
|
Resistance to 3 or more classes of antibiotics
|
MDR
|
n/a
|
39 (82.9)
|
35 (97.2)
|
27 (100)
|
101(91.8)
|
Prevalence of MDR E. coli in humans, chickens and poultry farm/LBM environment
The overall prevalence of E. coli from all sources was 26.8% (n=115), however, only 110 were further characterized due to viability. Of the 110 E. coli isolates from humans, chickens, and poultry farm or LBM environment, 91.8% (n=101) were MDR E. coli. Of these 38.6% (n=39) were human isolates, 34.7% (n=35) were chicken isolates and 26.7% (n=27) were isolates recovered from the poultry farm or LBM environment (Fig 1). Surprisingly, all the poultry environment isolates (27/27) were resistant to more than three classes of antimicrobials tested. Of the 101 MDR E. coli isolates 47.5% (n=48) were MDR5 or MDR6 (resistant to more than 5 antimicrobials) and 38.6% (n=39) of the isolates were classified as XDR (resistant to 8 or more classes of antimicrobials i.e. extensively drug-resistant isolates. Overall, 36.6% (n=37) of the isolates originated from the LBM while 63.4% (n=64) originated from the poultry farms. Of the 39 XDR E. coli isolates 41% (n=16) were from chickens, 33.3% (n=13) represented humans, while 25.6% (n=10) were recovered from the poultry farm or LBM environment.
Antimicrobial resistance phenotypes of E. coli isolates
The number of antimicrobials against which each isolate showed resistance was between one and thirteen. Surprisingly, a single E. coli isolate from a poultry farm was resistant to 13 out of 16 antimicrobials tested. E. coli isolates that exhibited resistance to only one antimicrobial drug was either resistant to ampicillin or streptomycin or colistin. The AMR phenotypes with AMP, CEP, CHL, CT, GEN, NAL, S, SXT, and TET profile had the highest frequency of 13.6 % (n=15). Fig 2 summarizes the multiple AMR patterns exhibited by the 110 E. coli isolates.
In silico AMR gene analysis of MDR E. coli isolates in humans, chickens and poultry environment
This study identified 57 different resistance determinants from 101 MDR E. coli isolates. Aminoglycosides accounted for the majority of these resistance determinants with about 14 different variants (aadA1, aadA2, aadA2b, aadA5, aadA16, armA, aac(3)-IIa, aac(3)-IId, aac(3)-Ib, aac(6)-Ib-cr, aph(3)-Ia, aph(3)-Ib, aph(6)-Id, ant(2)-Ia) detected. A high prevalence (70.3%) of aph(6)-Id, which is a plasmid-encoded gene, was also observed. About two-thirds of the isolates (67.3%) exhibited aph(3)-Ib gene, a metabolic enzyme that confers aminoglycoside resistance. The aac(3)-IId gene responsible for conferring gentamicin resistance was observed in 27.7% of the MDR E. coli isolates. We also detected aac(6)-Ib-cr gene, responsible for the reduction in ciprofloxacin activity in two MDR E. coli isolates. Six different variants of β-lactam resistance genes were detected (blaTEM-1, blaOXA-1, blaOXA-10, blaOXA-129, blaCTX-M-15, blaCTX-M-65) out of which blaCTX-M type was classical of the ESBL producing E. coli. Our study observed 10 different variants of fluoroquinolone resistance, an important antimicrobial on the WHO list, (qnrB1, qnrB19, qnrB52, qnrS1, qnrS2, qnrS3, qnrS7, qnrS11, qnrS13, aac(6)-Ib-cr) and associated with mutations in the gyrA, parC, and parE genes. We detected other important resistance determinants such as trimethoprim resistance (dfrA1, dfrA8, dfrA12, dfrA14, dfrA15, dfrA17, dfrA21, and dfrA27), macrolide resistance (mdfA, mphA, mefB, ermB, ereA, mphE and msrE), phenicol resistance (cmlA1, catA1, catA2, catB3, floR), rifampicin resistance (ARR-2 and ARR-3), sulphonamide resistance (sul1, sul2, sul3), tetracycline resistance (tetA, tetB, tetM) and plasmid-mediated colistin resistance gene (PMCR) - mcr-1.1.
Multi-locus Sequence Determination of MDR E. coli isolates
The 101 MDR E. coli isolates belonged to 66 different sequence types (ST), out of which one (1) was non-conclusive and eight (8) were new types. In the in silico MLST analysis of MDR E. coli, the following were the most prevalent: ST-155 (7.9%; n=8), ST-48 (7.9%; n=8), ST-10 (5.9%; n=6), ST-1638 (4%; n=4), ST-398 (3%; n=3), ST-216 (3%; n=3), ST-226 (3%; n=3), ST-101 (2%; n=2), ST-117 (2%; n=2), ST-165 (2%; n=2), ST-206 (2%; n=2), ST-4663 (2%; n=2), ST-1286 (2%; n=2), and ST-1196 (2%; n=2) as shown in Fig 3.
The entire ST-10 group (n=6), ST-218 group (n=3), ST-398 group (n=3) and ST-1638 group (n=4) belonged to phylogroup A. However, all but one isolate in both ST-48 (7/8) and ST-226 (2/3) also belonged to phylogroup A. A single isolate representing ST-155 (1/8) belonged to phylogroup A, with the rest (7/8) of the isolates placed in phylogroup B1. A majority of the novel ST (5/8) also belonged to phylogroup B1 while the non-conclusive isolate belonged to phylogroup F.
Phylogroups of E. coli isolates from humans, chickens and poultry farm/market environment
According to phylogenetic grouping, the majority of the E. coli isolates belonged to phylogroup A (n=61, 55.5%) followed by phylogroup B1 (n=36, 32.7%). The remaining isolates belonged to phylogroup G (n=3, 2.7%); D (n=2, 1.8%); E (n=2, 1.8%); F (n=2, 1.8%); B2 (n=1, 0.9%); C (n=1, 0.9%); clade I (n=1, 0.9%) and clade IV (n=1, 0.9%). The phylogroup A consisted of isolates from workers (n=36) and poultry farm/LBM environment (n=13). The E. coli isolates recovered from chickens most frequently belonged to phylogroup B1 (Fig 4). Of the 36 E. coli isolates, that belonged to phylogroup B1, 22.2% (n=8) were human isolates, 50% (n=18) were chicken isolates and 27.8% (n=10) were poultry farm/LBM environment isolates.
The peak of the blue triangle denotes the highest frequency of human E. coli isolates in phylogroup A. The orange triangle denotes the highest frequency of chicken E. coli isolates in phylogroup B1. The black triangle peaks in the same direction as the blue triangle indicating that the phylogroup A has the highest frequency for the E. coli isolates from the poultry farm/LBM environment.
Plasmid replicon profiles of MDR E. coli isolates from humans, chickens and poultry farm/LBM environment
Forty (40) different plasmid replicon types were detected among 97 MDR E. coli isolates however, 4% did not harbour any plasmid replicons. These were represented by two isolates from poultry farm workers (2%), 1% was a chicken isolate and 1% was recovered from the LBM environment. The most prevalent plasmid replicons detected in descending order were p0111 (36.6%, n=37); IncFIB(AP001918) (33.7%, n=34); IncFII (18.8%, n=19); ColpHAD28 (14.9%, n=15); IncQ1 (13.9%, n=14); IncFIB(K) (13.9%, n=14); ColpVC (12.9%, n=13); IncFIC(FII) (12.9%, n=13); IncR (9.9%, n=10); IncFII(pCoo) (9.9%, n=10); IncY (9.9%, n=10); IncX1 (8.9%, n=9) and IncI1-I(gamma) (8.9%, n=9). The plasmid replicons from human MDR E. coli isolates were more diverse than those from chicken and poultry farm/LBM environment isolates. Of the 40 different plasmid replicons detected from the MDR E. coli isolates 33 different types were detected in human MDR E. coli isolates, 26 in chicken isolates while 25 were detected in poultry farm/LBM environment isolates. Eighteen replicon types were common to isolates from all sources: p0111, IncFIB(AP001918), IncFII, ColpHAD28, IncQ1, IncFIB(K), ColpVC, IncFIC(FII), IncX1, IncFII(pCoo), IncI1-I (gamma), IncFII (29), IncFII(pHN7A8), IncFIA, Col156, IncHI2, IncHI2A and IncX4.
Of the 40 different replicon types identified, IncFIB(AP001918) was the most common among human isolates (n=12) while replicon type p0111 was the most commonly detected in both chicken (n=15) and poultry farm/LBM environment MDR E. coli isolates (n=14) as shown in Fig 5. Interestingly, IncFIB (pLF82), a phage plasmid was detected in one of the MDR E. coli isolates recovered from the LBM environment. Eight different plasmids were observed to harbour AMR genes. The following AMR genes were carried on plasmid replicons: mcr-1.1 + IncX4 (n = 2); tetA + IncX1 (n = 1); qnrB19 + Col440I (n = 7); sul2 + IncQ1 (n = 5); aph(3)-Ib + IncQ1 (n = 1); blaTEM-1 + IncFIC(FII) (n = 1); mdf(A) + IncFIB (n = 1); qnrS13 + IncFII (n = 1) and aac(3)-IIa + IncHI1B (n =1). The plasmid replicons harbouring the AMR genes were detected in commensal E. coli isolates recovered from poultry workers, chickens and the poultry environment (Table 2).
Table 2: Plasmid replicons harbouring AMR genes detected in MDR E. coli isolates recovered from humans, chickens, and poultry environments in Abuja-Nigeria, 2019
Isolate ID
|
Phylogroup
|
MLST
|
Plasmid replicons
|
AMR genes carried on plasmids
|
Origin
|
MA-125
|
A
|
ST-48
|
Col440I
|
qnrB19
|
Human (F)
|
MA-288
|
A
|
ST-48
|
Col440I
IncQ1
|
qnrB19
Sul2
|
Environment (F)
|
MA-174
|
A
|
ST-48
|
Col440I
IncQ1
|
qnrB19
Sul2
|
Chicken (F)
|
MA-158
|
B1
|
ST-48
|
IncQ1
IncFIB(pHCM2)
|
Sul2
mdf(A)
|
Chicken (F)
|
MA-249
|
D
|
ST-48
|
IncQ1
|
Sul2
|
Environment (M)
|
MA-163
|
A
|
ST-48
|
IncQ1
|
Sul2
|
Environment (F)
|
MA-019
|
B1
|
ST-155
|
Col440I
|
qnrB19
|
Chicken (M)
|
MA-069
|
B1
|
ST-155
|
IncFIC(FII)
|
blaTEM-1
|
Human (M)
|
MA-207
|
A
|
ST-10
|
IncFII
|
qnrS13
|
Environment (F)
|
MA-049
|
A
|
ST-10
|
IncX4
Col440I
|
mcr-1.1
qnrB19
|
Environment (M)
|
MA-293
|
A
|
ST-1638
|
IncX4
IncX1
|
mcr-1.1
tet(A)
|
Environment (F)
|
MA-287
|
A
|
ST-1638
|
IncHI1B(CIT)
|
aac(3)-IIa
|
Chicken (F)
|
MA-251
|
B1
|
ST-New
|
Col440I
|
qnrB19
|
+Environment (F)
|
MA-252
|
B1
|
ST-New
|
Col440I
|
qnrB19
|
+Human (F)
|
MA-258
|
D
|
ST-New
|
IncQ1
|
aph(3)-Ib
|
Human (M)
|
+These two isolates originated from the same farm. *Origin of isolates: F – Farm; M – Live bird market
Determination of pMLST for IncHI2 and IncF plasmid replicons
In silico pMLST identification and typing of IncHI2 and IncF plasmid replicons, were based on the combination of the alleles identified for the genes. For the IncHI2 the assigned ST was ST-4 for both isolates (MA_251 and MA_252 sourced from poultry farmworker and poultry litter on the same farm). The pMLST analysis also assigned the two IncF plasmids for isolates MA_251 and MA_252 with ST-F18:A-B1. It is interesting to note that although the plasmid structures of the two isolates were so similar, there was no clonal relationship between them.
Phylogenetic analysis of E. coli isolates from humans, chickens and poultry farm/market environment
In this study, all isolates were assigned a phylogenetic group and sequence type (ST) based on multilocus sequence typing. Isolates with the same phylogenetic group and ST were used to construct phylogenetic trees to determine if the isolates were related or very diverse. Two phylogenetic trees were constructed: one for all the isolates with prevalent STs (Fig 5) and the other focusing on isolates with new STs (Fig 6).
Overall, 34 isolates from this study were used to construct a phylogenetic tree. Some level of diversity was evident among these isolates. The phylogenetic tree showed that all the isolates were grouped into two main phylogroups and 6 STs. The isolates were grouped based on similarity among them. Whole-genome (wg) SNPs-based phylogenetic analysis showed that some isolates sharing the same ST and phylogroups were not clonally related. Two ST-155 (chicken and poultry environment with SNPs difference of 2468), two ST-155 (chicken and human with SNPs difference of 1419), and two ST-1638 (human and environment with SNPs difference of 1760) type isolates were observed not to be clonally related using wgSNP phylogeny (Fig 5).
Two isolates of human and environmental origin with SNPs difference of 6161 were not clonally related although the isolates shared a novel ST and belonged to the same phylogroup (Fig 6). The two isolates originating from the same farm had similar AMR gene profile (qnrB19, qnrS1, mdfA, mefB, sul 2, sul 3, blaTEM1, tetA, tetM, floR); as well as plasmid replicons (p0111, IncFIC(FII), IncHI2A, IncHI2, Col(pHAD28).