狐狸流产胎儿体内肺炎克雷伯菌的分离鉴定及耐药性分析

doi:10.11843/j.issn.0366-6964.2024.08.035

摘要/Abstract

摘要：

旨在了解狐狸流产的病因。本研究对河北省某狐狸养殖场流产胎儿进行解剖、病原分离培养、16S rRNA测序、毒力基因检测、血清型检测、药敏试验及致病性试验。结果显示，从狐狸流产胎儿体内分离获得6株肺炎克雷伯菌，检测出12种毒力基因(wabG、fimH、ent、icuA、icuB、mrkA、ycf、ecp、uge、ureA、iroB和ybtA)和3种血清型(K20、K54和K57)，其中, K57为本次检测过程的优势血清型；经K-B药敏纸片法分析，发现本次分离获得的肺炎克雷伯菌对氨基糖苷类、头孢菌素类、喹诺酮、多肽类等抗菌药物存在中度或高度敏感，对大环内酯类、四环素类、青霉素类等抗菌药物存在多重耐药；经致病性试验分析，本次分离获得的肺炎克雷伯菌对小鼠具有一定的致病性，半数致死量(LD₅₀)为1.2×10⁷ CFU·mL^-1。基于此，本研究分离得到的肺炎克雷伯菌具有一定的致病性和耐药性，且携带多种毒力基因，可能是诱发狐狸胎儿流产、死亡的因素之一，为狐狸胎儿流产的防控及诊断提供思路。

关键词: 狐狸, 肺炎克雷伯菌, 毒力基因, 分离鉴定

Abstract:

In order to understand the etiology of abortion in foxes, the aborted fetuses in a fox farm in Hebei Province was analyzed by dissection, pathogen isolation and cultivation, 16S rRNA sequencing, virulence gene detection, serotype detection, drug susceptibility analysisassay and pathogenicity analysis assay in this study. As a result, 6 strains of Klebsiella pneumoniae were isolated. Twelve virulence genes (wabG, fimH, ent, icuA, icuB, mrkA, ycf, ecp, uge, ureA, iroB and ybtA) and three serotypes (K20, K54 and K57) were determined, and the K57 was the dominant serotype. Through K-B drug susceptibility testing, it showed that the isolated K. pneumoniae was moderately or highly sensitive to aminoglycosides, cephalosporins, quinolones, polypeptides and other antibacterial drugs, and multiple drug resistance to macrolides, tetracycline, penicillin and other antibacterial drugs was found. According to the pathogenicity analysis, the K. pneumoniae isolate had certain pathogenicity to mice, and the median lethal dose (LD₅₀) was 1.2×10⁷ CFU·mL^-1. In general, K. pneumoniae isolate showed certain pathogenicity, drug resistance, and carrying a variety of virulence genes in this study, and it might be one of the factors causing abortion of fox fetuses. The findings might provide hints for prevention and control of fox fetal abortion.

Key words: fox, Klebsiella pneumoniae, virulence gene, solation and identification

中图分类号:

S852.611

王奇林, 曹润来, 王威阳, 张博, 刘志杰, 王晓旭. 狐狸流产胎儿体内肺炎克雷伯菌的分离鉴定及耐药性分析[J]. 畜牧兽医学报, 2024, 55(8): 3640-3648.

Qilin WANG, Runlai CAO, Weiyang WANG, Bo ZHANG, Zhijie LIU, Xiaoxu WANG. Isolation, Identification and Drug Resistance Analysis of Klebsiella pneumoniae in Aborted Fetuses of Fox[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3640-3648.

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参考文献 29

1	彭凤华. 吉林省毛皮产业发展对策研究[D]. 长春: 吉林大学, 2012.
	PENG F H. A study on development countermeasure of fur industry in Jilin[D]. Changchun: Jilin University, 2012. (in Chinese)
2	赵艳玲, 赵远, 刘长浩. 2021年狐狸病毒性疾病检测分析[J]. 山东畜牧兽医, 2023, 44 (7): 25-26, 30. doi: 10.3969/j.issn.1007-1733.2023.07.008
	ZHAO Y L , ZHAO Y , LIU C H . Analysis of viral disease detection in foxes in 2021[J]. Shandong Journal of Animal Science and Veterinary Medicine, 2023, 44 (7): 25-26, 30. doi: 10.3969/j.issn.1007-1733.2023.07.008
3	WANG W J , WEI Y T , CAO S H , et al. Divergent Cryptosporidium species and host-adapted Cryptosporidium canis subtypes in farmed minks, raccoon dogs and foxes in Shandong, China[J]. Front Cell Infect Microbiol, 2022, 12, 980917. doi: 10.3389/fcimb.2022.980917
4	WEI X Y , GAO Y , LV C , et al. The global prevalence and risk factors of Toxoplasma gondii among foxes: a systematic review and meta-analysis[J]. Microb Pathog, 2021, 150, 104699. doi: 10.1016/j.micpath.2020.104699
5	安富宇, 杨金雨, 冯涛, 等. 东北地区狐源大肠杆菌耐药性的检测[J]. 中国兽医杂志, 2019, 55 (2): 102-103, 107.
	AN F Y , YANG J Y , FENG T , et al. Detection of Escherichia coli resistance in fox source in Northeast China[J]. Chinese Journal of Veterinary Medicine, 2019, 55 (2): 102-103, 107.
6	韩坤, 孟祥玉, 白雪, 等. 狐狸源肺炎克雷伯菌的分离鉴定及毒力检测[J]. 中国兽医学报, 2019, 39 (4): 722- 727.
	HAN K , MENG X Y , BAI X , et al. Isolation, identification and virulence factors detection of Klebsiella pneumoniae isolated from fox[J]. Chinese Journal of Veterinary Science, 2019, 39 (4): 722- 727.
7	张瑞华, 高善颂, 庞天津, 等. 狐狸源停乳链球菌类马亚种的分离鉴定和耐药性分析[J]. 中国兽医杂志, 2022, 58 (12): 31- 35.
	ZHANG R H , GAO S S , PANG T J , et al. Isolation, identification and drug resistane analysis of Streptococcus dysgalactiae subsp. equisimilis from fox[J]. Chinese Journal of Veterinary Medicine, 2022, 58 (12): 31- 35.
8	WANG G Y , ZHAO G , CHAO X Y , et al. The characteristic of virulence, biofilm and antibiotic resistance of Klebsiella pneumoniae[J]. Int J Environ Res Public Health, 2020, 17 (17): 6278. doi: 10.3390/ijerph17176278
9	RIBEIRO M G , DE MORAIS A B C , ALVES A C , et al. Klebsiella-induced infections in domestic species: a case-series study in 697 animals (1997-2019)[J]. Braz J Microbiol, 2022, 53 (1): 455- 464. doi: 10.1007/s42770-021-00667-0
10	李超, 王玉英, 王冬冬, 等. 引起幼貂大量死亡的肺炎克雷伯菌的分离和鉴定[J]. 中国兽医杂志, 2015, 51 (11): 27- 29.
	LI C , WANG Y Y , WANG D D , et al. Isolation and identification on Klebsiella pneumoniae which was bacterial pathogen leading to disease of minks[J]. Chinese Journal of Veterinary Medicine, 2015, 51 (11): 27- 29.
11	张文举, 刘少杰, 张艳英, 等. 河北部分地区狐狸源肺炎克雷伯菌血清型鉴定、毒力基因及致病性检测[J]. 中国兽医学报, 2020, 40 (4): 735- 739.
	ZHANG W J , LIU S J , ZHANG Y Y , et al. Serotype identification, virulence genes and pathogenicity detection of Klebsiella pneumoniae from foxes in some areas of Hebei Province[J]. Chinese Journal of Veterinary Science, 2020, 40 (4): 735- 739.
12	李巧玲, 肖丽荣, 张文举, 等. 蓝狐源肺炎克雷伯菌的鉴定与药敏试验[J]. 畜牧与兽医, 2018, 50 (3): 103- 106.
	LI Q L , XIAO L R , ZHANG W J , et al. Isolation, identification and drug susceptibility of Klebsiella pneumoniae isolated from blue fox[J]. Animal Husbandry & Veterinary Medicine, 2018, 50 (3): 103- 106.
13	COLODNER R , ROCK W , CHAZAN B , et al. Risk factors for the development of extended-spectrum beta-lactamase-producing bacteria in nonhospitalized patients[J]. Eur J Clin Microbiol Infect Dis, 2004, 23 (3): 163- 167. doi: 10.1007/s10096-003-1084-2
14	EL-DOMANY R A , AWADALLA O A , SHABANA S A , et al. Analysis of the correlation between antibiotic resistance patterns and virulence determinants in pathogenic Klebsiella pneumoniae isolates from Egypt[J]. Microb Drug Resist, 2021, 27 (6): 727- 739. doi: 10.1089/mdr.2020.0236
15	TURTON J F , PERRY C , ELGOHARI S , et al. PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets[J]. J Med Microbiol, 2010, 59 (Pt 5): 541- 547.
16	BENGOECHEA J A , SA PESSOA J . Klebsiella pneumoniae infection biology: living to counteract host defences[J]. FEMS Microbiol Rev, 2019, 43 (2): 123- 144.
17	GIRI S , SHEKAR M , SHETTY A V , et al. Antibiotic resistance and random amplified polymorphic DNA typing of Klebsiella pneumoniae isolated from clinical and water samples[J]. Water Environ Res, 2021, 93 (11): 2740- 2753.
18	PACZOSA M K , MECSAS J . Klebsiella pneumoniae: going on the offense with a strong defense[J]. Microbiol Mol Biol Rev, 2016, 80 (3): 629- 661.
19	赵菲菲, 李杰, 韩宁, 等. 分离自屠宰场的肺炎克雷伯菌的耐药性分析[J]. 畜牧兽医学报, 2023, 54 (7): 3044- 3053. doi: 10.11843/j.issn.0366-6964.2023.07.035
	ZHAO F F , LI J , HAN N , et al. Antibacterial drug resistance analysis of Klebsiella pneumoniae isolated from slaughterhouse[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (7): 3044- 3053. doi: 10.11843/j.issn.0366-6964.2023.07.035
20	FURSOVA A D , FURSOV M V , ASTASHKIN E I , et al. Early response of antimicrobial resistance and virulence genes expression in classical, hypervirulent, and hybrid hvKp-MDR Klebsiella pneumoniae on antimicrobial stress[J]. Antibiotics (Basel), 2021, 11 (1): 7.
21	DI MARTINO P , CAFFERINI N , JOLY B , et al. Klebsiella pneumoniae type 3 pili facilitate adherence and biofilm formation on abiotic surfaces[J]. Res Microbiol, 2003, 154 (1): 9- 16.
22	贾志钰, 张海鹏, 宋志忠. 肺炎克雷伯菌毒力因子及耐药研究进展[J]. 中国地方病防治, 2022, 37 (4): 275- 277.
	JIA Z Y , ZHANG H P , SONG Z Z . Research progress on virulence factors and drug resistance of Klebsiella pneumoniae[J]. Chinese Journal of Control of Endemic Diseases, 2022, 37 (4): 275- 277.
23	REGUÉ M , HITA B , PIQUE N , et al. A gene, uge, is essential for Klebsiella pneumoniae virulence[J]. Infect Immun, 2004, 72 (1): 54- 61.
24	COGEN A L , MOORE T A . β2-microglobulin-dependent bacterial clearance and survival during murine Klebsiella pneumoniae bacteremia[J]. Infect Immun, 2009, 77 (1): 360- 366.
25	THEIL E C , CHEN H J , MIRANDA C , et al. Absorption of iron from ferritin is independent of heme iron and ferrous salts in women and rat intestinal segments[J]. J Nutr, 2012, 142 (3): 478- 483.
26	韩睿辉, 杜艳. 高毒力肺炎克雷伯菌致病机制中铁载体毒力基因的作用[J]. 华西医学, 2020, 35 (8): 991- 994.
	HAN R H , DU Y . The role of siderophore virulence genes in the pathogenic mechanism of hypervirulent Klebsiella pneumoniae[J]. West China Medical Journal, 2020, 35 (8): 991- 994.
27	RUSSO T A , OLSON R , FANG C T , et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae[J]. J Clin Microbiol, 2018, 56 (9): e00776.
28	FANG C T , LAI S Y , YI W C , et al. The function of wzy_K1 (magA), the serotype K1 polymerase gene in Klebsiella pneumoniae cps gene cluster[J]. J Infect Dis, 2010, 201 (8): 1268- 1269.
29	杨延, 刘梦, 赵丽丽, 等. 狐狸源肺炎克雷伯氏菌分离株生物学特征分析[J]. 中国预防兽医学报, 2018, 40 (11): 998- 1002.
	YANG Y , LIU M , ZHAO L L , et al. Analysis of biological characteristics of Klebsiella pneumonia isolated from fox[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40 (11): 998- 1002.

毒力基因 Virulence gene	引物序列(5′→3′) Primer sequence	片段大小/bp Product size	退火温度/℃ Annealing temperature
rmpA	F: ACCCTTTACAGCCAAATTTTCTTGT	468	52
	R: CTGGGCTACCTCTGCTTCATA
magA	F: TGATAAGTGGCGGAGATTCTGA	542	52
	R: TGATAAGTGGCGGAGATTCTGA
wabG	F: CTCTGGTGCGGCAGAAGTAC	931	52
	R: TGGCCGTCGACGATAAACTC
uge	F: GCGCACACCTATTCTCACCT	219	56
	R: GATCACATCCTGCACCCGAA
fimH	F: GTCTACGTTAACCTGACCCCG	781	56
	R: ATTGATAGACAAAGGTGATGCCGAT
mrkA	F: CGGCGGCCAGGTTAATTTCT	500	56
	R: CGTAGCTGTTAACCACACCG
entB	F: CTGCGCTTTGAGGAAGAGGAG	241	56
	R: GCAAGTGGTGATAACGCTGATAC
kfu	F: GTGCTGGCCTACTATCCGTT	520	56
	R: TATCGATACCGCCCAGCCAC
ybtA	F: ATGACGGAGTCACCGCAAAC	960	55
	R: TTACATCACGCGTTTAAAGG
aero	F: GCATAGGCGGATACGAACAT	556	50
	R: CACAGGGCAATTGCTTACCT
iutA	F: GGCTGGACATCATGGGAACTGG	300	55
	R: CGTCGGGAACGGGTAGAATCG
iroB	F: ACAACAACGCGGGCATTTAC	217	56
	R: TCCTGCATCTTTCGGCCAAT
icuA	F: GTAAACAGCGGCTTCAGCAC	1 230	56
	R: GCCAACTAAAACGTCAGCCC
icuB	F: ATGTCTAAGGCAAACATCGT	948	55
	R: TTACAGACCGACCTCCGTGA
ycf	F: TGGTCACGGATTATGTAACGGT	524	56
	R: ACGCTATGACAGAACCTGGT
ecp	F: ACAGCGCATCGGTCATATCC	1 319	56
	R: CAACTTTTTCGTCACCCCCG
wcaG	F: TGGTTGGGTCAGCAATCGT	161	54
	R: GCCAACTTTTGCAGCAGCTAAATAT
ureA	F: GCTGACTTAAGAGAACGTTATG	337	50
	R: GATCATGGCGCTACCTTA
alls	F: CTTCAGCAGATAAATGACGGGGTAG	244	56
	R: GTGGGTAAACCGCCATATTTTCC

菌株Bacterial strain	毒力基因Virulence gene
K.p-AF1	fimH、mrkA、iutA、ecp、ureA
K.p-AF2	fimH、mrkA、iutA、ecp、icuB、ureA
K.p-AF3	wabG、fimH、ent、mrkA、ecp、uge、ureA
K.p-AF4	wabG、fimH、ent、mrkA、iutA、ecp、uge、ybtA、icuB、ureA
K.p-AF5	wabG、fimH、ent、mrkA、ycf、ecp、uge、ureA
K.p-AF6	wabG、fimH、ent、mrkA、ycf、iroB、ecp、uge、ybtA、ureA

药物种类 Drug catalog	药物名称 Drug names	耐药 Drug resistance		中度敏感 Medium sensitivity		高度敏感 Highly sensitive
药物种类 Drug catalog	药物名称 Drug names	菌株数 Strain numbers	比例/% Proportion	菌株数 Strain numbers	比例/% Proportion	菌株数 Strain numbers	比例/% Proportion
氨基糖苷类	新霉素	6	100.0	—	—	—	—
Aminoglycosides	庆大霉素	2	33.3	4	66.7	—	—
	卡那霉素	5	83.3	1	16.7	—	—
大环内酯类	红霉素	6	100.0	—	—	—	—
Macrolides
四环素类	四环素	6	100.0	—	—	—	—
Tetracyclines	多西环素	6	100.0	—	—	—	—
头孢菌素类	头孢氨苄	4	66.7	2	33.3	—	—
Cephalosporins	头孢哌酮	3	50.0	1	16.7	2	33.3
	头孢曲松	1	16.7	—	—	5	83.3
喹诺酮类	诺氟沙星	4	66.7	1	16.7	1	16.7
Quinolones	环丙沙星	4	66.7	1	16.7	1	16.7
	氧氟沙星	4	66.7	1	16.7	1	16.7
青霉素类	青霉素	6	100.0	—	—	—	—
Penicillins	氨苄西林	6	100.0	—	—	—	—
多肽类	多黏菌素B	—	—	—	—	6	100.0
Polypeptides

组别 Groups	浓度/(CFU·mL^-1) Concentration	死亡数 Number of deaths	死亡率/% Death rate
1	2.1×10⁵	0	0
2	2.1×10⁶	2	25.0
3	2.1×10⁷	5	62.5
4	2.1×10⁸	7	87.5
5	2.1×10⁹	8	100.0
6	—	0	0

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