Exploration on Application Effectiveness of Microbial Capture Filter Membranes from Different Materials in Barrier Environment Facilities

doi:10.12300/j.issn.1674-5817.2024.004

Abstract

Abstract:

Objective To evaluate the effectiveness of filter membranes made from different materials in monitoring the health status of rodents in barrier environment facilities by investigating their microbial capture performance. Methods Pasteurella pneumotropica (Pp) and Staphylococcus aureus (Sa) were used as representative strains to simulate the process of microbial capture by filter membranes under laboratory conditions. The microbial capture effectiveness of five self-selected filter membranes (M1, M2, M3, M4, and M5) with adsorption and breathability properties and a commercial filter membrane (T1) were comprehensively evaluated based on captured dust mass, minimum detection limit, and differences in Ct values obtained through fluorescence quantitative PCR detection. The best-performing self-selected filter membrane was placed in the ventilation ducts of cage racks within the barrier facility, with sentinel mice in corresponding cage racks as the control group. Staphylococcus epidermidis and Escherichia coli were used as indicator bacteria to calculate the positive detection rate and coincidence rate, thereby exploring the feasibility of using microbial capture filter membranes to monitor the health status of experimental animals in barrier facilities. Results In terms of the captured dust mass, the self-selected filter membrane M3 (non-woven filter membrane with a diameter of 0.1 um)；showed a capture effectiveness second only to T1, with a capture mass of 0.126 g. For Sa, all filter membranes except M4 had a minimum detection limit of 102 CFU/g. For Pp, the minimum detection limit for all filter membranes was 10² CFU/g. However, the Ct value of the quantitative fluorescence PCR amplification results for M3 was significantly lower than that of other materials, indicating that M3 had the best capture performance among the five self-selected materials. In the filter detection verification experiment, the positive detection rate of Staphylococcus epidermidis in sentinel mouse feces and M3 was 50.00% (6/12) and 58.33% (7/12), respectively, with a coincidence rate of 92%. The positive detection rate of Escherichia coli in both sentinel mouse feces and M3 was 50.00% (6/12), with a coincidence rate of 100%. Conclusion Among the 5 self-selected filter membranes, M3 exhibits the best capturing performance. Within the barrier environment facilities, M3 outperforms sentinel mice in monitoring Staphylococcus epidermidis. Therefore, non-woven filter membrane with a diameter of 0.1 um； can be used as the material for microbial capture filter membranes, providing valuable insights for the selection and application of microbial capture filter membranes used in PCR monitoring of cage exhaust air dust.

Key words: Microbial capture filter membrane, Barrier environment facilities, Cage exhaust air dust, Fluorescence quantitative PCR

CLC Number:

Q95-33

KONG Chuiqin,TIAN Miaomiao,CAI Lidong,et al. Exploration on Application Effectiveness of Microbial Capture Filter Membranes from Different Materials in Barrier Environment Facilities[J]. Laboratory Animal and Comparative Medicine, 2024, 44(4): 436-444. DOI: 10.12300/j.issn.1674-5817.2024.004.

Figures/Tables 6

Table 1 Information of microbial capture filter membranes made from different materials

组别 Group	材质 Material	滤径/μm Filter diameter/μm	规格 Specification	厂家 Manufacturer
M1	玻纤滤材	0.1	H13高效	重庆再升科技股份有限公司
M2	PPK3熔喷布	0.1	90 g/m²	上海仁益制毡有限公司
M3	无纺布	0.1	80 g/m²	东莞市佳联达无纺布有限公司
M4	聚丙烯微孔滤膜	0.3	PP-T 0.22 μm	山东新华医疗器械股份有限公司
M5	脱脂纱布	0.2	7.5 cm×7.5 cm	徐州市徐卫卫生材料有限责任公司
T1	未知	未知	未知	泰尼百斯

Figure 1 Mixing effect of exhaust air dust (EAD) simulated samples contaminated by Staphylococcus aureus (Sa) (A) and Pasteurella pneumotropica (Pp) (B)Note：Ct， cycle threshold； CFU， colony forming unit.

Figure 2 Dust capture effectiveness of filter membranes made from different materialsNote：In the figure, M1, M2, M3, M4, and M5 were the materials selected for the experiment, and T1 was the purchased finished product. Based on visual observation, surfaces of M3, M5, and T1 had a large amount of dust attached, while surfaces of M1, M2, and M4 had relatively less dust attached. The mass differences of each filter membrane material, ranked from lowest to highest, were M4<M1<M2<M5<M3<T1.

Table 2 Evaluation of dust capturing effectiveness of filter membranes made from different materials

材质种类 Material	材质大小/cm Size/cm	粉尘捕捉前滤膜质量/g m₁/g	粉尘捕捉后滤膜质量/g m₂/g	质量差/g m（m₁－m₂）/g
M1	5×3	0.196±0.003	0.266±0.000	0.070±0.049
M2	5×3	0.070±0.000	0.164±0.000	0.094±0.067
M3	5×3	0.390±0.000	0.516±0.000	0.126±0.089^*
M4	5×3	0.202±0.000	0.221±0.000	0.019±0.013
M5	5×3	0.203±0.000	0.286±0.000	0.084±0.059
T1	5×3	0.617±0.000	0.760±0.000	0.143±0.101^*

Table 3 Detection sensitivity and coefficient of variation (CV) for Staphylococcus aureus (Sa) and Pasteurella pneumotropica (Pp) using filter membranes made from different materials

材质 Material Bacteria	细菌浓度/（CFU·g^-1） concentration/（CFU·g^-1）	金黄色葡萄球菌 Staphylococcus aureus (Sa)		嗜肺巴斯德杆菌 Pasteurella pneumotropica (Pp)
材质 Material Bacteria	细菌浓度/（CFU·g^-1） concentration/（CFU·g^-1）	循环阈值（ $x ¯$ ±s） Ct ( $x ¯$ ±s)	变异系数/% CV/%	循环阈值（ $x ¯$ ±s） Ct( $x ¯$ ±s)	变异系数/% CV/%
M1	10⁶	24.03±0.13	0.005	23.59±0.42	0.018
	10⁵	26.13±0.23	0.009	25.88±0.23	0.009
	10⁴	29.51±0.43	0.015	28.82±0.60	0.021
	10³	33.72±0.18	0.005	33.84±0.39	0.012
	10²	36.52±0.27	0.007	36.15±0.28	0.008
M2	10⁶	23.42±1.06	0.045	19.50±0.17	0.009
	10⁵	26.50±0.47	0.018	22.15±0.22	0.010
	10⁴	28.22±0.13	0.005	25.67±0.33	0.013
	10³	32.30±0.28	0.009	30.33±0.34	0.011
	10²	35.28±0.42	0.012	35.97±0.25	0.007
M3	10⁶	24.74±0.17	0.007	19.78±0.43	0.022
	10⁵	26.08±0.00	0.000	21.46±0.10	0.005
	10⁴	29.60±0.23	0.008	25.25±0.28	0.011
	10³	35.01±0.03	0.001	27.39±0.19	0.007
	10²	35.14±0.28^*	0.008	32.91±0.80^*	0.024
M4	10⁶	28.40±0.05	0.002	24.96±0.43	0.017
	10⁵	29.59±0.02	0.001	28.39±0.34	0.012
	10⁴	35.70±0.62	0.017	30.83±0.51	0.017
	10³	36.24±0.21	0.006	33.64±0.45	0.013
	10²	/	/	36.56±0.00	0.012
M5	10⁶	23.31±0.41	0.018	18.04±0.70	0.039
	10⁵	25.96±0.16	0.006	21.76±0.49	0.023
	10⁴	28.85±0.25	0.009	25.08±0.23	0.009
	10³	33.23±0.36	0.011	28.11±0.46	0.016
	10²	35.56±0.45	0.013	32.94±0.40	0.012
T1	10⁶	22.92±0.31	0.014	16.88±0.43	0.025
	10⁵	25.36±0.23	0.009	21.62±0.34	0.016
	10⁴	28.93±0.13	0.004	23.68±0.44	0.019
	10³	31.98±0.37	0.012	27.46±0.75	0.027
	10²	33.14±0.00^*	0.024	32.10±0.28^*	0.009

Table 3 Detection sensitivity and coefficient of variation (CV) for Staphylococcus aureus (Sa) and Pasteurella pneumotropica (Pp) using filter membranes made from different materials

材质 Material Bacteria	细菌浓度/（CFU·g^-1） concentration/（CFU·g^-1）	金黄色葡萄球菌 Staphylococcus aureus (Sa)		嗜肺巴斯德杆菌 Pasteurella pneumotropica (Pp)
材质 Material Bacteria	细菌浓度/（CFU·g^-1） concentration/（CFU·g^-1）	循环阈值（ $x ¯$ ±s） Ct ( $x ¯$ ±s)	变异系数/% CV/%	循环阈值（ $x ¯$ ±s） Ct( $x ¯$ ±s)	变异系数/% CV/%
M1	10⁶	24.03±0.13	0.005	23.59±0.42	0.018
	10⁵	26.13±0.23	0.009	25.88±0.23	0.009
	10⁴	29.51±0.43	0.015	28.82±0.60	0.021
	10³	33.72±0.18	0.005	33.84±0.39	0.012
	10²	36.52±0.27	0.007	36.15±0.28	0.008
M2	10⁶	23.42±1.06	0.045	19.50±0.17	0.009
	10⁵	26.50±0.47	0.018	22.15±0.22	0.010
	10⁴	28.22±0.13	0.005	25.67±0.33	0.013
	10³	32.30±0.28	0.009	30.33±0.34	0.011
	10²	35.28±0.42	0.012	35.97±0.25	0.007
M3	10⁶	24.74±0.17	0.007	19.78±0.43	0.022
	10⁵	26.08±0.00	0.000	21.46±0.10	0.005
	10⁴	29.60±0.23	0.008	25.25±0.28	0.011
	10³	35.01±0.03	0.001	27.39±0.19	0.007
	10²	35.14±0.28^*	0.008	32.91±0.80^*	0.024
M4	10⁶	28.40±0.05	0.002	24.96±0.43	0.017
	10⁵	29.59±0.02	0.001	28.39±0.34	0.012
	10⁴	35.70±0.62	0.017	30.83±0.51	0.017
	10³	36.24±0.21	0.006	33.64±0.45	0.013
	10²	/	/	36.56±0.00	0.012
M5	10⁶	23.31±0.41	0.018	18.04±0.70	0.039
	10⁵	25.96±0.16	0.006	21.76±0.49	0.023
	10⁴	28.85±0.25	0.009	25.08±0.23	0.009
	10³	33.23±0.36	0.011	28.11±0.46	0.016
	10²	35.56±0.45	0.013	32.94±0.40	0.012
T1	10⁶	22.92±0.31	0.014	16.88±0.43	0.025
	10⁵	25.36±0.23	0.009	21.62±0.34	0.016
	10⁴	28.93±0.13	0.004	23.68±0.44	0.019
	10³	31.98±0.37	0.012	27.46±0.75	0.027
	10²	33.14±0.00^*	0.024	32.10±0.28^*	0.009

Table 4 Detection validation results for filter membrane M3

检测项目 Testing item	M3滤膜 M3 filter	哨兵鼠粪便样品 Sentinel mouse faeces			符合率/% Coincidence rate/%
检测项目 Testing item	M3滤膜 M3 filter	阳性 Positive	阴性 Negative	汇总 Summary	符合率/% Coincidence rate/%
表皮葡萄球菌 Staphylococcus epidermidis	阳性	6	1	7	92
	阴性	0	5	5
	汇总	6	6	12
大肠埃希菌 Escherichia coli	阳性	6	0	6	100
	阴性	0	6	6
	汇总	6	6	12

References 29

1	LIPMAN N S, HOMBERGER F R. Rodent quality assurance testing: use of sentinel animal systems[J]. Lab Anim, 2003, 32(5):36-43. DOI: 10.1038/laban0503-36 .
2	LIVINGSTON R S, RILEY L K. Diagnostic testing of mouse and rat colonies for infectious agents[J]. Lab Anim, 2003, 32(5):44-51. DOI: 10.1038/laban0503-44 .
3	O'CONNELL K A, TIGYI G J, LIVINGSTON R S, et al. Evaluation of in-cage filter paper as a replacement for sentinel mice in the detection of murine pathogens[J]. J Am Assoc Lab Anim Sci, 2021, 60(2):160-167. DOI: 10.30802/AALAS-JAALAS-20-000086 .
4	于灵芝, 魏晓锋, 黎明, 等. 啮齿类实验动物健康监测用脏垫料哨兵动物法和排风粉尘PCR法比较[J]. 实验动物与比较医学, 2024, 44(3): 321-327. DOI:10.12300/j.issn.1674-5817.2023.168 .
	YU L Z, WEI X F, LI M, et al. Comparison of methods between soiled bedding sentinels and exhaust air dust PCR for health monitoring of rodent laboratory animals[J]. Lab Anim Comp Med, 2024, 44(3): 321-327. DOI:10.12300/j.issn.1674-5817.2023.168 .
5	MILLER M, BRIELMEIER M. Environmental samples make soiled bedding sentinels dispensable for hygienic monitoring of IVC-reared mouse colonies[J]. Lab Anim, 2018, 52(3):233-239. DOI: 10.1177/0023677217739329 .
6	COMPTON S R, HOMBERGER F R, PATURZO F X, et al. Efficacy of three microbiological monitoring methods in a ventilated cage rack[J]. Comp Med, 2004, 54(4):382-392.
7	HENDERSON K S, PERKINS C L, HAVENS R B, et al. Efficacy of direct detection of pathogens in naturally infected mice by using a high-density PCR array[J]. J Am Assoc Lab Anim Sci, 2013, 52(6):763-772. DOI: 10.1136/vr.f6532 .
8	BESSELSEN D G, MYERS E L, FRANKLIN C L, et al. Transmission probabilities of mouse parvovirus 1 to sentinel mice chronically exposed to serial dilutions of contaminated bedding[J]. Comp Med, 2008, 58(2):140-144. DOI: 10.1111/j.1751-0813.2008.00260.x .
9	JENSEN E S, ALLEN K P, HENDERSON K S, et al. PCR testing of a ventilated caging system to detect murine fur mites[J]. J Am Assoc Lab Anim Sci, 2013, 52(1):28-33. DOI: 10.1016/j.jinsphys.2013.01.001 .
10	KÖRNER C, MILLER M, BRIELMEIER M. Detection of Murine Astrovirus and Myocoptes musculinus in individually ventilated caging systems: investigations to expose suitable detection methods for routine hygienic monitoring[J]. PLoS One, 2019, 14(8): e0221118. DOI: 10.1371/journal.pone.0221118 .
11	MILLER M, RITTER B, ZORN J, et al. Exhaust air dust monitoring is superior to soiled bedding sentinels for the detection of Pasteurella pneumotropica in individually ventilated cage systems[J]. J Am Assoc Lab Anim Sci, 2016, 55(6):775-781.
12	NIIMI K, MARUYAMA S, SAKO N, et al. The Sentinel^TM EADR program can detect more microorganisms than bedding sentinel animals[J]. Jpn J Vet Res, 2018, 66(2): 125-129. DOI: 10.14943/jjvr.66.2.125 .
13	PETTAN-BREWER C, TROST R J, MAGGIO-PRICE L, et al. Adoption of exhaust air dust testing in SPF rodent facilities[J]. J Am Assoc Lab Anim Sci, 2020, 59(2):156-162. DOI: 10.30802/AALAS-JAALAS-19-000079 .
14	WHARY M T, CLINE J H, KING A E, et al. Monitoring sentinel mice for Helicobacter hepaticus, H rodentium, and H bilis infection by use of polymerase chain reaction analysis and serologic testing[J]. Comp Med, 2000, 50(4):436-443.
15	DUBELKO A R, ZUWANNIN M, MCINTEE S C, et al. PCR testing of filter material from IVC lids for microbial monitoring of mouse colonies[J]. J Am Assoc Lab Anim Sci, 2018, 57(5):477-482. DOI: 10.30802/aalas-jaalas-18-000008 .
16	GERWIN P M, RICART ARBONA R J, RIEDEL E R, et al. PCR testing of IVC filter tops as a method for detecting murine pinworms and fur mites[J]. J Am Assoc Lab Anim Sci, 2017, 56(6):752-761.
17	ZORN J, RITTER B, MILLER M, et al. Murine norovirus detection in the exhaust air of IVCs is more sensitive than serological analysis of soiled bedding sentinels[J]. Lab Anim, 2017, 51(3):301-310. DOI: 10.1177/0023677216661586 .
18	MAILHIOT D, OSTDIEK A M, LUCHINS K R, et al. Comparing mouse health monitoring between soiled-bedding sentinel and exhaust air dust surveillance programs[J]. J Am Assoc Lab Anim Sci, 2020, 59(1):58-66. DOI: 10.30802/aalas-jaalas-19-000061 .
19	LUCHINS K R, MAILHIOT D, THERIAULT B R, et al. Detection of lactate dehydrogenase elevating virus in a mouse vivarium using an exhaust air dust health monitoring program[J]. J Am Assoc Lab Anim Sci, 2020, 59(3):328-333. DOI: 10.30802/AALAS-JAALAS-19-000107 .
20	BAUER B A, BESCH-WILLIFORD C, LIVINGSTON R S, et al. Influence of rack design and disease prevalence on detection of rodent pathogens in exhaust debris samples from individually ventilated caging systems[J]. J Am Assoc Lab Anim Sci, 2016, 55(6):782-788.
21	LUPINI L, BASSI C, GUERRIERO P, et al. Microbiota and environmental health monitoring of mouse colonies by metagenomic shotgun sequencing[J]. World J Microbiol Biotechnol, 2022, 39(1):37. DOI: 10.1007/s11274-022-03469-0 .
22	王立鹏, 李永旺, 王晨娟, 等. Dole qPCR检测嗜肺巴斯德杆菌的可靠性研究及在啮齿类实验动物质量监测中的应用[J]. 现代检验医学杂志, 2019, 34(5):109-114. DOI: 10.3969/j.issn.1671-7414.2019.05.027 .
	WANG L P, LI Y W, WANG C J, et al. Reliability research of Dole qPCR method on Pasteurella pneumotropica and application in health screening of laboratory rodents[J]. J Mod Lab Med, 2019, 34(5):109-114. DOI: 10.3969/j.issn.1671-7414.2019.05.027 .
23	于灵芝, 谢建芸, 冯丽萍, 等. 金黄色葡萄球菌荧光定量PCR检测方法的建立及其在大鼠、小鼠粪便检测中的应用[J]. 实验动物与比较医学, 2023(5):566-573. DOI: 10.12300/j.issn.1674-5817.2023.022 .
	YU L Z, XIE J Y, FENG L P, et al. Establishment of fluorescence qPCR method for detection of Staphylococcus aureus and its application in feces detection of rats and mice[J]. Lab Anim Comp Med, 2023(5):566-573. DOI: 10.12300/j. issn.1674-5817.2023.022 .
24	RAGLAND N H, MIEDEL E L, GOMEZ J M, et al. Staphylococcus xylosus PCR-validated decontamination of murine individually ventilated cage racks and air handling units by using 'active-closed' exposure to vaporized hydrogen peroxide[J]. J Am Assoc Lab Anim Sci, 2017, 56(6):742-751.
25	WON Y S, KWON H J, OH G T, et al. Identification of Staphylococcus xylosus isolated from C57BL/6J-Nos2(tm1Lau) mice with dermatitis[J]. Microbiol Immunol, 2002, 46(9):629-632. DOI: 10.1111/j.1348-0421.2002.tb02744.x .
26	史志远, 陈璐萍, 李博星, 等. 不同粪便DNA提取方法比较分析[J]. 生物工程学报, 2022, 38(9):3542-3550. DOI: 10.13345/j.cjb.220085 .
	SHI Z Y, CHEN L P, LI B X, et al. Comparative analysis of different fecal DNA extraction methods[J]. Chin J Biotechnol, 2022, 38(9):3542-3550. DOI: 10.13345/j.cjb.220085 .
27	WINN C B, ROGERS R N, KEENAN R A, et al. Using filter media and soiled bedding in disposable individually ventilated cages as a refinement to specific pathogen-free mouse health monitoring programs[J]. J Am Assoc Lab Anim Sci, 2022, 61(4):361-369. DOI: 10.30802/AALAS-JAALAS-22-000013 .
28	MANUEL C A, PUGAZHENTHI U, SPIEGEL S P, et al. Detection and elimination of Corynebacterium bovis from barrier rooms by using an environmental sampling surveillance program[J]. J Am Assoc Lab Anim Sci, 2017, 56(2):202-209.
29	KIM E, YANG S M, WON J E, et al. Real-time PCR method for the rapid detection and quantification of pathogenic Staphylococcus species based on novel molecular target genes[J]. Foods, 2021, 10(11):2839. DOI: 10.3390/foods10112839 .

[1]	Fangni LIU, Junping LU, Yuehua KE, Changjun WANG, Jinpeng GUO. Prevalence of Mouse Norovirus in Experimental Mice in Beijing [J]. Laboratory Animal and Comparative Medicine, 2023, 43(2): 205-212.
[2]	WANG Rui-qi, LIAN Chuan-jiang, YI Cheng, HAN Ling-xia, YANG Chun-wen, CHEN Hong-yan. Differential Expression Analysis of miR-200b-3p and miR-200b-5p in Marek’s Disease Resistant and Susceptible SPF Chickens [J]. Laboratory Animal and Comparative Medicine, 2017, 37(3): 244-248.