| The spread of multidrug-resistant bacteria and drug-resistant genes carried by captive wild animals poses a serious threat to wildlife conservation as well as public health security.The spread of antibiotic resistance and antibiotic resistance genes(ARGs)from bacteria in the ex-situ population of giant pandas has become increasingly prominent.Studies had confirmed that Escherichia coli and Klebsiella pneumonia(KP)isolated from captive giant pandas were resistant to antibiotics,however the research on the antibiotic resistance of the gut microflora of giant pandas of different age groups still remains unknown.Furthermore,the multidrug resistance,antibiotic resistance mechanisms and horizontal transmission mechanism of the opportunistic pathogen KP has also yet to be investigated in this species.Based on this,the research on antibiotic resistance in this study which chose the captive giant pandas in the Chengdu Research Base of Giant Panda Breeding as the research objectwas divided into two main topics.First,the differences in the ARGs of the gut microbiota of four different age groups in giant pandas,as well as the association between ARGs and the gut microbiota structure,was analyzed through metagenomics.This method could objectively evaluate the health risks caused by the ARGs in captive giant pandas.Second,the antibiotic resistance spectrum,ARGs and mobile genetic elements(MGEs)of multi-drug-resistant(MDR)KP were analyzed,and the mechanism of multi-drug resistance and horizontal transmission was investigated.This research aimed to provide a reference for preventing and slowing the spread of antibiotic resistant bacteria and ARGs in the population of captive giant pandas.1.Metagenomics-based study of antibiotic resistance genes in the gut microbiota of giant pandasThrough metagenomic sequencing,a comprehensive study was carried out on the ARGs carried by the gut microbiota of 60 giant pandas in four different age groups(juvenile,sub-adult,adult,and geriatric)from the Chengdu Research Base of Giant Panda Breeding.Results:(1)The total relative abundance of ARGs,as well as 16 of the top 20 ARGs subtypes,and the abundance of ARGs that mediate resistance to different antibiotics(except fosfomycin)were significantly higher in the geriatric and juvenile groups compared to the adult and sub-adult groups.This finding was consistent with the changes in the abundance of Proteobacteria in the gut microbiota from giant pandas in the four age groups.(2)There was no significant difference in the total number of MGEs among the four different age groups(P > 0.05).Procrustes analysis was then performed based on the two sets of obtained non-metric multidimensional scaling(NMDS)coordinates,and it was determined that ARGs were significantly associated with the microbial community in the gut in different age groups(P < 0.05).(3)Antibiotic efflux was the main resistance mechanism,mainly dominated by Proteobacteria.The above results showed that the gut microbiota of giant pandas of different age groups carried many diverse ARGs,which were significantly related to the composition of the microbiota,and there was a possibility of horizontal transmission.2.Study on antibiotic resistance and molecular typing of multidrug-resistant Klebsiella pneumoniae from giant pandaAcross four seasons,376 fresh feces were collected from 94 captive giant pandas of different ages and sex housed at the Chengdu Research Base of Giant Panda Breeding.KP was isolated and identified from fresh fecal samples by differential culture in medium,gram staining and microscopy,biochemical identification and 16 S r DNA sequencing.The MDR strains were screened by the Kirby-Bauer(K-B)disk diffusion method.We further evaluated the ARGs of MDR strains by high-throughput quantitative PCR.In addition,we then analyzed MGEs,integron gene cassettes,and the multi-locus sequence typing of MDR KP strains by PCR.Results:(1)A total of 182 KP strains were isolated from 376 fresh fecal samples,and 30 MDR KP strains were further screened.(2)The 30 MDR KP isolates were mainly resistant to β-lactams antibiotics,the proportion of which was significantly related to the season,and the resistance to ceftriaxone and other antibiotics was significantly correlated with age and season.(3)Among the 30 MDR KP strains,one ESBLs-producing strain was identified with genotype bla TEM+bla CTX-M-1+bla SHV;three carbapenemase-producing strains were identified with genotypes bla KPC(2 strains)and bla KPC+bla NDM-1(1 strain).(4)Fifty different types of antibiotic resistance genes were found,which included a total of 671 ARGs,in the 30 MDR isolates.The top ten ARGs were: van TC-02,aac C,bla CTX-M-04,bla SHV-01,bla SHV-02,amp C-04,bla OXY,tet D,bla TEM and tet A-02.Thirteen MGEs were detected,of which IS26(96.67%)and int I1(96.67%)had the highest frequency.The thirty MDR KP isolates were negative for the tra A,tra F,tns A,IS1133,ISpa7,ISkpn6,int I2 and int I3 genes.(5)A further investigation of integrons revealed that two types of specific gene cassettes(dfr A12 + orf F +aad A2 and dfr A12 + orf F)were identified in class 1 integrons.Multi-locus sequence typing results showed that 22 STs in the thirty MDR KP isolates were identified,the main type was ST37(5/30),and ST37 had two single-site variants(SLVs).The above results showed that MDR KP had appeared in the giant panda population,and the antibiotic resistance varies with season,age,and the class of antibiotics;MDR KP also carried a large number of diverse ARGs,including some ARGs that produce Extended-Spectrum β-lactamases(ESBLs)or carbapenemases,these ARGs may spread horizontally in giant panda populations,so more rational use of antibiotics is needed in clinical practice,and effective monitoring and strict biosecurity strategies should be adopted to prevent the spread of multidrug-resistant strains.3.Multidrug resistance and horizontal transmission mechanism of Klebsiella pneumoniae isolated from giant panda based on whole genome sequencingWhole-genome sequencing was used to compare and analyze the types,numbers,and locations of ARGs in four isolates of ESBLs-MDR-KP(KP1),carbapenem-MDR-KP(KP2),MDR-KP(KP3),and non-MDR-KP(KP4)isolates.Results:(1)The ARGs carried on the chromosome genomes of the four strains were the same,with 24 species and 25 ARGs.In terms of the number of plasmids,KP4 carried two plasmids,while KP1,KP2,and KP3 carried three,one,and one,respectively.In terms of ARGs carried by plasmids,KP1,KP2 and KP3 had 8-11 types,8-21 ARGs subtypes in the plasmid genomes,while KP4 had only 1 ARG in the plasmid genomes,indicating that the multidrug resistance of KP1,KP2 and KP3 was mainly determined by the ARGs carried by the plasmid.(2)The drug-resistant phenotype and genotype of the four KP strains were further compared,and it was found that the drug-resistant phenotype and drugresistant genotype were consistent with each other.The production of ESBLs enzymes on the plasmid was found to be mediated by the ARGs CTX-M-1 and TEM-1,while the resistance to aminoglycoside antibiotics was mediated by the ARGs AAC(3)-IIa and/ or APH(3’’)-Ib,and lastly,the resistance to macrolide antibiotics was mediated by the ARG mph A.The resistance to tetracycline antibiotics on the plasmid was mediated by the ARGs tet(59)and/or tet(C),while the resistance to sulfonamide antibiotics was mediated by the ARGs sul1 and/or sul2.The above results show that the MDR phenotype and ESBLs production of KP from giant pandas were mainly mediated by the ARGs on the plasmid,which exist a threat to horizontal transmission.Conclusion :(1)There was a significant correlation between the ARGs and the composition of gut microbiota in the giant panda.(2)Multi-drug resistance strains had existed in KP isolated from giant pandas,which were mainly resistant to β-lactam antibiotics;The proportion of MDR KP strains isolated from giant pandas was significantly related to season,and the resistance to antibiotics such as ceftriaxone was significantly related to age and season.(3)The MDR KP strains isolated from giant pandas carried many diverse ARGs and MGEs,which posed a risk of horizontal transmission of ARGs in the giant panda;and some strains produced ESBLs or carbapenemases,which may be the reson for multi-drug resistance mechanisms.(4)The multidrug-resistant phenotype and ESBLs production of KP isolates were mainly mediated by ARGs on the plasmids,which existed a threat to horizontal transmission. |
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