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.