Whole Genome Sequencing of Multi-drug Resistant Escherichia coli and Its Drug Resistance Analysis

doi:10.13304/j.nykjdb.2023.0169

Abstract

Abstract:

In order to understand colistin resistance genes carried by Escherichia coli， screen sensitive plant drugs， and solve the dilemma of multiple drug resistance and no drug option in animal clinic， the resistance genes of Escherichia coli were identified by 16S rRNA sequencing and PCR. The antimicrobial susceptibility of 78 antibiotics were detected， and the bacteriostatic and bactericidal effects of 4 kinds of natural plant extracts （palmatine， berberine， baicalin and macleaya cordata） were tested. The results showed that 145 strains of E.coli were isolated and identified from intestinal tract of swine clinical diarrhea cases in 2021 （from January to December） and 2022 （from January to June）， and a clinical strain carrying colistin resistance genes （mcr-4，mcr-5） and β-lactamases blaTEM and AmpC was identified， which named HN2149. The antimicrobial susceptibility of 78 antibiotics showed that the HN2149 strains was sensitive to cefepime， cefodizime， fosfomycin， cefixime， meropenem， cefoxitin， cefazolin， cefoperazone， cefotaxime， ceftazidime， cefuroxime， ceftriaxone， aztreonam， piperacillin-tazobactam， ticacillin/clavulanic acid， cefoperazone sulbactam， ceftazidime/clavulanic acid， cefotaxime/clavulanic acid， ceftizoxime， cefmetazole， cefetamet， and was resistant to 57 antibiotics. The drug sensitivity of 4 plant extracts showed that the macleaya cordata had the best bacteriostatic and bactericidal effect on HN2149 strain， while the other 3 extracts had no effects on HN2149 strain. Above results provided reference for the prevention and control of swine colibacillosis.

Key words: carbapenemase, β-lactamase, mcr-4, mcr-5, blaTEM, AmpC, Escherichia coli, bacterial drug resistance

摘要：

为了解大肠杆菌携带的粘菌素耐药基因并筛选敏感药物，解决多重耐药和动物临床无药可选的困局，采用16S rRNA菌种全基因组测序，PCR筛查mcr-4、mcr-5、blaTEM和AmpC酶耐药基因，应用BLAST和MEGA软件进行生物信息学和系统进化树分析，并对该菌株进行了78种抗生素抗菌药物敏感性测试及4种天然植物提取物（黄藤素、黄连素、黄芩苷和博落回）的抑菌、杀菌效果试验。结果表明，从2021年（1—12月）和2022年（1—6月）猪临床腹泻病例肠道中分离鉴定的145株大肠埃希菌（Escherichia coli，E.coli）中，鉴定出1株同时携带粘菌素耐药基因（mcr-4，mcr-5）和β内酰胺酶blaTEM、AmpC基因的猪源大肠埃希氏菌临床菌株HN2149。78种抗生素药敏试验结果显示，HN2149菌株对头孢吡肟、头孢地嗪、磷霉素、头孢克肟、头孢唑林、头孢哌酮、美洛培南、头孢西丁、头孢呋辛、头孢曲松、头孢噻肟、头孢他啶、替卡西林/克拉维酸、头孢哌酮/舒巴坦、氨曲南、哌拉西林/他唑巴坦、头孢噻肟/克拉维酸、头孢唑肟、头孢美唑、头孢他啶/克拉维酸和头孢他美敏感，对其余57种抗菌药物表现为耐药。4种植物提取物的药敏结果显示，博落回对菌株HN2149的抑菌、杀菌效果较好，其余3种提取物均对该菌株无抑菌和杀菌效果。以上研究结果为猪大肠杆菌病的防控提供参考。

关键词: 碳青霉烯酶, β-内酰胺酶, mcr-4, mcr-5, blaTEM, AmpC, 大肠埃希氏菌, 细菌耐药

CLC Number:

S852

Haili LI, Yindi XU, Zhifang WANG, Wenhao ZHU, Lixian ZHANG, Chunjiang MA. Whole Genome Sequencing of Multi-drug Resistant Escherichia coli and Its Drug Resistance Analysis[J]. Journal of Agricultural Science and Technology, 2024, 26(6): 113-121.

李海利, 徐引弟, 王治方, 朱文豪, 张立宪, 马春江. 一株多重耐药大肠杆菌全基因组测序及其耐药性分析[J]. 中国农业科技导报, 2024, 26(6): 113-121.

Figures/Tables 7

References 34

1	HUR J, STEIN B D, LEE J H. A vaccine candidate for post-weaning diarrhea in swine constructed with a live attenuated Salmonella delivering Escherichia coli K88ab, K88ac, FedA, and FedF fimbrial antigens and its immune responses in a murine model [J]. Can. J. Vet. Res., 2012, 76(3):186-194.
2	CASEY T A, BOSWORTH B T. Design and evaluation of a multiplex polymerase chain reaction assay for the simultaneous identification of genes for nine different virulence factors associated with Escherichia coli that cause diarrhea and edema disease in swine [J]. J. Vet. Diagn. Invest., 2009, 21(1):25-30.
3	BEIER R C, BISCHOFF K M, ZIPRIN R L, et al.. Nisbet DJ: chlorhexidine susceptibility, virulence factors, and antibiotic resistance of beta-hemolytic Escherichia coli isolated from neonatal swine with diarrhea [J]. Bull. Environ. Contam. Toxicol., 2005, 75(5):835-844.
4	WADA Y, KATO M, YAMAMOTO S, et al.. Invasive ability of Escherichia coli O18 isolated from swine neonatal diarrhea [J]. Vet. Pathol., 2004, 41(4):433-437.
5	STAHL C H, CALLAWAY T R, LINCOLN L M, et al.. Inhibitory activities of colicins against Escherichia coli strains responsible for postweaning diarrhea and edema disease in swine [J]. Antimicrob. Agents Chemother., 2004, 48(8):3119-3121.
6	THOMAS P W, CHO E J, BETHEL C R, et al.. Discovery of an effective small-molecule allosteric inhibitor of New Delhi metallo-beta-lactamase (NDM) [J]. ACS Infect. Dis., 2022, 8(4):811-824.
7	QUAN J, DAI H, LIAO W, et al.. Etiology and prevalence of ESBLs in adult community-onset urinary tract infections in East China: a prospective multicenter study [J]. J. Infect., 2021, 83(2):175-181.
8	XIONG Y, ZHANG C, GAO W, et al.. Genetic diversity and co-prevalence of ESBLs and PMQR genes among plasmid-mediated AmpC beta-lactamase-producing Klebsiella pneumoniae isolates causing urinary tract infection [J]. J. Antibiot. (Tokyo), 2021, 74(6):397-406.
9	JOMEHZADEH N, AHMADI K, JAVAHERIZADEH H, et al.. The first evaluation relationship of integron genes and the multidrug-resistance in class a ESBLs genes in enteropathogenic Escherichia coli strains isolated from children with diarrhea in Southwestern Iran [J]. Mol. Biol. Rep., 2021, 48(1):307-313.
10	BABAZADEH F, TEIMOURPOUR R, ARZANLOU M, et al.. Phenotypic and molecular characterization of extended-spectrum beta-lactamase/AmpC- and carbapenemase-producing Klebsiella pneumoniae in Iran [J]. Mol. Biol. Rep., 2022, 49(6):4769-4776.
11	FAVIER P, RAFFO C, TORRES D, et al.. Third-generation cephalosporins programmed restriction in the context of an outbreak of AmpC beta-lactamase-producing gram-negative bacilli in critical units: a real-life experience [J]. Rev. Chilena Infectol., 2021, 38(5):597-604.
12	MUDDASSIR M, MUNIR S, RAZA A, et al.. Epidemiology and high incidence of metallo-beta-lactamase and AmpC-beta-lactamases in nosocomial [J] Iran. J. Basic Med. Sci., 2021, 24(10):1373-1379.
13	FETAHAGIC M, IBRAHIMAGIC A, UZUNOVIC S, et al.. Detection and characterisation of extended-spectrum and plasmid-mediated AmpC beta-lactamase produced by Escherichia coli isolates found at poultry farms in Bosnia and Herzegovina [J]. Arh. Hig. Rada Toksikol., 2021, 72(4):305-314.
14	MATHILDE L, LAURENT P, PATRICE N. Rapid multiplex polymerase chain reaction for detection of mcr-1 to mcr-5 genes [J]. Diag. Microbiol. Infect. Dis., 2018, 92(4):267-269.
15	DONG F, LU J, WANG Y, et al.. A five-year surveillance of carbapenemase-producing Klebsiella pneumoniae in a pediatric hospital in China reveals increased predominance of NDM-1 [J]. Biomed. Environ. Sci., 2017, 30(8):562-569.
16	DOMINGUEZ-PEREZ R A, DE LA TORRE-LUNA R, AHUMADA-CANTILLANO M, et al.. Detection of the antimicrobial resistance genes blaTEM-1, cfxA, tetQ, tetM, tetW and ermC in endodontic infections of a Mexican population [J]. J. Glob. Antimicrob. Resist., 2018, 15:20-24.
17	WAGNER K, MANCINI S, RITTER C, et al.. Evaluation of the AID AmpC line probe assay for molecular detection of AmpC-producing Enterobacterales [J]. J. Glob. Antimicrob. Resist., 2019, 19:8-13.
18	李海利,方剑玉,游一,等.中草药提取物对多重耐药猪胸膜肺炎放线杆菌体外抑菌效果研究[J].中国兽药杂志,2022,56(2):70-77.
	LI H L, FANG J Y, YOU Y, et al.. In vitro antibacterial experiment and antibacterial effect of Chinese herbal medicine extracts on multi-drug resistant Actinobacillus pleuropneumoniae of pigs [J]. Chin. J. Vet. Drug, 2022, 56(2):70-77.
19	ABDEL AAL A M, KHALIL N O, RASHED H G, et al.. Genetic detection of AmpC beta-lactamase among gram negative isolates “a single center experience” [J]. Egypt. J. Immunol., 2021, 28(4):195-205.
20	SHAH A, ALAM S, KABIR M, et al.. Migratory birds as the vehicle of transmission of multi drug resistant extended spectrum beta lactamase producing Escherichia fergusonii, an emerging zoonotic pathogen [J]. Saudi J. Biol. Sci., 2022, 29(5):3167-3176.
21	BAKR K I, ABDUL-RAHMAN S M, MUHAMMAD HAMASALIH R. Molecular detection of beta-lactamase genes in Klebsiella pneumoniae and Escherichia coli isolated from different clinical sources [J]. Cell Mol. Biol. (Noisy-le-grand), 2022, 67(4):170-180.
22	LU J, WANG L, WEI Y, et al.. Trends and risk factors of extended-spectrum beta-lactamase urinary tract infection in Chinese children: a nomogram is built and urologist should act in time [J]. Transl. Pediatr., 2022, 11(6):859-868.
23	GHARAIBEH M H, ALYAFAWI D A, ELNASSER Z A, et al.. Emergence of mcr-1 gene and carbapenemase-encoding genes among colistin-resistant Klebsiella pneumoniae clinical isolates in Jordan [J]. J. Infect. Public Health., 2022, 15(8):922-929.
24	XU T, XUE C X, CHEN Y, et al.. Frequent convergence of mcr-9 and carbapenemase genes in Enterobacter cloacae complex driven by epidemic plasmids and host incompatibility [J]. Emerg. Microbes Infect., 2022, 11(1):1959-1972.
25	TALAT A, USMANI A, KHAN A U. Detection of E. coli IncX1 plasmid-mediated mcr-5.1 gene in an Indian hospital sewage water using shotgun metagenomic sequencing: a first report [J]. Microb. Drug Resist., 2022, 28(7):759-764.
26	KASSEM, I I, ASSI A, OSMAN M, et al.. Letter to the editor: first report of the detection of the plasmid-borne colistin resistance gene, mcr-1.26, in multidrug-resistant Escherichia coli isolated from a domesticated pigeon [J]. Microb. Drug Resist., 2022, 28(7):821-823.
27	OTEO J, MENCIA A, BAUTISTA V, et al.. Colonization with enterobacteriaceae-producing ESBLs, AmpCs, and OXA-48 in wild avian species, Spain 2015-2016 [J]. Microb. Drug Resist., 2018, 24(7):932-938.
28	SALAMANDANE A, MALFEITO-FERREIRA M, BRITO L. A high level of antibiotic resistance in Klebsiella and Aeromonas isolates from street water sold in Mozambique, associated with the prevalence of extended-spectrum and AmpC ss-lactamases [J]. J. Environ. Sci. Health B., 2022, 57(7):561-567.
29	GARCIA-FIERRO R, DRAPEAU A, DAZAS M, et al.. Comparative phylogenomics of ESBL-, AmpC- and carbapenemase-producing Klebsiella pneumoniae originating from companion animals and humans [J]. J. Antimicrob. Chemother., 2022, 77(5):1263-1271.
30	MUNTEAN M M, MUNTEAN A A, GUERIN F, et al.. Optimization of the rapid carbapenem inactivation method for use with AmpC hyperproducers-authors’ response [J]. J. Antimicrob. Chemother., 2022, 77(4):1210-1211.
31	GONZALEZ MESA L, RAMOS MORI A, NADAL BECERRA L, et al.. Phenotypic and molecular identification of extended-spectrum beta-lactamase (ESBL) TEM and SHV produced by clinical isolates Escherichia coli and Klebsiella spp. in hospitals [J]. Rev. Cubana Med. Trop., 2007, 59(1):52-58.
32	BROWN R P, APLIN R T, SCHOFIELD C J, et al.. Mass spectrometric studies on the inhibition of TEM-2 beta-lactamase by clavulanic acid derivatives [J]. J. Antibiot. (Tokyo), 1997, 50(2):184-185.
33	BROWN R P, APLIN R T, SCHOFIELD C J. Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry [J]. Biochemistry, 1996, 35(38):12421-12432.
34	BRET L, CHANAL C, SIROT D, et al.. Characterization of an inhibitor-resistant enzyme IRT-2 derived from TEM-2 beta-lactamase produced by Proteus mirabilis strains [J]. J. Antimicrob. Chemother., 1996, 38(2):183-191.

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	产物长度 Product length/bp	退火温度 Annealing temperature/℃
Mcr4-F	TTGCAGACGCCCATGGAATA	207	57
Mcr4-R	GCCGCATGAGCTAGTATCGT	207	57
Mcr5-F	GGACGCGACTCCCTAACTTC	608	58
Mcr5-R	ACAACCAGTACGAGAGCACG	608	58
blaTEM-F	CCAATGCTTAATCAGTGAGG	400	57
blaTEM-R	ATGAGTATTCAACATTTCCG	400	57
AmpC-F	GATCGTTCTGCCGCTGTG	270	57
AmpC-R	GGGCAGCAAATGTGGAGCAA	270	57

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	产物长度 Product length/bp	退火温度 Annealing temperature/℃
Mcr4-F	TTGCAGACGCCCATGGAATA	207	57
Mcr4-R	GCCGCATGAGCTAGTATCGT	207	57
Mcr5-F	GGACGCGACTCCCTAACTTC	608	58
Mcr5-R	ACAACCAGTACGAGAGCACG	608	58
blaTEM-F	CCAATGCTTAATCAGTGAGG	400	57
blaTEM-R	ATGAGTATTCAACATTTCCG	400	57
AmpC-F	GATCGTTCTGCCGCTGTG	270	57
AmpC-R	GGGCAGCAAATGTGGAGCAA	270	57