3.1 MDR Klebsiella pneumoniae M297-1 isolates from Red Kangaroo
Overall, thirty-four isolates were isolated from animal fecal samples in
Zhengzhou Zoo, including 33 isolates of Escherichia coli and 1
isolate of Klebsiella pneumoniae . The susceptibility profiles
indicated that most of the isolates were susceptible to some of the
common antimicrobial drugs in clinical use, while the antimicrobial
resistances of the isolates from Red Kangaroo (n=4) were serious (Table
1). Among them, compared with other isolates, the isolate ofKlebsiella pneumoniae (named as M297-1) had high resistance to
major groups antimicrobial drugs including
group A (Ampicillin ), group
B (Cefuroxime, Cefepime,
Ceftriaxone, Ciprofloxacin, Levofloxacin, Trimethoprim/
Sulfamethoxazole, Ampicillin/Sulbactam ), group C (Aztreonam,
Chloramphenicol ), group U (Nitrofurantoin ). Furthermore, this
isolate was subjected to whole genome sequencing analysis.
3.2 Genomic structure of
the Chr-M297-1 harboring blaDHA-3,blaSHV-1 and blaCTX-M-14
The chromosomal DNA of M297-1
(named as Chr-M297-1) is 5,750,384
bp in size, and exhibits 100% identity with a query coverage of 100%
to the K.pneumoniae by
Ribosomal Multilocus Sequence Typing (rMLST). Isolate M297-1 belongs to
ST 290)(https://pubmlst.org/rmlst/) ,
has the highest homology with Klebsiella pneumoniae isolate
(99.93%) from human clinical samples based on phylogenetic tree of 16s
rRNA (Figure S1). For ST290, only five isolates were recorded in the
database, of which one strain was from dairy cow and the other four
strains were from human clinical samples. Chr-M297-1 possesses 9 gene
islands and 1 prophage, 169 antimicrobial genes most importantly
carrying 3 β-lactamase genes
(bla DHA-3, bla SHV-1, andbla CTX-M-14), FosA5, dfrA3, sul3 etc.(Table 2). In addition, the resistance pump was mainly composed of ABC
family genes, as well as MFS, SMR and MATE family genes.
3.3 Genomic structure of
plasmid carrying important antimicrobial genes
K.pneumoniae isolate M297-1 contains two plasmids
pM297-1.1(222,864bp) and
pM297-1.2(225,763bp), which belong to IncFIB(K) and
IncFII(K),respectively. pM297-1.1
contains five Genomic_islands, two β-lactamase genes
(bla CTX-M-14and bla TEM-191),
two aminoglycoside resistant genes
(APH(3”)-Ib and APH(6)-Id), quinolone
resistance gene
QnrS1 and others. According to
PLSDB database, only three plasmids were similar to pM297-1.1 focusing
on multidrug resistant region, which is
TnpA-other-blaCTX-M-14-TnpA/Tn903-blaTEM-191 – TnpA/IS2-other-
QnrS1 -tnpR/Tn552-ISKpn19- tnpR/Tn4653 (Figure 1).
Specially, conjugal transfer protein genes (traABCDEGHIKLMNPQTUVX-
trbBCEFI- FinO ), Lactose permease and two aminoglycoside resistant
genes (transposase- APH(3”)-Ib and APH(6)-Id ) co-located
in the same larger Genomic_island
11 ( 42,044bp-101,359bp).
pM297-1.2 contains 7
Genomic_islands and 1 prophage. Comparing to pM297-1.1, pM297-1.2
harbors much more antimicrobial genes including 2
extended-spectrum β-lactamase
(ESBLs) gene
(bla CTX-M-3and bla TEM-1), 5 aminoglycoside resistance genes
(APH(6)-I,APH(3’)-Ia, AAC(3)-IIa,aadA16, AAC(6’)-Ib-cr ) ,
fluoroquinolones resistant genes
(QnrB2 and QnrS1 ), mphA, sul1, QacEdelta1
multidrug exporter, etc . Notably, pM297-1.2 carries
chloramphenicol resistant gene (floR), tetracycline resistant gene
(tetG), sulfonamide resistance gene (sul1,sul2) (Table 2), but pM297-1.1
does not contain these genes.
Furthermore, the genomic region of
pM297-1.1 and pM297-1.2 were
divided into different Multidrug Resistance regions according to
composition of antimicrobial genes and related antimicrobial genes. The
pM297-1.1possessed one Multidrug resistance gene region
(TnpA-other-bla CTX-M-14-InsC/Tn903-bla TEM-191-insD-other-insC21-QnrS1 -
tnpR/Tn552 -other-tnpR/Tn4653) which was entirely conserved in
p911021-tetA, p1_020098, pLAP2_020009, NZ_CP040176.1
(similarity,>99.86%). This genomic region was flanked by InsC/Tn903
and insC21 sequences and co-harbored other genes encoding
extended-spectrum β-lactamases and
one gene conferring resistance to
fluoroquinolones.
The multidrug resistance region of pM297-1.2 appeared to consists of
three parts, which are named MDR1, MDR2 and MDR3. MDR1
was bracketed by derivatives of
Tn903, Tn3 and Tn1721(Tn21 subfamily) and harbored resistance genes
against aminoglycoside, sulfonamide and chloramphenicol. MDR2 carried
one class I integron (intl1) and co-harbored other genes resistance to
aminoglycoside, sulfonamide, rifampicin, and
fluoroquinolones. Notably, MDR3
was bracketed by derivatives of Tn3 harboring two β-lactamases genesbla CTX-M-3 and bla TEM-1and Tn4653 gene carrying one gene resistance to fluoroquinolones. Some
plasmid, including pKp21774-135, pKPNH54.1, pKF3-94, pL22-5, p2,
pR210-2-CTX, pCTXM15_020019, pSCM96-1, pNH25.2, were found to possess
the MDR3 by a database search. Obviously, these two plasmids evolved
from other plasmids by inserting, deleting or replacing.