不同耐药类型结核分枝杆菌毒力变化的初步研究

doi:10.3969/j.issn.1000-6621.2021.09.016

摘要/Abstract

摘要：

目的初步评价结核分枝杆菌(Mycobacterium tuberculosis,MTB)临床耐药分离株和实验室诱导耐药株的毒力变化表征。方法选取MTB标准株H37Rv和减毒株H37Ra各1株、4株临床耐药分离株(15833、16030、17080和30744,各1株)和8株实验室诱导耐药株[分别为单耐Pretomanid(PA-824)菌株(P1)、单耐利奈唑胺(Lzd)菌株(L1)、单耐PBTZ-169菌株(169-1、169-2、169-3,各1株),以及同时耐PA-824和Lzd的LPDR菌株(PL1、PL2、PL3,各1株)]。测定临床耐药分离株对一、二线抗结核药物和实验室诱导耐药株对Lzd、PA-824、PBTZ-169的最低抑菌浓度(minimum inhibitory concentration,MIC)值。绘制各研究菌株的体外生长曲线及测量感染巨噬细胞2d后的胞内菌落形成单位(colony forming units,CFU),并测定感染2d后巨噬细胞释放乳酸脱氢酶(lactate dehydrogenase,LDH)的含量。动物实验选取BALB/c小鼠168只,6~7周龄,体质量16~18g/只,分为14组,每组12只。将各耐药菌株以尾静脉注射的方式感染BALB/c小鼠,记录小鼠半数生存期,进行生存分析。结果体外实验表明,MTB临床耐药分离株30744生长曲线相比于其他耐药株的生长平台期提前,菌株体外适应性较低,其余各耐药菌株生长曲线趋势基本一致。临床耐药分离株(15833、16030、30744、17080)和对Lzd和PA-824同时耐药的实验室诱导耐药株(PL1、PL2、PL3)感染巨噬细胞2d后的胞内CFU计数[分别为(5.180±0.074)、(5.571±0.029)、(5.550±0.073)、(5.446±0.099)、(5.763±0.197)、(5.907±0.053)、(5.661±0.083)log₁₀CFU/ml]明显低于标准株H37Rv[(6.207±0.028)log₁₀CFU/ml],差异均有统计学意义(t=17.932、12.758、12.034、10.241、4.796、7.042、9.113,P值均<0.05)。另外,菌株15833、16030、30744、1708、P1、L1、PL1、PL2、PL3、169-1、169-2、169-3感染巨噬细胞2d后LDH释放量的吸光度A₄₉₀值比值(分别为0.252±0.039、0.412±0.078、0.247±0.022、0.358±0.054、0.329±0.015、0.483±0.017、0.328±0.046、0.455±0.075、0.283±0.041、0.258±0.044、0.374±0.080、0.311±0.097)均明显低于标准株H37Rv(0.958±0.025),差异均有统计学意义(t=27.269、16.788、38.244、24.238、44.005、32.698、27.713、15.171、31.798、31.472、16.515、15.570,P值均<0.01)。体内毒力实验显示:(1)所有菌株感染小鼠后,半数生存期(≥15d)均明显长于H37Rv标准株(12d)。(2)小鼠生存曲线显示:准广泛耐药菌株感染小鼠的存活时间长于耐多药菌株,即30744>17080>16030>15833;双重耐药菌株LPDR组小鼠存活时间>PBTZ-169耐药菌株组=单耐Lzd组>单耐PA-824耐药菌株组。结论 MTB耐药株的毒力均较野生型标准株下降,毒力的大小与该菌株耐药的数量可能呈负相关。

关键词: 分枝杆菌,结核, 毒力, 生存率分析, 评价研究

Abstract:

Objective To evaluate the virulence of clinical drug-resistant isolates and laboratory induced drug-resistant strains of Mycobacterium tuberculosis (MTB). Methods MTB standard strain H37Rv and attenuated strain H37Ra, four clinical drug-resistant isolates (15833, 16030, 17080 and 30744) and eight laboratory induced drug-resistant strains, including one pretomanid(PA-824)resistant strain (P1), one Lzd resistant strain (L1), three PBTZ-169 resistant strain (169-1, 169-2, 169-3), three PA-824 and Lzd dual resistant strains (PL1, PL2, PL3), were selected. The minimum inhibitory concentration (MIC) of clinical drug-resistant isolates against first-line and second-line anti-tuberculosis drugs and laboratory induced drug-resistant strains against Lzd, PA-824 and PBTZ-169 were measured. The in vitro growth curves of the studied strains and the intracellular colony forming units (CFU) of macrophages 2 days after infection were drawn, and the content of lactate dehydrogenase (LDH) released by macrophages 2 days after infection was measured.One hundred and sixty-eight BALB/c mice, 6-7 weeks old and 16-18 g/mouse, were randomly divided into 14 groups with 12 mice in each group. BALB/c mice were infected with drug-resistant strains by tail vein injection. The half survival time of mice was recorded and analyzed. Results In vitro experiments showed that the plateau phase of growth curve of MTB clinical drug-resistant isolate 30744 was earlier than that of the other drug-resistant strains, the adaptability of the strain in vitro was lower, and the growth curve trend of the other drug-resistant strains was basically identical. The intracellular CFU counts of clinical drug resistant isolates (15833, 16030, 30744, 17080) and laboratory induced drug resistant strains (PL1, PL2, PL3) resistant to Lzd and PA-824 were (5.180±0.074), (5.571±0.029), (5.550±0.073), (5.446±0.099), (5.763±0.197), (5.907±0.053), and (5.661±0.083) log₁₀ CFU/ml, which was significantly lower than that of standard strain H37Rv ((6.207 ± 0.028) log₁₀ CFU/ml), and the difference was statistically significant (t=17.932, 12.758, 12.034, 10.241, 4.796, 7.042, 9.113, Ps<0.05). In addition, the absorbance A₄₉₀ values of LDH release from macrophages infected by strains 15833, 16030, 30744, 1708, P1, L1, PL1, PL2, PL3, 169-1, 169-2 and 169-3 were 0.252±0.039, 0.412±0.078, 0.247±0.022, 0.358±0.054, 0.329±0.015, 0.483±0.017, 0.328±0.046, 0.455±0.075, 0.283±0.041, 0.258±0.044, 0.374±0.080, and 0.311±0.097, which was significantly lower than that of standard strain H37Rv (0.958±0.025), and the difference was statistically significant (t=27.269, 16.788, 38.244, 24.238, 44.005, 32.698, 27.713, 15.171, 31.798, 31.472, 16.515, 15.570, Ps<0.01). In vivo virulence test showed that: (1) The half surviors’ alive time (≥15 days) of all strains infected mice was significantly longer than that of H37Rv (12 days). (2) The survival curve of mice showed that the survival time of mice infected with pre-XDR strain was longer than that of MDR strains, namely 30744>17080>16030>15833; (3)The survival time of mice in the dual drug-resistant strain LPDR group>PBTZ-169 drug-resistant strain group=single Lzd resistant group>single PA-824 resistant strain group. Conclusion The virulence of MTB resistant strains was lower than that of wild-type standard strains, and the virulence might be negatively correlated with the number of drug-resistance.

Key words: Mycobacterium tuberculosis, Virulence, Survival analysis, Evaluation studies

周莹宇, 付雷, 张炜焱, 王彬, 陈曦, 陆宇, 陈效友. 不同耐药类型结核分枝杆菌毒力变化的初步研究[J]. 中国防痨杂志, 2021, 43(9): 952-960. doi: 10.3969/j.issn.1000-6621.2021.09.016

ZHOU Ying-yu, FU Lei, ZHANG Wei-yan, WANG Bin, CHEN Xi, LU Yu, CHEN Xiao-you. Analysis and comparative study on the virulence of several drug-resistant Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2021, 43(9): 952-960. doi: 10.3969/j.issn.1000-6621.2021.09.016

图/表 14

表1

表2

表3

图1

图2

图3

表4

不同类型结核分枝杆菌感染巨噬细胞2d后胞内CFU计数情况

菌株种类	CFU计数 (log₁₀CFU/ml, $x ¯$ ±s)	t值	P值
H37Rv	6.207±0.028	-	-
L1	6.134±0.057	-1.506	0.207
P2	6.125±0.095	1.144	0.316
PL1	5.763±0.197	4.796	0.009
PL2	5.907±0.053	7.042	0.020
PL3	5.661±0.083	9.113	0.001
169-1	5.800±0.385	1.501	0.271
169-2	5.933±0.221	1.710	0.225
169-3	5.938±0.147	2.541	0.064
15833	5.180±0.074	17.932	<0.01
16030	5.571±0.029	12.758	<0.01
30744	5.550±0.073	12.034	<0.01
17080	5.446±0.099	10.241	0.001
H37Ra	4.634±0.286	7.715	0.020

表4

表5

不同类型结核分枝杆菌感染巨噬细胞2d后巨噬细胞释放乳酸脱氢酶情况

菌株种类	吸光度值(A₄₉₀) 比值( $x ¯$ ±s)	t值	P值
H37Rv	0.958±0.025	-	-
L1	0.329±0.015	44.005	<0.01
P2	0.483±0.017	32.698	<0.01
PL1	0.328±0.046	27.713	<0.01
PL2	0.455±0.075	15.171	<0.01
PL3	0.283±0.041	31.798	<0.01
169-1	0.258±0.044	31.472	<0.01
169-2	0.374±0.080	16.515	<0.01
169-3	0.311±0.097	15.570	<0.01
15833	0.252±0.039	27.269	<0.01
16030	0.412±0.078	16.788	<0.01
30744	0.247±0.022	38.244	<0.01
17080	0.358±0.054	24.238	<0.01
H37Ra	0.233±0.009	54.247	<0.01

表5

表6

图4

图5

表7

表8

图6

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菌株	耐药类型	INH	RFP	EMB	Sm	Km	Mfx	Lfx
H37Rv	-	0.09	0.01	1.82	0.06	0.73	0.05	0.12
15833	耐多药	>6.80^a	>17.00^a	1.02	0.08	0.34	0.03	0.03
16030	耐多药	>6.80^a	>17.00^a	6.21^a	0.16	2.56	0.03	0.06
17080	准广泛耐药	>6.80^a	>17.00^a	25.32^a	24.7^a	0.34	0.61	3.34^a
30744	准广泛耐药	>6.80^a	>17.00^a	7.00^a	31.9^a	22.03^a	0.03	0.18

菌株	第0天	第3天	第7天	第14天	第21天	第28天
H37Rv	5.148±0.121	6.856±0.084	6.675±0.065	7.044±0.264	7.370±0.142	7.256±0.113
H37Ra	5.090±0.079	6.556±0.126	6.439±0.239	6.694±0.058	7.027±0.046	7.137±0.221
L1	5.029±0.011	6.077±0.031	6.854±0.072	7.118±0.035	7.233±0.021	7.011±0.161
P1	5.128±0.070	5.671±0.093	6.826±0.090	7.016±0.039	7.221±0.026	7.255±0.037
PL1	5.039±0.036	5.951±0.024	7.019±0.026	7.113±0.024	7.155±0.043	7.260±0.074
PL2	5.008±0.030	6.259±0.027	7.159±0.068	7.103±0.009	7.423±0.002	7.212±0.085
PL3	5.018±0.240	6.223±0.037	6.892±0.087	6.841±0.044	7.126±0.026	7.430±0.126
169-1	5.013±0.039	6.503±0.175	6.969±0.276	7.190±0.082	7.783±0.147	7.287±0.081
169-2	5.079±0.115	6.598±0.069	6.826±0.127	7.015±0.065	7.537±0.168	7.327±0.261
169-3	5.227±0.077	6.865±0.174	7.024±0.082	6.948±0.059	7.483±0.072	7.270±0.201
15833	5.112±0.097	6.207±0.019	6.998±0.069	7.328±0.073	7.530±0.078	7.470±0.115
16030	5.363±0.044	6.146±0.036	7.243±0.038	7.428±0.036	7.429±0.062	7.183±0.154
17080	5.502±0.394	6.361±0.019	7.125±0.020	7.409±0.038	7.709±0.142	7.223±0.386
30744	5.246±0.205	6.411±0.108	6.243±0.074	6.548±0.103	6.663±0.230	6.660±0.104

组别	15833		16030		30744		17080
组别	χ²值	P值	χ²值	P值	χ²值	P值	χ²值	P值
H37Rv	25.721	0.000	25.721	0.000	25.721	0.000	22.208	0.000
H37Ra	26.767	0.000	26.134	0.000	14.488	0.000	25.545	0.000
15833	-	-	1.185	0.276	22.557	0.000	7.704	0.006
16030	1.185	0.276	-	-	25.011	0.000	8.974	0.003
30744	22.557	0.000	25.011	0.000	-	-	11.240	0.001
17080	7.704	0.006	8.974	0.003	11.240	0.001	-	-

组别	L1		P1		PL1		PL2		PL3		169-1		169-2		169-3
组别	χ²值	P值	χ²值	P值	χ²值	P值	χ²值	P值	χ²值	P值	χ²值	P值	χ²值	P值	χ²值	P值
H37Rv	25.721	0.000	4.602	0.032	25.721	0.000	21.149	0.000	25.721	0.000	25.721	0.000	25.721	0.000	23.868	0.000
H37Ra	27.212	0.000	26.103	0.000	5.223	0.022	26.769	0.000	7.703	0.006	27.039	0.000	17.607	0.000	26.145	0.000
L1	-	-	14.175	0.000	15.402	0.000	0.002	0.963	15.098	0.000	0.439	0.507	2.144	0.143	0.484	0.487
P1	14.175	0.000	-	-	26.103	0.000	15.980	0.000	26.103	0.000	18.040	0.000	17.070	0.000	14.357	0.000
PL1	15.402	0.000	26.103	0.000	-	-	21.881	0.000	0.192	0.661	16.684	0.000	6.905	0.009	26.145	0.000
PL2	0.002	0.963	15.980	0.000	21.881	0.000	-	-	19.102	0.000	0.208	0.648	1.475	0.225	4.356	0.037
PL3	15.098	0.000	26.103	0.000	0.192	0.661	19.102	0.000	-	-	16.830	0.000	5.352	0.021	23.622	0.000
169-1	0.439	0.507	18.040	0.000	16.684	0.000	0.208	0.648	16.830	0.000	-	-	1.145	0.285	1.672	0.196
169-2	2.144	0.143	17.070	0.000	6.905	0.009	1.475	0.225	5.352	0.021	1.145	0.285	-	-	3.600	0.058
169-3	0.484	0.487	14.357	0.000	26.145	0.000	4.356	0.037	23.622	0.000	1.672	0.196	3.600	0.058	-	-