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, trimethoprim/ sulfamethoxazole, streptomycin, ampicillin, nalidixic acid and gentamicin. On the contrary resistance to colistin, imipenem, ceftazidime, amoxicillin/clavulanic acid, cefuroxime, cefotaxime and ceftriaxone were quite low although colistin resistance rate of 11.8% in commensal E. coli is quite worrisome (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). The number of antimicrobials against which each isolate showed resistance was between one and thirteen. Surprisingly, a single isolate from a poultry farm was resistant to 13 out of 16 antimicrobials tested. 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 1 summarizes the multiple AMR patterns exhibited by the isolates.
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 4thGeneration
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 as the remaining five isolates were mistakenly discarded. Of the 110 E. coli isolates, 91.8% (n=101) were MDR E. coli. Of these MDR E. coli isolates 38.6% (n=39), 34.7% (n=35), and 26.7% (n=27) were recovered from humans, chickens and poultry environment respectively (Fig 2). Surprisingly, all the poultry environment isolates were MDR. Of the 101 MDR E. coli isolates 47.5% (n=48) were MDR5 (resistant to more than 5 classes) and 38.6% (n=39) were classified as XDR (resistant to 8 or more classes i.e. extensively drug-resistant isolates). Overall, 36.6% (n=37) of the isolates originated from the LBMs while 63.4% (n=64) originated from farms. Of the 39 XDR E. coli isolates 41% (n=16), 33.3% (n=13), and 25.6% (n=10) were recovered from chickens, humans and the poultry environment respectively.
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. Genes encoding resistance to aminoglycosides accounted for the majority with about 14 different determinants (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. Ten different fluoroquinolone resistance determinants were observed, 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 E. coli isolates, the following were observed to appear more than once: ST155 (7.9%; n=8), ST48 (7.9%; n=8), ST10 (5.9%; n=6), ST1638 (4%; n=4), ST398 (3%; n=3), ST216 (3%; n=3), ST226 (3%; n=3), ST101 (2%; n=2), ST117 (2%; n=2), ST165 (2%; n=2), ST206 (2%; n=2), ST4663 (2%; n=2), ST1286 (2%; n=2), and ST1196 (2%; n=2). The most prevalent STs are shown in Fig 3.
Phylogroups of E. coli isolates from humans, chickens and poultry environment
A majority of the isolates belonged to phylogroup A (n=61, 55.5%) followed by phylogroup B1 (n=36, 32.7%) while the rest 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%). Isolates with phylogroup A originated from workers (n=36) and poultry environment (n=13) while isolates recovered from chickens mostly belonged to phylogroup B1 (Fig 4). Of the 36 E. coli isolates, belonging to phylogroup B1, 22.2% (n=8); 50% (n=18) and 27.8% (n=10) were recovered from humans, chickens and the poultry environment respectively.
All isolates assigned ST10 (n=6), ST218 (n=3), ST398 (n=3) and ST1638 (n=4) belonged to phylogroup A. However, all but one isolate assigned ST48 (7/8) and ST226 (2/3) also belonged to phylogroup A while a majority with ST155 (7/8) and novel ST (5/8) belonged to phylogroup B1.
Plasmid replicon profiles of MDR E. coli isolates from humans, chickens and poultry environment
Forty (40) different plasmid replicon types were detected among 97 MDR E. coli isolates however, 4% (n = 4) did not harbour any plasmid replicons. 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 recovered from human isolates were more genetically diverse than those recovered from chickens and the poultry environment. 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.
IncFIB(AP001918) was the most common among human isolates (n=12) while p0111 was commonly detected in both chicken (n=15) and poultry environment isolates (n=14). Interestingly, IncFIB (pLF82), a phage plasmid was detected in one isolate 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 was commonly detected in commensal E. coli isolates recovered from poultry workers, chickens and the poultry environment.
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 ST4 for isolates (MA_251 and MA_252) originating from a poultry farmworker and poultry litter on the same farm. The pMLST analysis 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 environment
All isolates assigned a phylogenetic group and ST were used to construct phylogenetic trees to determine if the isolates were genetically related or very diverse. Three phylogenetic trees were constructed: one for all the isolates (Fig 5), one focusing on isolates with novel STs (Fig 6A) and one with isolates of different origins assigned the same ST (Fig 6B).
Overall, 110 isolates used to construct a maximum likelihood phylogenetic tree showed that the isolates were genetically diverse. 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. The strains were in completely different clades in the SNP tree, separated by strains belonging to other STs. Three isolates with ST-1638 recovered from human, chicken and poultry environment were clustered together on the same clade. Pairwise SNP differences between the genomes of the isolates showed that they were not clonally related (Fig 6B).
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 phylogroup B1 (Fig 6A). 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).