4 DISCUSSION
In this study, we reported firstly the multidrug-resistantKlebsiella pneumoniae M297-1 carried by healthy Red Kangaroo, which is suggested that asymptomatic animal hosts were more likely to be ignored and may become an important reservoir for drug-resistant pathogens, such as Salmonella entericus (Perron, Quessy, & Bell, 2008). Salmonella isolated from captive wild animals in Ibadan, western Nigeria, has been observed to be resistant to sulfadiazine and penicillin (Falade & Durojaiye, 1976). Among the 232 isolates of Gram-negative bacteria isolated from mammals, reptiles and birds raised in Asakusa Zoo, Hiroshima Prefecture, Japan, 21.1% of Gram-negative bacteria carry at least one drug-resistant gene and have multiple drug-resistant phenotypes (Ahmed et al., 2007). ESBLs andfluoroquinolone resistance genes detected in Czech zoos were associated with the transmission of specific E.coil clones and plasmids of specific incompatible groups between different animal species (Dobiasova et al., 2013). S.aureus from a zoo and wildlife in Germany from 2008 to 2016, two isolates from juvenile red squirrels showed multiple drug resistance phenotypes (Fessler et al., 2018). The frequency of AMRs detected in animals living in human settlements is significantly higher than that in animals living in the wild environment (Grall et al., 2015; Kock et al., 2018). The isolates from wild animals show a similar pattern of drug resistance toE.coil from human clinical sources in their study areas (Jobbins & Alexander, 2015). another some studies, drug-resistant Salmonella isolates identified from zoo environments, animals and their keepers have been confirmed to be clone-related (Farias et al., 2015; Milton et al., 2018). Therefore, wild animals may be important hosts and storage hosts for the spread of drug-resistant bacteria, and human activities significantly affect the microbial community of captive wild animals in zoo environments that are easy to be ignored.
In the present study, the ST290 sequence types K.pneumoniaeM297-1 from Red Kangaroo is closely related to that of human clinical isolates, but only 5 cases of this sequence type have been reported worldwide, reported by the United States, Australia and China (Figure 2). The plasmid of isolates carrying bla KPC-2,bla IMP-8, bla TEM-1 andbla CTX-M-15 genes transmission have been found in neonatal infections (Jin et al., 2017; Kong et al., 2020). The first case of nosocomial epidemics infection caused by NDM-5 metallo-β-lactamase ST290 isolates was reported in China (Wang et al., 2019). Therefore, our study further showed that zoo-derived MDR bacteria are closely related to human-derived MDR.
bGWAS provided comprehensive information about isolate M297-1. The whole genome analysis of M297-1 confirmed that it‘s plasmids carried clinically related ESBLs genes bla TEM-1, bla TEM-191, bla CTX-M-3 andbla CTX-M-14 (Table 2 and Figure 1). Since the late 1990s, the multidrug-resistant Enterobacteriaceae that produces ESBLs has become an important cause of urinary tracts and bloodstream infections in the community (Pitout & Laupland, 2008; Wyres et al., 2020). This is a rapidly evolving class of β-lactamases, usually from bla TEM-1, bla TEM-2 orbla SHV-1 genes, which have the ability to hydrolyze third-generation cephalosporins and aztreonamand can be inhibited by clavulanic acid (Paterson & Bonomo, 2005). Although new members of the ESBLs family are often found, the earlierbla CTX-M-14 andbla CTX-M-15 enzymes are prevalent in world (Bush & Bradford, 2020; Bush & Fisher, 2011), whilebla CTX-M-3 enzymes are prevalent mainly in Europe (Canton et al., 2008). Resistance to extended-spectrum cephalosporins such as ceftazidime, cefotaxime and cefepime was often observed when ESBLs appeared in Klebsiella sp. (Babic, Hujer, & Bonomo, 2006). ESBLs, was also detected on M297-1 chromosome, but it also stably carried ESBLs positive plasmids pM297-1.1 and pM297-1.2, indicating that drug resistance itself are not the only selection criterion for maintaining ESBLs cod plasmids. In addition, in this study, other drugs resistance genes of sulfonamides, fluoroquinolones, aminoglycosides and tetracyclines on chromosomes and plasmids suggest that it may have evolved under the pressure of many antibiotics.
Mobile genetic elements, such as insertion sequence, transposon, integron and prophage, can mobilize antibiotic resistance genes. The detection of bla TEM andbla CTX-M type β-lactamases genes in a variety of genetic backgrounds suggests that their mobilization may involve multiple mechanisms. In this study, for plasmid pM297-1.1, IS1380 is located at upstream of bla CTX-M-14, and downstream detected IS903 is also located at upstream ofbla TEM-191. bla TEM-191 may also be related to its downstream truncated IS2 gene, and similar structures also exist in similar plasmids retrieved (p911021-tetA,MG288679.1;p1_020098,NZ_CP036307.1; pLAP2_020009, CP038004.1) (Figure 1). This seems to imply that the β-lactamase gene carried by pM297-1.1 may form a tandem structure of drug resistance gene after multiple homologous recombination. For MDR 3 region of plasmid pM297-1.2, the resistance gene box of β-lactamase genes in Tn3-like transposon wastnpA-tnpR-bla TEM-1-other-bla CTX-M-3, and also located on the prophage. We found that the similar plasmid (pKF3-94, pL22-5, p2, pCTXM15_ 020019, pSCM96-1) also had a complete or truncated similar structure (Figure 1). There are usually two IS431mec insertion sequence genes from Staphylococcus aureus at upstream and downstream of aminoglycosides, fluoroquinolones, sulfonamidesand tetracyclines in the pM297-1.2 MDR region. The structure of the intI1- gene cassette carried by the plasmid pM297-1.2 supports the concept of mobile elements to transfer antimicrobial genes between different bacteria. The drug resistance gene cassette (aac(6’)-1b-cr-arr-3-dfrA27-aadA16) is derived from the plasmid (ACC_NUCCORE : EU675686) carried by the multi-drug resistanceE.coil isolated from the urine of patients in Huashan Hospital (Wei et al., 2009). This structure is completely preserved in pM297-1.2 from Klebsiella pneumoniae M297-1 (Figure 1) and is located on the Genomic Island. The drug resistance gene cassette mainly encodes β-lactamases, acetyltransferases and nucleoside transferases, which do not to require significant cell interaction and integrated into the metabolic network. Therefore, the interference from the existing genome is minimal, and it is the best example of a single gene corresponding to a single phenotype (Ghaly, Geoghegan, Tetu, & Gillings, 2020), but in any case, this genetic factor will help bacteria evolve multiple determinants of antibiotic resistance in different habitats (Edge & Hill, 2005).
Asia is one of the centers of antibiotic resistance, there is a surprising number of drug-resistant strains, includingK.pneumoniae , a large number of acquired gram-negative MDR strains had been found (Jean & Hsueh, 2011). The prevalent STs in Asia include ST15, ST23, ST14 and ST231. Among them, ST15 is very common, ST23 is significantly related to Southeast Asia, while ST14 and ST231 are significantly related to South Asia (Wyres et al., 2020). In this study, most of the plasmids retrieved from PLSDB database were carried by K.pneumoniae . Among the K.pneumoniae isolates carrying highly similar plasmids to pM297-1.2, only two isolates of ST15 were isolated from Thailand and China, and one strain of ST23 and one strain of ST14 from China. The isolates carrying highly similar plasmids to pM297-1.1 did not detect the above four sequence types (Table S1). As a result, the two plasmids in our study belong to IncF, a narrow host spectrum plasmid widely in Enterobacteriaceae , and can carry a variety of AMR genes and play a major role in the spread of specific antimicrobial genes (Carattoli, 2011; Rozwandowicz et al., 2018). Some studies showed that IncFIB and IncFII plasmids are effective vectors of β-lactamases. These plasmids can promote the transfer of antimicrobial genes when exposed to antibiotics (Rooney et al., 2019).bla NDM, bla OXA and other genes spread in the Enterobacteriaceae flora of medical facilities by relying on these plasmids (Simner et al., 2018; Strydom et al., 2020; Wu et al., 2019), while bla CTX-M can become the dominant gene carried by IncFII plasmids in France and China (Dahmen, Haenni, Chatre, & Madec, 2013; Du et al., 2012), but it seems that bla CMY-42 is replacingbla CTX-M-15 in some areas (Paul, Babenko, & Toleman, 2020). In particular, some IncFIB can not only express a high level of drug resistance, but also enhance the virulence ofK.pneumoniae after conjugation with K.pneumoniae (Yang, Wai-Chi Chan, Zhang, & Chen, 2019). To sum up, our results suggested that the co-prevalence of plasmid IncFIB and IncFII in the same isolates may lead to serious public health problems.
Similar plasmids were found by Sweden, Thailand and China respectively. Except for one isolate in Sweden and two isolates in Thailand, the other isolates carrying similar plasmids mainly distributed among the southern cities of Sichuan province, China (Figure 3, Table S1). From 2010 to 2019, it is suggested that the epidemic distribution of this drug-resistant plasmid is gradually spreading in China, and the difference in carrying drug-resistant genes indicates that it is evolving. However, the phylogenetic analysis of the related isolates showed that M297-1 located on different branch with otherK.pneumoniae isolates carrying similar plasmids (Figure 4). The possible interpretation may be that it comes from samples in Red Kangaroo, other reference isolates from clinical samples in human.
In conclusion, our study demonstrates the high resistance of 13 drugs including Ceftriaxone and Cefepimeon MDR K. pneumoniaeisolate from healthy red kangaroos in Zhengzhou zoo, China. Most importantly, this is the first report on a K. pneumoniae M297-1 (ST290) from wild animal carrying bla DHA-3,bla SHV-1, bla CTX-M-14,bla TEM-191, bla TEM-1, andbla CTX-M-3 in China and two conjugal transferable plasmids co-harboring other antimicrobial genes APH(3’)-Ia, APH(3”)-Ib, APH(6)-Id, AAC(3)-IIa, AAC(6’)-Ib-cr, aadA16, QnrB2, QnrS1, QacEΔ1, mphA, sul1 and dfrA27 etc. our research confirmed that there is a close relationship between drug-resistant strains carried by wild animals in zoos and human clinical isolates.it is suggested that the zoo may be becoming an important reservoir of clinically important MDR isolate, which pose a potential public health risk that can be ignored.