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.