ABSTRACT
The prevalence of colistin-resistant bacteria limited the usage of colistin in the treatment of clinical multidrug-resistant Gram-negative bacterial infections. Here, we aimed to investigate the prevalence and molecular characterization of mcr-1-carrying isolates from pigeons close to humans following the ban on the use of colistin as an animal feed additive in China. Methods, including PCR, antimicrobial susceptibility testing, conjugation experiments, plasmid replicon typing, genome sequencing, bioinformatics analysis, measurement of growth curves, competition experiments, and plasmid stability assays were used to identify and characterize mcr-1-positive isolates. In total, 45 mcr-1-positive E. coli isolates were acquired from 100 fecal samples, and MICs of colistin ranged from 4 to 8 mg/L. The prevalence of mcr-1-positive E. coli isolates from pigeons was mainly mediated by IncX4 plasmids (39/45), including transferable mcr-1-bearing IncX4 plasmids with fitness advantage in 21 isolates, and nontransferable mcr-1-bearing IncX4 plasmids with fitness disadvantage in 18 isolates. There is a similar structure among the 6 mcr-1-bearing nontransferable IncX4 plasmids and 10 mcr-1-bearing transferable IncX4 plasmids in 16 E. coli isolates that have been sequenced. Plasmid transferability evaluation indicated that the same IncX4 plasmid has different transferability in different E. coli isolates. In conclusion, this study demonstrates that pigeons could act as potential reservoirs for the spread of mcr-1-positive E. coli in China. Transferability of IncX4 plasmids may be influenced by host chromosome in the same bacterial species. Additional research on the factors influencing the transferability of IncX4 plasmids in different bacterial hosts is required to help combat antimicrobial resistance.
IMPORTANCE The emergence of plasmid-mediated colistin resistance gene mcr-1 incurs great concerns. Since the close proximity of pigeons with humans, it is significant to understand the prevalence and molecular characterization of mcr-1-positive isolates in pigeons, to provide a rationale for controlling its spread. Here, we found that the prevalence of mcr-1-positive E. coli from pigeons was mainly mediated by IncX4 plasmids. However, different transferability and fitness of mcr-1-bearing IncX4 plasmids in E. coli were observed, which demonstrated that transferability of IncX4 plasmids could be affected not only by genes on plasmids, but also by chromosomal factors in the same bacterial species. Our finding provided a new insight on studying the factors influencing the transferability of plasmids.
KEYWORDS: Escherichia coli, mcr-1, pigeons, IncX4, transferability, fitness
INTRODUCTION
Colistin is applied as one of the last-resort therapies to treat carbapenem-resistant Enterobacteriales infections (1, 2). Due to the emergence of the plasmid-mediated mobile colistin resistance gene mcr-1 in China and the rapid spread of mcr-1-positive isolates between humans and animals (3, 4), the Chinese government banned colistin as an animal growth promoter on 1 May 2017 (5). Withdrawal of colistin from animal feed may contribute to the decline of mcr-1-positive isolates, but they are still prevalent in humans, animals, and the environment, posing a serious threat to public health (6).
It is known that a large number of pigeons are living in close proximity to humans and animals all around the world. Numerous studies have indicated that pigeon feces is a possible reservoir spreading antibiotic-resistant bacteria (7 to 9). The prevalence of mcr-1-carrying isolates from pigeons likely contributes to its prevalence in the environment, humans, and animals. Therefore, it is significant to understand the prevalence characteristics of mcr-1-positive isolates in pigeons to provide a rationale for controlling its spread. Pigeon, harboring high nutritious value, is one important food animal in China (10). The prevalence of mcr-1-positive isolates from pigeons before 2017 in China has been reported (11), but the prevalence and genomic features of the mcr-1-carrying isolates were not fully explored after the ban of colistin as a feed additive. In this study, we aim to investigate the prevalence and molecular characteristics of mcr-1-bearing isolates from pigeons close to humans after China banned the use of colistin as an animal feed additive.
RESULTS AND DISCUSSION
Bacterial identification and resistance phenotypes.
A total of 45 mcr-1-bearing isolates were recovered from 100 collected fecal samples from pigeons in Jiangsu, China. All mcr-1-carrying isolates were identified as E. coli. This mcr-1 prevalence of pigeon origin in Jiangsu (45/100, 45%) is much higher than previously reported in 2016 (11/46, 23.9%) (11). This indicated that there was no significant decrease in the prevalence of mcr-1-positive isolates from pigeons after China banned the use of colistin as an animal feed additive. Antimicrobial susceptibility testing revealed that MICs of colistin for all mcr-1-bearing isolates ranged from 4 to 8 mg/L (Table 1). These 45 isolates exhibited a high rate of resistance to doxycycline (66.67%, 30/45) and amoxicillin (64.44%, 29/45). In addition, partial isolates showed resistance to enrofloxacin (42.22%, 19/45), florfenicol (33.33%, 15/45), ceftiofur (28.89%, 13/45), and aztreonam (24.44%, 11/45). One isolate LP5-1 conferred resistance to meropenem with the MIC 16 mg/L (Table 1). Further PCR and Sanger sequencing proved that LP5-1 was a blaNDM-5-bearing isolate using primers described earlier (12).
TABLE 1.
The MICs of 45 mcr-1-positive E. coli isolates against 10 antimicrobialsa
| Strain IDs | CST | FFC | CFF | DOX | ATM | STR | AMX | ENR | MEM | RIF |
|---|---|---|---|---|---|---|---|---|---|---|
| LP53-1 | 8 | >64 | >64 | 8 | 32 | >64 | >64 | 2 | ≤0.125 | 8 |
| LP63-1 | 8 | >64 | 1 | >64 | ≤0.25 | 8 | >64 | 1 | ≤0.125 | 8 |
| LP23-1 | 4 | >64 | >64 | 16 | 16 | >64 | >64 | 2 | ≤0.125 | 8 |
| LP15-1 | 8 | >64 | 0.5 | 8 | ≤0.25 | >64 | 4 | ≤0.25 | ≤0.125 | 8 |
| LP21-1 | 8 | >64 | ≤0.25 | 8 | ≤0.25 | >64 | 4 | 0.5 | ≤0.125 | 8 |
| LP98-1 | 8 | >64 | >64 | 8 | 16 | >64 | >64 | 0.5 | ≤0.125 | 4 |
| LP12-1 | 8 | >64 | >64 | 16 | 8 | >64 | >64 | 1 | ≤0.125 | 8 |
| LP45-1 | 8 | 4 | ≤0.25 | 16 | ≤0.25 | 8 | 4 | ≤0.25 | ≤0.125 | 8 |
| LP82-1 | 8 | 8 | >64 | 1 | 16 | 4 | >64 | 1 | ≤0.125 | 4 |
| LP37-1 | 8 | 4 | 0.5 | 16 | 0.5 | 16 | 4 | 0.5 | ≤0.125 | 8 |
| LP20-1 | 8 | 4 | ≤0.25 | 2 | ≤0.25 | 8 | >64 | ≤0.25 | ≤0.125 | 16 |
| LP7-1 | 8 | 4 | ≤0.25 | 1 | ≤0.25 | 8 | >64 | ≤0.25 | ≤0.125 | 4 |
| LP41-1 | 8 | 2 | ≤0.25 | 16 | ≤0.25 | 8 | 4 | 0.5 | ≤0.125 | 8 |
| LP4-1 | 8 | 2 | ≤0.25 | 1 | ≤0.25 | 4 | >64 | ≤0.25 | ≤0.125 | 4 |
| LP31-1 | 4 | 2 | ≤0.25 | 8 | ≤0.25 | 4 | 2 | ≤0.25 | ≤0.125 | 8 |
| LP75-1 | 8 | 8 | ≤0.25 | 2 | ≤0.25 | 8 | 4 | ≤0.25 | ≤0.125 | 8 |
| LP3-1 | 8 | 4 | ≤0.25 | 1 | ≤0.25 | 8 | >64 | ≤0.25 | ≤0.125 | 4 |
| LP8-1 | 4 | 8 | ≤0.25 | 32 | ≤0.25 | >64 | >64 | 1 | ≤0.125 | >256 |
| LP39-1 | 4 | 4 | 0.5 | 32 | ≤0.25 | >64 | >64 | 1 | ≤0.125 | 16 |
| LP65-1 | 4 | 8 | 0.5 | 32 | ≤0.25 | >64 | >64 | 1 | ≤0.125 | >256 |
| LP81-1 | 4 | 8 | 0.5 | 16 | ≤0.25 | >64 | >64 | 1 | ≤0.125 | >256 |
| LP95-1 | 4 | >64 | 1 | 64 | ≤0.25 | 8 | >64 | 2 | ≤0.125 | 8 |
| LP50-1 | 4 | 4 | >64 | 32 | 64 | 16 | >64 | 32 | ≤0.125 | 8 |
| LP92-1 | 4 | 4 | >64 | 32 | 32 | 32 | >64 | 16 | ≤0.125 | 8 |
| LP66-1 | 4 | 8 | 1 | 64 | ≤0.25 | 16 | 1 | 2 | ≤0.125 | 8 |
| LP48-1 | 4 | 8 | 2 | 64 | ≤0.25 | 8 | 1 | 2 | ≤0.125 | 8 |
| LP24-1 | 4 | >64 | 0.5 | 32 | ≤0.25 | 16 | >64 | 32 | ≤0.125 | 8 |
| LP55-1 | 4 | 8 | ≤0.25 | 64 | ≤0.25 | 16 | 1 | 2 | ≤0.125 | 8 |
| LP51-1 | 4 | 8 | >64 | 32 | 32 | >64 | >64 | 16 | ≤0.125 | 8 |
| LP91-1 | 8 | 4 | >64 | 32 | 64 | >64 | >64 | 32 | ≤0.125 | 8 |
| LP85-1 | 4 | 8 | >64 | 32 | 32 | >64 | >64 | 16 | ≤0.125 | 8 |
| LP84-1 | 4 | 4 | 0.5 | 4 | ≤0.25 | 8 | 8 | ≤0.25 | ≤0.125 | 16 |
| LP10-1 | 4 | 4 | 0.5 | 4 | ≤0.25 | 8 | 4 | ≤0.25 | ≤0.125 | 16 |
| LP49-2 | 4 | >64 | 1 | 32 | ≤0.25 | >64 | >64 | 64 | ≤0.125 | 8 |
| LP62-2 | 8 | >64 | 2 | 32 | ≤0.25 | >64 | 8 | 32 | ≤0.125 | >256 |
| LP43-1 | 4 | >64 | 0.5 | 32 | ≤0.25 | >64 | >64 | 16 | ≤0.125 | 8 |
| LP80-1 | 8 | 8 | 0.5 | 32 | ≤0.25 | 8 | 4 | ≤0.25 | ≤0.125 | 16 |
| LP94-1 | 4 | 4 | 0.5 | 2 | ≤0.25 | 16 | 4 | 16 | ≤0.125 | 8 |
| LP87-1 | 8 | 4 | 0.5 | 64 | ≤0.25 | 16 | 4 | ≤0.25 | ≤0.125 | 16 |
| LP64-1 | 4 | >64 | ≤0.25 | 32 | ≤0.25 | >64 | >64 | 1 | ≤0.125 | >256 |
| LP71-1 | 4 | 4 | 1 | 4 | ≤0.25 | 8 | 2 | ≤0.25 | ≤0.125 | 16 |
| LP62-1 | 4 | 4 | 0.5 | 32 | ≤0.25 | 8 | 2 | 16 | ≤0.125 | 4 |
| LP25-1 | 8 | 8 | >64 | 32 | 16 | 16 | >64 | 16 | ≤0.125 | 8 |
| LP68-1 | 8 | >64 | >64 | 16 | 2 | >64 | >64 | 16 | ≤0.125 | 8 |
| LP5-1 | 4 | 32 | >64 | 64 | ≤0.25 | 16 | >64 | ≤0.25 | 16 | 16 |
| ATCC 25922 | 0.25 | 4 | 0.5 | 1 | ≤0.25 | 4 | 4 | ≤0.25 | ≤0.125 | 4 |
CST, colistin; FFC, florfenicol; CFF, ceftiofur; DOX, doxycycline; ATM, aztreonam; STR, streptomycin; AMX, amoxicillin; ENR, enrofloxacin; MEM, meropenem; RIF, rifampicin.
Transferability of mcr-1 and mcr-1-associated plasmid types.
To investigate the transmissibility of the mcr-1 gene, all mcr-1-harboring isolates were subjected to conjugation experiments. The genetic structures carrying the mcr-1 gene of 23 E. coli isolates with colistin resistance phenotypes were successfully transferred into recipient E. coli C600 or J53. To learn the location of mcr-1, PCR-based replicon typing (PBRT) was performed for all 45 mcr-1-positive isolates and 23 transconjugants obtained by conjugation assays. Replicon types of 45 mcr-1-positive isolates and 23 transconjugants are shown in Table 2. We confirmed that mcr-1 was located on IncX4-type plasmids in 39 isolates (Table 2) using specific primers IncX4-F and MCR1-RC-F targeting the IncX4-type plasmid replicon and the mcr-1 gene with a product length of 2,854 bp (13). This indicated that mcr-1-bearing IncX4-type plasmid is the main vector for prevalence of mcr-1 in pigeons from Jiangsu. It is well known that mcr-1-harboring IncX4-type plasmid is one of the most prevalent conjugative plasmids worldwide (13 to 15). However, mcr-1-bearing IncX4-type plasmids in 18 out of 39 isolates were nontransferable in this study. In the remaining 6 isolates carrying no mcr-1-harboring IncX4-type plasmid, we found that mcr-1 is located on the IncI2 plasmids in two isolates, LP64-1 and LP71-1, by comparing the replicon types of parent isolates and their corresponding transconjugants (Table 2). The mcr-1 in the other four isolates were nontransferable; therefore, we could not confirm the location of the mcr-1 by PBRT (Table 2).
TABLE 2.
Transferability of mcr-1 and PBRT of 45 mcr-1-carrying isolates and corresponding transconjugants
| Strain IDs | Replicon types | Recipient of conjugation | Transferability of mcr-1 | Replicon types of transconjugants | Location of mcr-1 |
|---|---|---|---|---|---|
| LP53-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP63-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP23-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP15-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP21-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP98-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP12-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP45-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP82-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP37-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP20-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP7-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP41-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP4-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP31-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP75-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP3-1 | IncX4, IncFIB, IncF | Nontransferable | IncX4 | ||
| LP8-1 | IncX4, IncFIB, IncF, IncI1 | J53 | Transferable | IncX4 | IncX4 |
| LP39-1 | IncX4, IncFIB, IncF, IncI1 | C600 | Transferable | IncX4 | IncX4 |
| LP65-1 | IncX4, IncFIB, IncF, IncI1 | J53 | Transferable | IncX4 | IncX4 |
| LP81-1 | IncX4, IncFIB, IncF, IncI1 | J53 | Transferable | IncX4 | IncX4 |
| LP95-1 | IncX4, IncFIB, IncF, IncI1 | C600 | Transferable | IncX4 | IncX4 |
| LP50-1 | IncX4, IncFIB, IncF, IncI2 | J53 | Transferable | IncX4 | IncX4 |
| LP92-1 | IncX4, IncFIB, IncF, IncI2 | J53 | Transferable | IncX4 | IncX4 |
| LP66-1 | IncX4, IncFIB, IncF, IncI2 | J53 | Transferable | IncX4 | IncX4 |
| LP48-1 | IncX4, IncFIB, IncF, IncI2 | J53 | Transferable | IncX4 | IncX4 |
| LP24-1 | IncX4, IncFIB, IncF, IncI2 | C600 | Transferable | IncX4 | IncX4 |
| LP55-1 | IncX4, IncFIB, IncF, IncI2 | C600 | Transferable | IncX4 | IncX4 |
| LP51-1 | IncX4, IncFIB, IncF, IncI1, IncI2 | C600 | Transferable | IncX4 | IncX4 |
| LP91-1 | IncX4, IncFIB, IncF, IncI1, IncI2 | C600 | Transferable | IncX4 | IncX4 |
| LP85-1 | IncX4, IncFIB, IncF, IncI1, IncI2 | C600 | Transferable | IncX4 | IncX4 |
| LP84-1 | IncX4, IncI1, IncI2 | C600 | Transferable | IncX4 | IncX4 |
| LP10-1 | IncX4, IncI1, IncI2 | C600 | Transferable | IncX4 | IncX4 |
| LP49-2 | IncX4 | C600 | Transferable | IncX4 | IncX4 |
| LP62-2 | IncX4, IncN | J53 | Transferable | IncX4 | IncX4 |
| LP43-1 | IncX4, IncI1 | C600 | Transferable | IncX4 | IncX4 |
| LP80-1 | IncX4, IncFIB | J53 | Transferable | IncX4 | IncX4 |
| LP94-1 | IncX4, IncI2, IncI1, IncY | C600 | Transferable | IncX4 | IncX4 |
| LP87-1 | IncX4, IncFIB, IncI1 | Nontransferable | IncX4 | ||
| LP64-1 | IncI2, IncI1 | J53 | Transferable | IncI2 | IncI2 |
| LP71-1 | IncI2, IncI1, IncHI1, IncY | C600 | Transferable | IncI2 | IncI2 |
| LP62-1 | IncI2, IncFIB, IncF | Nontransferable | IncI2 | ||
| LP25-1 | IncI2, IncHI2, IncFIB, IncF | Nontransferable | IncI2 | ||
| LP68-1 | IncHI2, IncFIB, IncF | Nontransferable | IncHI2 | ||
| LP5-1 | IncFIB, IncF | Nontransferable | Chromosome |
Whole-genome sequencing analysis.
Twenty-one mcr-1-carrying isolates were selected to perform the genome sequencing, including 16 isolates carrying mcr-1-bearing IncX4 plasmids (based on their MICs, replicon typing, and transferability of IncX4 plasmids), 1 isolate carrying mcr-1-bearing IncI2 plasmid, and 4 isolates (LP25-1, LP62-1, LP68-1, and LP5-1) with undetermined location of mcr-1 by conjugation and PBRT. By analyzing draft genomes, it was determined that the mcr-1-bearing contig also carried the IncHI2A replicon gene in isolate LP68-1, while the mcr-1 gene and the IncI2 replicon gene were on the same contig in isolates LP25-1 and LP62-1. Therefore, we confirmed that isolates LP25-1 and LP62-1 both contained mcr-1-harboring IncI2 plasmids, and that LP68-1 carried an mcr-1-harboring IncHI2 plasmid. However, we are still unable to determine the localization of mcr-1 in LP5-1 based on the draft genome.
The phylogenetic tree showed that 21 E. coli isolates were grouped into two major clusters and were diverse. These distinct 21 mcr-1-positive isolates were assigned into 11 known sequence types (STs) (Fig. 1a), including ST646 (n = 5), ST155 (n = 4), ST38 (n = 2), ST224 (n = 1), ST8024 (n = 1), ST7153 (n = 1), ST939 (n = 1), ST648 (n = 1), ST2351 (n = 1), ST6164 (n = 1), and ST6775 (n = 1). Isolates LP81-1 and LP84-1 belonged to novel STs (Fig. 1a). Three isolates (PT62, PT76, and PT77) coharboring mcr-1, tet(X4), and blaNDM-1 were also acquired from this pigeon farm, and we had reported this previously (16). Isolate LP5-1 was very closely related to these three isolates and belonged to ST6775 (Fig. 1a). Han et al. reported that mcr genes were present among fosA3-carrying E. coli at the proportion of 77.8%. These fosA3-carrying E. coli were also isolated from pigeons but from Guangdong, China in 2016 (17). These mcr-bearing isolates in their study were categorized into the highly abundant STs that differed from the STs in our study, except for ST155. The phylogenetic relationship and multiple STs suggest that mcr-1-carrying isolates in pigeons from China are diverse.
FIG 1.
The genetic relationship of 21 mcr-1-carrying isolates from pigeons and the distribution of replicons and antimicrobial resistance genes. (a) Phylogenetic tree of 21 mcr-1-positive E. coli isolates and the distribution of replicons. The presence or lack of replicons is colored in purple or light gray, respectively. The legend shows the similarity (%) of the replicons. (b) The distribution of antimicrobial resistance genes. The blue rectangles indicate the presence of resistance genes. The legend indicates the similarity (%) of the antimicrobial resistance genes. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article. Three isolates (PT62, PT76 and PT77) co-harboring mcr-1, tet(X4) and blaNDM-1, that we had reported previously, were presented in this figure.
Prevalence of acquired antibiotic resistance genes.
Thirty-three resistance genes, conferring resistance to diverse antibiotics, were identified in these 21 isolates that have been sequenced. LP68-1, containing 12 resistance genes, was the isolate with the largest number of resistance genes among 21 isolates. With the exception of mcr-1, the tetracycline resistance gene tet(A) was the most prevalent and was present in 17 isolates. A total of nine genes conferring resistance to aminoglycoside were detected, including aac(3)-IId (n = 1), aadA16 (n = 1), aadA1 (n = 3), aadA2 (n = 1), aadA5 (n = 3), aph(3′)-Ia (n = 2), aph(3′)-Ic (n = 2), strA (n = 6), and strB (n = 7). Additionally, three sulfonamides resistance genes, sul1 (n = 3), sul2 (n = 6), and sul3 (n = 1), and five trimethoprim resistance genes, dfrA1 (n = 1), dfrA12 (n = 1), dfrA14 (n = 3), dfrA17 (n = 4), and dfrA27 (n = 1), were identified. Furthermore, macrolide resistance genes mph(A) (n = 1) and mef(B) (n = 1), phenicol resistance genes cmlA1 (n = 2) and floR (n = 7), quinolone resistance genes oqxA (n = 1), oqxB (n = 1), and qnrS1 (n = 3), fosfomycin resistance gene fosA (n = 1), and rifampicin resistance gene arr-3 (n = 2) were found. Importantly, CTX-M-type extended-spectrum-β-lactamase-encoding genes blaCTX-M-14 (n = 1), blaCTX-M-15 (n = 1), and blaCTX-M-65 (n = 3), OXA-type extended-spectrum-β-lactamase-encoding gene blaOXA-10 (n = 1), and carbapenems resistance gene blaNDM-5 (n = 1) were also found (Fig. 1b).
Genetic environments of mcr-1.
To investigate the genomic characterization of prevalent mcr-1-bearing IncX4 plasmids in the pigeon farm and the localization of mcr-1 in LP5-1, isolates LP50-1 and LP5-1 were selected to be sequenced with the nanopore sequencing. Genetic analysis showed that isolate LP50-1 harbored a chromosome and seven plasmids consisting of pLP50-1-101kb, pLP50-1-87kb, pLP50-1-MCR1, pLP50-1-8kb, pLP50-1-4kb, pLP50-1-3kb, and pLP50-1-1kb. The mcr-1 gene was located on the pLP50-1-MCR1 that was a typical IncX4-type plasmid. Sequence analysis revealed that the pLP50-1-MCR1 shared 99.99% identity at 100% coverage with plasmid p2017.19.01CC (LC511660) in E. coli isolated from humans in Vietnam and plasmid pEC7-mcr-1 (CP060967) in E. coli isolated from humans in China. Additionally, it exhibited 99.98% identity at 100% coverage with plasmid pMCR-NMG38 (MK836307) in E. coli isolated from swine in China (Fig. 2a). These three similar plasmids are also mcr-1-positive plasmids. High similarity of these mcr-1-harboring IncX4 plasmids indicated the prevalence of E. coli carrying mcr-1-harboring IncX4 plasmids between animals and humans in different regions. Comparing pLP50-1-MCR1 and draft assembly sequences of another 15 isolates carrying mcr-1-bearing IncX4 plasmids that have been sequenced with Illumina HiSeq 2500 platform, we confirmed that all the mcr-1-harboring IncX4 plasmids have a similar structure (Fig. 2b). LP5-1 harbored a chromosome and six plasmids consisting of pLP5-1-106kb, pLP5-1-NDM-47kb, pLP5-1-37kb, pLP5-1-9kb, pLP5-1-3kb, and pLP5-1-1kb. The mcr-1 gene was located on the chromosome and mediated by the ISApl1-mcr-1-pap2-ISApl1, which was called Tn6330 mobile element and responsible for transfer of mcr-1 (18). The pLP5-1-NDM-47kb was a typical IncX3 plasmid-carrying blaNDM-5. Isolates LP5-1, PT62, PT76, and PT77 had identical chromosomes and plasmids (Fig. 1a), but PT62, PT76, and PT77 additionally carried a transferable tet(X4)-bearing plasmid (16), indicating that the formation of PT62, PT76, and PT77 may be caused by horizontal transfer of tet(X4)-harboring plasmids.
FIG 2.
Circular comparison of mcr-1-bearing IncX4 plasmids. (a) Circular comparison of mcr-1-bearing plasmid pLP50-1-MCR1 with three similar IncX4 plasmids in the NCBI database. The outmost circle indicates the plasmid pLP50-1-MCR1 with genes annotated. (b) Circular comparisons between pLP50-1-MCR1 and draft assembly sequences of another 15 isolates carrying mcr-1-bearing IncX4 plasmids that have been sequenced with the Illumina HiSeq 2500 platform.
Characterization of nontransferable mcr-1-bearing IncX4 plasmids.
In total, the mcr-1-bearing IncX4 plasmids in 18 out of 39 isolates were nontransferable (Table 2). Six isolates carrying nontransferable IncX4 plasmids were sequenced with the Illumina HiSeq 2500 platform, and they belonged to ST646 (n = 5) and ST6164 (n = 1). However, nontransferable IncX4 plasmids had a highly similar structure to transferable IncX4 plasmids, including type IV secretion systems that are responsible for plasmid conjugative transfer. Therefore, we speculated that it may be factors other than the mcr-1-bearing IncX4 plasmids that cause this type of plasmid to fail to transfer horizontally. According to the results of PBRT, we found that all isolates carrying nontransferable IncX4 plasmids had the same replicons (IncX4, IncFIB, and IncF), except the ST6164 E. coli LP87-1. It is possible that all the isolates except LP87-1, carrying nontransferable IncX4 plasmids, were the ST646 E. coli (Fig. 1a and Table 2). This indicated that the ST646 E. coli isolates could spread mcr-1-bearing IncX4 plasmids by clonal dissemination rather than horizontal transfer. In fact, the phenomenon that commonly conjugative plasmids cannot be transferred into the recipient appeared in other studies as well (14, 19, 20).
Fitness effects of mcr-1-bearing IncX4 plasmids.
Isolates LP53-1 and LP63-1 carrying nontransferable IncX4 plasmids and isolates LP39-1 and LP51-1 carrying transferable IncX4 plasmids were selected to study the fitness of IncX4 plasmids. We first acquired the mcr-1-positive transconjugant CLP39-1 of LP39-1, and eliminated mcr-1-bearing IncX4 plasmids by the CRISPR-Cas9 system in four isolates to acquire strains LP53-1ΔIncX4, LP63-1ΔIncX4, LP39-1ΔIncX4, and LP51-1ΔIncX4. Then we assessed plasmid fitness effects by measurement of growth curves and competition assays. Growth curves indicated that the growth rate of LP39-1ΔIncX4 was slower than that of LP39-1 (Fig. 3a). No significant differences in growth rates were observed between LP51-1 and LP51-1ΔIncX4 (Fig. 3b), and between C600 and CLP39-1 (Fig. 3c). However, the growth rate of LP53-1 was slower than that of LP53-1ΔIncX4 (Fig. 3d). The growth rate of LP63-1 was slower than that of LP53-1ΔIncX4 in the logarithmic growth phase, and there was no obvious difference between the two after the logarithmic growth phase (Fig. 3e). Competition assays showed that LP39-1, LP51-1, and CLP39-1 were more competitive than LP39-1ΔIncX4, LP51-1ΔIncX4, and C600, but LP53-1 and LP63-1 were less competitive than LP53-1ΔIncX4 and LP63-1ΔIncX4 (Fig. 3f). The above results suggested that carriage of transferable mcr-1-bearing IncX4 plasmids improves host fitness, but carriage of nontransferable mcr-1-bearing IncX4 plasmids imposed a burden on the host. A previous report indicated that the mcr-1-carrying IncX4 plasmid increased fitness of E. coli DH5α (19), which was consistent with our findings on strains in which mcr-1-carrying IncX4 plasmid was transferable. In addition, the transferability may contribute to increased fitness of IncX4 plasmids on host bacteria (21). The transferable mcr-1-bearing IncX4 plasmids make a significant contribution to the prevalence of mcr-1 from the fitness perspective. We electroporated the nontransferable mcr-1-positive IncX4 plasmid of LP63-1 into LP39-1ΔIncX4 to acquire LP39-1ΔIncX4::IncX4, and found that the mcr-1-positive IncX4 plasmid could be transferred from LP39-1ΔIncX4::IncX4 into C600 again. We speculated that the transferability of these plasmids was related to genes of chromosome. Further study on the inhibitor to the transferability of IncX4 plasmids is required to contribute to the control of antimicrobial resistance. Eight isolates, including four isolates carrying transferable IncX4 plasmids and four isolates carrying nontransferable IncX4 plasmids, were randomly selected to assess stability of mcr-1-bearing IncX4 plasmids. The results showed that the transferable IncX4 plasmids and nontransferable IncX4 plasmids remained relatively stable after long-term passages (Fig. 3g). It is possible that the fitness advantage of transferable mcr-1-bearing IncX4 plasmids and the stability of mcr-1-bearing IncX4 plasmids allow mcr-1 to remain prevalent without selection of colistin after colistin was banned as an animal feed additive.
FIG 3.
Fitness effects of mcr-1-bearing IncX4 plasmids on host. (a) Growth curves of LP39-1ΔIncX4 and LP39-1. (b) Growth curves of LP51-1ΔIncX4 and LP51-1. (c) Growth curves of C600 and CLP39-1. (d) Growth curves of LP53-1ΔIncX4 and LP53-1. (e) Growth curves of LP63-1ΔIncX4 and LP63-1. (f) Dynamics of competition experiments between plasmid-free E. coli isolates and E. coli isolates carrying mcr-1-harboring plasmids. (g) Stability of four transferable mcr-1-bearing IncX4 plasmids and four nontransferable mcr-1-bearing IncX4 plasmids.
Conclusions.
To conclude, this study clearly illustrates that pigeons could act as reservoirs of mcr-1-positive E. coli in China. The prevalence of mcr-1-positive E. coli isolates from pigeons was mainly mediated by IncX4 plasmids, including transferable mcr-1-bearing IncX4 plasmids with fitness advantage in some E. coli isolates, and nontransferable mcr-1-bearing IncX4 plasmids with fitness disadvantage in some E. coli isolates. Plasmid transferability evaluation indicated that the same IncX4 plasmid has different transferability in different E. coli isolates. The transferability of these plasmids may be influenced by host chromosome in the same bacterial species. Additional research on the factors influencing the transferability of IncX4 plasmids in different bacterial hosts is required to help combat antimicrobial resistance. Continuous monitoring of mcr-1-positive E. coli from pigeons is necessary to understand its prevalence trends, and effective strategies to prevent such prevalence are urgently needed.
MATERIALS AND METHODS
Sample collection and identification of mcr-1.
In June 2021, 100 fresh fecal samples were collected from a pigeon farm located in Jiangsu, China. Samples were incubated in brain heart infusion (BHI) broth for 4 h at 37°C with continuous shaking (200 rpm). Then 100-μL cultures were transferred into fresh BHI broth containing colistin (2 mg/L) and incubated for 12 to 16 h at 37°C with shaking. Subsequently, colistin-resistant samples were plated on MacConkey agar plates and incubated for 12 to 16 h at 37°C. One or more colonies with varied morphological traits per sample were chosen and subsequently screened for the presence of mcr-1 using PCR and Sanger sequencing (22). Bacterial species of all mcr-1-positive isolates were identified by 16S rRNA sequencing.
Antimicrobial susceptibility testing.
Antimicrobial susceptibility profiles were determined for all the mcr-1-positive E. coli isolates by the broth microdilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines (23). We used the following 10 antimicrobial agents: doxycycline, enrofloxacin, ceftiofur, streptomycin, amoxicillin, rifampicin, florfenicol, meropenem, colistin, and tigecycline. Results were interpreted according to the CLSI standards (M100-S30 and M31-A3) and the European Committee on Antimicrobial Susceptibility Testing breakpoints (EUCAST, version 12.0) (http://www.eucast.org/clinical_breakpoints/).
Conjugation experiments and plasmid replicon typing.
Conjugation experiments were done using mcr-1-bearing isolates as donors and E. coli C600 (rifampicin resistance) as the recipient strain. E. coli J53 (sodium azide resistance) was employed as the recipient if the isolate was resistant to rifampicin or failed to conjugate with E. coli C600. Briefly, the donor and recipient cultures were mixed in a 1:1 proportion, and 0.1 mL of the mixed cultures were plated onto solid lysogeny broth (LB) medium and incubated at 37°C overnight. Subsequently, conjugation mixtures on LB agar plates were collected and diluted in sterile saline. Transconjugants were selected by streaking the conjugation mix on LB agar plates supplemented with colistin (2 mg/L) and rifampicin (300 mg/L) or sodium azide (200 mg/L). The presence of mcr-1 in transconjugants was checked by PCR and colistin resistance phenotypes. The PBRT (24) and the specific primers targeting the IncX4, IncHI2, and IncI2 replicons (13) were used to identify the plasmid replicons of mcr-positive isolates and corresponding transconjugants.
Genome DNA sequencing and bioinformatic analysis.
According to MICs, transferability of mcr-1, and replicon types, 21 mcr-1-carrying isolates were selected to perform the genome sequencing. Genome DNA extractions were performed using the FastPure Bacteria DNA Isolation minikit (Vazyme, China) following the manufacturer’s instructions. The sequencing was conducted using the Illumina HiSeq 2500 platform to generate accurate short raw reads. SPAdes was used to assemble the short-read Illumina raw sequences to acquire draft genomes (25). The multilocus sequence typing (MLST), plasmid replicons, and antimicrobial resistance genes were analyzed using tools MLST (26), PlasmidFinder (27) and ResFinder (28) (https://www.genomicepidemiology.org/services/). Two representative isolates were further sent out for QitanTech nanopore single-molecule long-read sequencing (16, 29). To acquire the complete sequence of chromosome and plasmids, Unicycler was used to perform de novo assembly with the hybrid strategy based on the Illumina short-read data and QitanTech nanopore long-read data (30, 31). The complete genome sequences were then annotated using Rapid Annotation using the Subsystems Technology annotation website server (https://rast.nmpdr.org/rast.cgi) (32). The draft genomes were annotated using Prokka (33), and a pan-genome analysis was conducted using Roary (34). The phylogenetic tree was constructed using FastTree based on single nucleotide polymorphisms of core genomes (35) and visualized by iTOL (36). TBtools was used to visualize the distributions of antimicrobial resistance genes (37). Circular comparisons between mcr-1-harboring plasmids were performed using the BRIG (38). Comparisons between plasmids and draft genome sequences were performed using the website server (https://server.gview.ca/).
Curing of mcr-1-bearing IncX4 plasmids.
Plasmid pCure-rif was employed to cure mcr-1-bearing IncX4 plasmids in E. coli. pCure-rif was constructed using the ClonExpress II One Step Cloning kit (Vazyme, China) as follows. First, we replaced the kanamycin resistance gene in pSGKP-km (Addgene plasmid ID: 117233) (39) with the rifampicin resistance gene arr-2 from the clinical isolate PK8215 (CP080122) to generate plasmid pSGKP-rif. Then the gene encoding the Cas9 nuclease was amplified from plasmid pCasKP-hph (Addgene plasmid ID: 117232) (39) and cloned into pSGKp-rif, creating the plasmid pSGKp-Cas9-rif. Finally, the araC gene and l-arabiinducible promoter araBAD were amplified from pCasKP-hph and subsequently inserted into the plasmid pSGKp-Cas9-rif as the promoter for Cas9, resulting in pCure-rif. The 20-nt base-pairing region of an sgRNA targeting the mcr-1 gene was designed through the online tool CHOPCHOP (http://chopchop.cbu.uib.no/). The paired oligonucleotides were randomly selected (mcr-spacer-F: tagtAAAGCTGTTTGATGTCACCG and mcr-spacer-R: aaacCGGTGACATCAAACAGCTTT), then annealed and ligated to the BsaI-digested pCure-rif using T4 DNA ligase (NEB, USA), resulting in pCure-rif-mcr, and then electroporated into competent cells of isolates carrying mcr-1-bearing IncX4 plasmids, followed by selection on agar plates supplemented with 100 mg/L rifampicin. Overnight cultures of strains carrying pCure-rif-mcr were diluted in 1 mL LB broth containing 0.1% l-arabinose in combination with 100 mg/L rifampicin and incubated for 12 h at 37°C. Then, the culture was plated on LB agar plates, and colonies lacking mcr-1-bearing IncX4 plasmid were selected and confirmed by PCR. For curing plasmid pCure-rif-mcr, colonies lacking mcr-1-bearing IncX4 plasmid were streaked onto LB agar plates containing 5% sucrose at 37°C for 12 h.
Measurement of growth curves.
Overnight cultures from single colonies were adjusted to the 0.5 McFarland standard. Then, 5 μL adjusted cultures were diluted into 5 mL LB broth and incubated at 37°C, 200 rpm for 12 h. Bacterial growth was monitored by measuring the OD620 every 1 h using Multuskan FC (Thermo Fisher Scientific) (40).
Competitive fitness.
The relative competitive fitness of plasmid-carrying clones was determined in pairwise serial competition experiments with the isogenic plasmid-free strain. Briefly, overnight cultures of each competitor were adjusted to the 0.5 McFarland standard and mixed in a 1:1 ratio, and 0.05 mL mixed competitors were transferred into 5 mL fresh LB broth (day 0). After 24 h of growth at 37°C, 0.05 mL cultures were transferred into 5 mL fresh LB broth (day 1), and then two transfers were performed (days 2 and 3). The colony-forming unit (CFU) of each competitor were determined by plating serial dilutions on antibiotic-free LB plates and selective plates containing 2 mg/L colistin. loge ratio was calculated as follow: loge ratio = loge R(t)–loge R(0), where “R” represents the ratio of the CFU of plasmid-bearing and plasmid-free cells in the competing cultures and “t” represents the time in days. If there is no difference in fitness between competing strains, the loge ratio will be considered 0. If plasmid carriage improves host fitness relative to that of plasmid-free strains, loge Ratio is positive, and it is negative if plasmid carriage reduces host fitness (40, 41).
Plasmid stability.
To learn the stability of mcr-1-bearing IncX4 plasmids, amounts of 5 μL overnight cultures from single colonies were transferred into 5 mL antibiotic-free LB broth on the first day. Then, serial transfers of 5 μL cultures to 5 mL fresh LB broth were performed every 12 h and passaged for 30 days (60 passages; ~600 generations). Cultures from 60 passages were serially diluted in 0.9% saline and plated onto LB plates without antibiotics and LB plates supplemented with colistin (2 mg/L). The stability frequency was calculated as follow: (CFU on LB plate containing colistin/CFU on antibiotic-free LB plate) × 100% (40).
Data availability statement.
The nucleotide sequences acquired in this study have been deposited under the NCBI BioProject with the accession number PRJNA861424.
ACKNOWLEDGMENTS
This work was supported by the Postgraduate Research & Practice Innovation Program of Jiangsu Province (no. KYCX22_3537), the National Natural Science Foundation of China (no. 31872526), Jiangsu Agricultural Science and Technology Innovation Fund (no. CX(21)2010), the China Postdoctoral Science Foundation (no. 2020M671632), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
We declare no conflicts of interest.
Contributor Information
Zhiqiang Wang, Email: zqwang@yzu.edu.cn.
Ruichao Li, Email: rchl88@yzu.edu.cn.
Daria Van Tyne, University of Pittsburgh School of Medicine.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The nucleotide sequences acquired in this study have been deposited under the NCBI BioProject with the accession number PRJNA861424.