The antimicrobial susceptibility
Antimicrobial susceptibility test using microbroth dilution method showed that this strain was resistant to ertapenem (16 µg/ml), imipenem (8 µg/ml), meropenem (> 16 µg/ml), cefepime (> 32 µg/ml), ceftazidime (> 32 µg/ml), cefotaxime (> 32 µg/ml), cefuroxime (> 64 µg/ml), cefazolin (> 32 µg/ml), cefmetazole (> 64 µg/ml), piperacillin/tazobactam (> 256 µg/ml), amikacin (8 µg/ml), gentamicin (64 µg/ml), funantuoyin (128 µg/ml), trimethoprim and sulphame-thoxazole (> 32 µg/ml), aztreonam (> 128 µg/ml), piperacillin (> 256 µg/ml), ciprofloxacin (8 µg/ml), levofloxacin (4 µg/ml), and ceftazidime/avibactam (> 32 µg/ml), while remained susceptible to aztreonam/avibactam (< 0.25 µg/ml), tigecycline (1 µg/ml), colistin B (0.25 µg/ml) and fosfomycin (32 µg/ml).
Genome content
We found a chromosome with size of 4,584,517 bp, and 4 plasmids (pECL-90-1, pECL-90-2, pECL-90-3, and pECL-90-4) ranging in sizes from 6,364 bp to 348,891 bp (Table 1). The sizes of 4 plasmids were was in accordance with the results of S1-PFGE analysis, and the completeness of these plasmids were further verified by PCR loop experiment (Primers and the sizes of products were shown in Table S1). fosA and blaACT-16 were identified in the chromosome, and the other resistance determinants were carried by pECL-90-2 and pECL-90-3.
Table 1
The key features of the Enterobacter hormaeche isolate
|
Size (bp)
|
G + C(%)
|
Resistant determinants
|
plasmid
|
Accession No
|
Chromosome
|
4584517
|
55.58
|
fosA and blaACT−16
|
ST418
|
CP061744
|
pECL-90-1
|
106756
|
51.02
|
NA
|
IncFIB
|
CP061745
|
pECL-90-2
|
348891
|
48.59
|
blaIMP−4, mcr-9.1, aac(3)-IId, aac(6')-IIc,
aac(6')-Ib3,
aph(3'')-Ib, aph(3')-Ia,
aph(6)-Id, blaSFO−1, blaSHV−12,
blaTEM−1B, ere(A), mph(A), catA2, aac(6')-Ib-cr, sul1, tet(D), dfrA19
|
IncHI2/2A
|
CP061746
|
pECL-90-3
|
44961
|
46.49
|
blaNDM-1
|
IncX3
|
CP061747
|
pECL-90-4
|
6364
|
51.21
|
NA
|
Untypeable
|
CP061748
|
NA: not applicable |
MLST analysis assigned ECL-90 to sequence type (ST) 418 (allelic profile 53-35-154-44-45-4-6) (18), which is known as one of the predominant epidemic clones of carbapenemase-producing E. cloacae in China (19, 20).
Antimicrobial Resistant Genes
Antibiotic resistance genes including genes conferring beta-lactam resistance (blaNDM−1, blaIMP−4, blaSFO−1, blaSHV−12, blaTEM−1B, blaACT−16), colistin resistance (mcr-9.1), aminoglycoside resistance [aac(3)-IId, aac(6')-IIc, aac(6')-Ib3, aph(3'')-Ib, aph(3')-Ia, aph(6)-Id, aph(6)-Id], fluoroquinolone and aminoglycoside resistance [aac(6')-Ib-cr], macrolide resistance (ereA, mphA), phenicol resistance (catA2), sulphonamide resistance (sul1), tetracycline resistance (tetD), and trimethoprim (dfrA19) were identified. Overall, the genotypes identified were consistent with the resistance phenotypes.
PCR detection of resistance genes also identified blaNDM−1, blaIMP−4 and mcr-9. Broth conjugation assays using a sodium azide-resistant E. coli J53 isolate as a recipient showed that blaNDM−1 was transferable, but not for mcr-9.1 and blaIMP−4.
Characterization of mcr-9.1
ECL-90 was susceptible to colistin although mcr-9.1 was detected. The IPTG-induced expression of mcr-9.1 in E. coli BL21(DE3) containing pET28a-mcr-9.1 did not confer resistance to colistin, and the resistance to colistin could not be induced by using sub-MIC concentration of colistin. This is consistent with the previous report that mcr − 9.1 is inactive in colistin resistance (21). A recent study with global data revealed that Enterobacter spp., was the predominant host of mcr-9 (37%) (22).
The genetic environment of IMP-4 and mcr-9.1
bla IMP−4 and mcr-9.1 were co-identified on an IncHI2/2A-type plasmid pECL-90-2, which was 348,891 bp in length with an average GC content of 48.59% (Fig. 1). Consistently, a recent report identified that IncHI2-type plasmids may serve as a critical reservoir of mcr-9 (22). Blasting the sequence of pECL-90-2 in GenBank database showed the best matches were plasmid pGW1 carried by a Cronobacter sakazakii strain GZcsf-1 (CP028975, 86.8% query coverage and 99.99% sequence identity) and plasmid p17277A_477 carried by a Klebsiella quasipneumoniae subsp. quasipneumoniae strain M17277 (CP043927, 79.54% query coverage and 99.99% sequence identity). This suggests that the plasmid could widely disseminate among Enterobacteriaceae. The downstream regulatory genes (qseC and qseB) detected in p17277A_477 (CP043927) were replaced by an IS26 here (Fig. 1).
The blaIMP−4 gene was carried by a class I integron designated as In823b, which located in an IS6100-IS26 transposon-like structure (23, 24). The structure has previously been identified as being prevalent mediating the dissemination of blaIMP−4 gene in China (24). The intl1 gene of In823b was disrupted by the insertion of IS26, and a single resistance gene cassette blaIMP−4-attCblaIMP−4 adjacent to a group IIc intron Kl.pn.I3 was identified (Fig. 1). The typical 3’-conserved segment of In823b was absent.
NDM-1 genetic environment
The blaNDM−1 gene was carried on a 44,961-bp IncX3 plasmid (pECL-90-3) (Fig. 2). IncX3-type plasmids mediating the dissemination of blaNDM−1 among these homologous strains have been previously evidenced intensively (25). Query against GenBank showed that pECL-90-3 shared the highest similarity with pNDM5-L725 carried by E. coli strain L725 (CP036205, 99.73% query coverage and 99.99% sequence identity) and pBM527-2 carried by Citrobacter sp. strain CF971 (CP041048, 99.73% query coverage and 99.99% sequence identity).