|Table of Contents|

Establishment of an in vitro model of bacteria adhesion to respiratory epithelial cells



Research Field:
Publishing date:



Establishment of an in vitro model of bacteria adhesion to respiratory epithelial cells


WANG Ruoxi WANG Qian SUN Fengjun LIU Yao ZHANG Lei LI Xiaoyu XIA Peiyuan

Department of Pharmacy, First Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400038, China


flow chamber bacterium epithelial cells adhesion

R322.3; R329-33; R378

Objective    To establish an in vitro model to study the adhesion of bacteria to respiratory epithelial cells based on the construction of flow-chamber. Methods    After 1×105 HBE cells were seeded in the flow chamber precoated with 2 mg/mL bovine collagen, the chamber was cultured in RPMI 1640 medium containing 20% serum and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. Once the bottom was covered with HBE cells, the chamber could be used for further experiments. When the number of cells in the flow chamber was taken as the evaluation index, the effects of the flow rate, the flow duration and the mobile phase composition on the cellular model were investigated by orthogonal design, and the optimal experimental conditions were screened. Then under the obtain optimal conditions, after the bacteria were adhered to the cells at 2×108, 108, 5×107, and 2.5×107 CFU/mL inoculated concentrations respectively, the amount of bacterial adhesion was taken as an index, and the results were compared with the conventional plate culture for bacterial adhering to cells, so as to determine whether the flow chamber is a viable model of bacteria adhesion to cells. The amount of bacteria adhering to cells was characterized by SYTO9 fluorescently labeled bacteria. Results    The influencing factors in optimal experimental conditions in order of their impacts were as follows: flow rate>flow duration>mobile phase composition. The ANOVA study showed that the impacts of flow rate and flow duration were significant (P<0.05), and while those of mobile phase composition were not. After fluorescent staining, the model realized the real-time fluorescence observation of the bacteria adhesion to the respiratory epithelial cells. According to the quantitative results of bacterial adhesion, with the increase of multiplicity of infection, both the flow chamber and the conventional culture plate detected the elevated amount of adhering bacteria, in a linear manner. However, with the same multiplicity of infection, the conventional culture plate model detected significantly larger amount of adhering bacteria when compared to the flow chamber (P<0.05). Conclusion   Under appropriate flow rate and flow duration, our model is an alternative to the conventional plate for studying the adhesion of bacteria to the airway epithelial cells, with the advantages of being more close to the in vivo environment, high accuracy and real-time observation


[1]CHOI C H, LEE J S, LEE Y C, et al. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells[J]. BMC Microbiol, 2008, 8(1): 216. DOI:10.1186/1471-2180-8-216.
[2]SMANI Y, MCCONNELL M J, PACHON J. Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells[J]. PLoS ONE, 2012, 7(4): e33073. DOI:10.1371/journal.pone.0033073.
[3]PEREZ A, MERINO M, RUMBOFEAL S, et al. The FhaB/FhaC twopartner secretion system is involved in adhesion of Acinetobacter baumannii AbH12OA2 strain[J]. Virulence, 2017, 8(6): 959-974. DOI:10.1080/21505594.2016.1262313.
[4]VAN DER FLIER M, CHHUN N, WIZEMANN T M, et al. Adherence of Streptococcus pneumoniae to immobilized fibronectin[J]. Infect Immun, 1995, 63(11): 4317-4322.
[5]ANDERSEN T E, KINGSHOTT P, PALARASAH Y, et al. A flow chamber assay for quantitative evaluation of bacterial surface colonization used to investigate the influence of temperature and surface hydrophilicity on the biofilm forming capacity of uropathogenic Escherichia coli[J]. J Microbiol Methods, 2010, 81(2): 135-140. DOI:10.1016/j.mimet.2010.02.009.
[6]ARAI T, OCHIAI K, SENPUKU H. Actinomyces naeslundii GroELdependent initial attachment and biofilm formation in a flow cell system[J]. J Microbiol Methods, 2015, 109: 160-166. DOI:10.1016/j.mimet.2014.12.021.
[7]YU C, LI X, ZHANG N, et al. Inhibition of biofilm formation by Dtyrosine: effect of bacterial type and Dtyrosine concentration[J]. Water Res, 2016, 92: 173-179. DOI:10.1016/j.watres.2016.01.037.
[8]QIN L, KIDA Y, ISHIWADA N, et al. The relationship between biofilm formations and capsule in Haemophilus influenzae[J]. J Infect Chemother, 2014, 20(3): 151-156. DOI:10.1016/j.jiac.2013.06.001.
[9]NEIL R B, SHAO J Q, APICELLA M A. Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B[J]. Microbes Infect, 2009, 11(2): 281-287. DOI:10.1016/j.micinf.2008.12.001.
[10]黎皓思, 潘频华. 鲍曼不动杆菌黏附相关机制研究进展[J]. 中国感染与化疗杂志, 2015, 15(5): 496-500.
LI H S, PAN P H. Research advances on adherence mechanism of Acinetobacter baumannii.[J]. Chin J Infect Chemother, 2015, 15(5): 496-500.
[11]GUZEK A, RYBICKI Z, KORZENIEWSKI K, et al. Etiological factors causing lower respiratory tract infections isolated from hospitalized patients[J]. Adv Expl Med Biol, 2015,835:37-44. DOI:10.1007/5584_2014_23.
[12]AMENDYSSILVA S A, CORREAGARCA P, GARCAGUILLN F J, et al. Outcomes of critically ill cancer patients with Acinetobacter baumannii infection[J]. World J Crit Care Med, 2015, 4(3): 258-264. DOI:10.5492/wjccm.v4.i3.258.
[13]MCCONNELL M J, ACTIS L, PACHN J. Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models[J]. FEMS Microbiol Rev, 2013, 37(2): 130-155. DOI:10.1111/j.15746976.2012.00344.x.
[15]BAKKER D P, VAN DER PLAATS A, VERKERKE G J, et al. Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces[J]. Appl Environ Microbiol, 2003, 69(10): 6280-6287. DOI:10.1128/aem.69.10.6280-6287.2003.
[16]KHAN O F, CHAMBERLAIN M D, SEFTON M V. Toward an in vitro vasculature: differentiation of mesenchymal stromal cells within an endothelial cell-seeded modular construct in a microfluidic flow chamber[J]. Tissue Eng Part A, 2012, 18(7/8): 744-756. DOI:10.1089/ten.TEA.2011.0058.


Last Update: 2018-06-14