A total of 45 E. coli isolates were isolated from 21 sloth bear faecal
samples and the antibiogram showed that the isolates were resistant to gentamicin (62.2%, 28/45), cefotaxime (60.0%, 27/45), ceftazidime (60.0 %, 27/45), cefotaxime/clavulanic acid (57.8%, 26/45), ceftriaxone (55.6 %, 25/45), ceftazidime /clavulanic acid (55.6%, 25/45), norfloxacin (55.6%, 25/45), cefpodoxime (53.3%, 24/45), cefoxitin (53.3%, 24/45), piperacillin/tazobactam (53.3%, 24/45), cefexime (51.1%, 23/45), ciprofloxacin (51.1%, 23/45), tetracycline (51.1%, 23/45), sulphadimidine/trimethoprim combination (46.7%, 21/45), cefepime (46.7%, 21/45), chloramphenicol (42.2%, 19/45), nitrofurantoin (40.0%, 18/45), aztreonam (35.6%, 16/45), meropenem (11.1%, 5/45), imipenem (8.9%, 4/45) and ertapenem (8.9%, 4/45). Of the 45 isolates, 51.1 % (23/45) were resistant to more than two classes of screened antibiotics and classified as MDR. The ESBL screening by combined disc method revealed 37.8% (17/45) isolates as ESBL producers (Table 1).
Table 1
Antibiotic susceptibility pattern, virulence and antibiotic resistance determinants of E. coli isolates (n = 45) from rescued sloth bear
S. no
|
Isolates
|
Antibacterial resistance pattern
|
Minimum inhibitory concentration
( µg/mL)
|
Virulence and antibiotic resistance determinants
|
Accession number
|
AMX
|
AMC
|
CZ
|
ATZ
|
COT
|
CX
|
CTX
|
CFM
|
CPD
|
CIP
|
CAZ
|
CTR
|
K
|
C
|
GEN
|
TE
|
FOX
|
F/M
|
TZP
|
NX
|
IMP
|
MRP
|
ERP
|
CTX
|
CTR
|
MRP
|
1
|
SB-1
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
-
|
nd
|
2
|
SB-2 ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
8
|
12
|
nd
|
CTX-M-1,qnrA,AmpC,catI
|
nd
|
3
|
SB-3 ESBL
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
S
|
R
|
R
|
S
|
S
|
S
|
12
|
18
|
nd
|
sulI,tetA,ac (3)-IV, eaeA
|
nd
|
4
|
SB-4
|
R
|
S
|
R
|
S
|
R
|
R
|
S
|
S
|
R
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
-
|
nd
|
5
|
SB-5
|
R
|
S
|
R
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
R
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
floR
|
nd
|
6
|
SB-6
|
R
|
R
|
R
|
S
|
R
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
Stx2
|
nd
|
7
|
SB-7
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
12
|
16
|
nd
|
CTX-M-15, qnrS, Stx2
|
MK559368
|
8
|
SB-8ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
S
|
R
|
S
|
S
|
S
|
S
|
8
|
12
|
nd
|
AmpC, sulI, floR, ac (3)-IV
|
nd
|
9
|
SB-9
|
R
|
S
|
S
|
S
|
R
|
R
|
S
|
R
|
S
|
R
|
S
|
S
|
R
|
R
|
S
|
S
|
S
|
S
|
R
|
R
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
floR,TEM-1
|
nd
|
10
|
SB-10
|
R
|
S
|
S
|
S
|
R
|
S
|
R
|
S
|
S
|
R
|
S
|
S
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
floR
|
nd
|
11
|
SB-11
|
R
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
R
|
S
|
S
|
R
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
floR
|
nd
|
12
|
SB-12 ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
S
|
12
|
16
|
nd
|
CTX-M,qnrS, ac (3)-IV, eae A
|
nd
|
13
|
SB-13
ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
32
|
64
|
nd
|
floR,dhfrI
|
nd
|
14
|
SB-14
|
R
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
R
|
S
|
R
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
catI
|
nd
|
15
|
SB-15ESBL
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
16
|
32
|
nd
|
CTX-M,qnrA,sulI,floR
|
nd
|
16
|
SB-16 ESBL
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
8
|
12
|
nd
|
CTX-M,qnrB,tetA,hlyA
|
nd
|
17
|
SB-17 ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
12
|
16
|
nd
|
CTX-M,qnrS,tetB, dhfrI,TEM-1
|
MK559374
|
18
|
SB-18
|
R
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
catI
|
nd
|
19
|
SB-19ESBL
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
8
|
12
|
nd
|
CTX-M,qnrB,sulII,catI, ac (3)-IV
|
nd
|
20
|
SB-20 ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
S
|
R
|
S
|
R
|
R
|
S
|
S
|
S
|
16
|
18
|
nd
|
CTX-M,sulI, dhfrI
|
nd
|
21
|
SB-22 ESBL
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
S
|
R
|
S
|
S
|
S
|
S
|
12
|
16
|
nd
|
CTX-M,qnrA,TEM-1
|
nd
|
22
|
SB-23 ESBL
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
32
|
64
|
nd
|
CTX-M,TEM-1,qnrS,sulI,.AmpC,tetB,catI, ac (3)-IV,dhfrI
|
MK559371, MK559373
|
23
|
SB-24
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
hlyA
|
nd
|
24
|
SB-25
|
S
|
R
|
S
|
S
|
S
|
S
|
R
|
S
|
R
|
R
|
S
|
S
|
R
|
S
|
R
|
R
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
ac (3)-IV
|
nd
|
25
|
SB-26
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
-
|
nd
|
26
|
SB27/ 17-CRE
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
8
|
16
|
16
|
NDM-5, TEM-1,qnrS,sulII,catI, ac (3)-IV
|
MF136747,MK559369, MK559372
|
27
|
SB28/ 12-CRE
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
16
|
32
|
32
|
NDM-5,CTX-M,TEM-1,AmpC
|
MK559365, MK559367, MK559370, MK559375
|
28
|
SB28/ 16-CRE
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
16
|
8
|
8
|
NDM-5, CTX-M, Stx1,tetB,catI, aph (3′)-IIa
|
MK559366
|
29
|
SB-29/18-CRE
|
R
|
R
|
R
|
S
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
8
|
32
|
16
|
tetB
|
Efflux pump
|
30
|
SB-30 CRE
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
16
|
16
|
32
|
CTX-M-,qnrS,floR, dhfrI,stx2
|
Efflux pump
|
31
|
SB-31 ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
16
|
32
|
nd
|
CTX-M,sulI,tetB, dhfrI
|
nd
|
32
|
SB-32 ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
8
|
8
|
nd
|
CTX-M,qnrB,catI
|
nd
|
33
|
SB-33 ESBL
|
R
|
R
|
R
|
S
|
S
|
R
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
S
|
R
|
R
|
S
|
S
|
S
|
32
|
32
|
nd
|
CTX-M,tet A, eaeA
|
nd
|
34
|
SB-34 ESBL
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
S
|
S
|
16
|
16
|
nd
|
CTX-M,sulI,tetA, aph (3′)-IIa
|
nd
|
35
|
SB-35
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
sulII
|
nd
|
36
|
SB-36
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
-
|
nd
|
37
|
SB-37
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
R
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
-
|
nd
|
38
|
SB-38
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
aph (3′)-IIa
|
nd
|
39
|
SB-39
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
-
|
nd
|
40
|
SB-40
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
ac (3)-IV
|
nd
|
41
|
SB-41
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
hlyA
|
nd
|
42
|
SB-42
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
eaeA
|
nd
|
43
|
SB-43
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
aph (3′)-IIa
|
nd
|
44
|
SB-44
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
-
|
nd
|
45
|
SB-45
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
nd
|
nd
|
nd
|
-
|
nd
|
R: resistant ; S: sensitive; AMX: amoxicillin; AMC: amoxicillin–clavulanic acid; ATZ: aztreonam; C: chloramphenicol; CTR: ceftriaxone; CPD: cefpodoxime; CAZ: ceftazidime;CZ- cefazolin, CFM: cefixime; CTX: cefotaxime; CPM: cefepime; CIP: ciprofloxacin; COT: co-trimoxazole; ERP: ertapenem; F/M: nitrofurantoin; FOX: cefoxitin; GEN : gentamicin; IMP: imipenem; K-kanamycin, NX: norfloxacin; MRP- meropenem; TE: tetracycline; TZP: piperacillin–tazobactam,; nd- not done. |
Citations are available as wild birds as reservoirs of extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase producing E. coli (Alcala et al. 2016). Interestingly, a significant percentage of MDR E. coli isolates detected in black-headed gulls (3.3%), starlings (19.2%), herring gulls (3.3%), deer (3.3%) of Ireland (Carroll et al. 2015). Screening for carbapenem resistance showed that 11.11% (5/45) isolates were resistant to any carbapenem drugs. Three carbapenem-resistant isolates showed keyhole between EDTA and carbapenem drugs, indicating metallo beta-lactamase production (MBL). A conservative approach was utilized for genotypic screening, and only isolated classified as resistant were analysed for AMR encoding genes. All the ESBL producing isolates were MDR type and genotypic screening of the ESBL positive isolates (n = 17) revealed blaCTX-M-1 (n = 10), blaCTX-M-15 (n = 7) and other antibiotic resistance genes like blaTEM (n = 6), blaAmpC (n = 4), qnrS (n = 6), qnrA (n = 3), qnrB (n = 3), sulI (n = 7), sulII (n = 3), tetA (n = 4), tetB (n = 5). Similarly, most of the ESBL E. coli isolates in wild birds were multi-drug resistant, and the most common resistant phenotype are beta-lactams, quinolones, tetracycline, and sulfamethoxazole/trimethoprim (Alcala et al. 2016). The dominant extended-spectrum cephalosporin resistance genes in animals were blaCTX-M-1, while in humans were blaCTX-M-15 and blaCTX-M-14. However, recent studies showed that the prevalence of CTX-M-1 genes in humans (Madec et al. 2015) and rare identification of CTX-M-15 in animal isolates (Dahmen et al. 2013). In the present study, E. coli isolated from sloth bear harbored both CTX-M-1 and CTX-M-15 variants indicated sharing of gene pool. Screens for carbapenemase genes revealed three isolates were positive for the blaNDM-5 gene. All five carbapenem resistant isolates were MDR ESBL producers. The bla NDM positive isolates also co-harboured blaCTX-M1/15, blaTEM-1, qnrS, qnrA, qnrB, sul1, tetA and tetB genes.
The efflux pump-mediated carbapenem resistance was noticed in two (40 %, 2/5) isolates. Efflux pump mediated carbapenem resistance was noticed in E. coli isolated from piglets and dairy calves (Pruthvishree et al. 2017; Murugan et al. 2019). Chloramphenicol (catI, floR), aminoglycoside (ac (3)-IV, aph (3′)-IIa), quinolone (qnrS, qnrA, qnrB), trimethoprim (dhfrI), sulphadimidine (sulI, sulII) and tetracycline resistance genes (tetA, tetB) were commonly detected in the phenotypic resistance isolates. Furness et al. (2017) reported E. coli from the faeces of small free-living mammals were resistant to trimethoprim, ampicillin, ciprofloxacin, and cefotaxime. Screens for virulence genes revealed that five ESBL producing and one carbapenem-resistant isolates harbored any of the virulence genes screened (Table 1). Reports were available in India on ESBL producing and carbapenem resistant E. coli harboring virulence genes in animals (Pruthvishree et al. 2017; Nirupama et al. 2018; Pruthvishree et al. 2018). Plasmid replicon typing of the three blaNDM-5gene-positive E. coli revealed that the NDM gene was on Incl1 plasmid in two isolates (SB27/ 17-CRE, SB28/ 12-CRE) and IncF plasmid was on one isolate (SB28/ 16-CRE). The Incl1 and IncF plasmids also co-harbored PMQR, extended-spectrum cephalosporin, sulphonamide, and tetracycline resistance genes. The IncF plasmids have been reported worldwide among Enterobacteriaceae and are one of the most prevalent incompatibility types involved in the transfer of resistance determinants (Yang et al. 2015). Recently, IncF plasmid carrying multi-drug resistant genes were reported in Shigella flexneri isolates from India (Sethuvel et al. 2019). In our study, the Incl1 and IncF plasmids also co-harbored blaCTX-M-15, blaTEM-1, tetA, and sulI AMR genes. Similarly, IncF plasmid encoding beta-lactamase (NDM-1, OXA-1), aminoglycoside (aacA4, aadA2, and aacC2), and extended-spectrum cephalosporin resistance (CTX-M-15) genes from E.coli ST131 was reported (Bonnin et al. 2012). The plasmid multilocus sequence typing (pMLST) of the blaNDM isolates showed ST 297. Recently we reported blaVIM positive E.coli to isolate with ST 297 (Murugan et al. 2019).
The ESBL producing, carbapenem and multidrug resistant E. coli were divided into three groups in the principal component analysis (PCA). The first two dimensions which included showed the most variance of data (first: 62.5%; second: 7.3%). The third and fourth dimensions, despite being significant, were not plotted due to the complexity of the corresponding output (Fig. 1). Similar PCA analysis of ESBL producing E. coli isolates were carried out in piglets and pig farm workers (Tamta et al. 2020).
In conclusion, the study documented the presence of multi-drug resistant (MDR), ESBL producing, and carbapenem resistant E. coil carrying virulence genes. The isolates co-harboring antibiotic resistant with virulence genes are critically important in public health point of view. To the best of our knowledge, this is the first report of IncF and Incl1 type plasmids carrying multidrug-resistant and NDM carbapenamse producing genes in sloth bear.