Comparative Study On Microbial Sampling Methods Of Esophageal Squamous Cell Carcinoma Biopsy And Mucosal Swab

BackgroundHistological and cytological samplings are the main types of method for sampling microbiota of upper gastrointestinal(UGI)tract.However,there is no consensus regarding the optimal sampling method for UGI tract microbiota.In order to compare tissue biopsy and mucosal swab specimens for assessment of the UGI microbiota,paired tissue biopsy and mucosal swab specimens were collected from patients diagnosed with esophageal squamous cell carcinoma(ESCC),who were scheduled for surgery with the organ removal.Without a true gold-standard method,we systematically compared biopsies and mucosal swab specimens for microbial sampling from ESCC patients in order to provide insight for planning future microbiota collections from the UGI tract.MethodsA total of 67 ESCC participants were recruited in the Linxian Cancer Hospital,Henan province,China.Sterile biopsy forceps and swabs were used to collect paired tissue specimens and mucosal swab specimens from the surgical resection specimens of ESCC carcinoma and adjacent normal tissue.All the specimens were stored at-80 Celsius after collection until DNA extraction.The V4 region of the 16S rRNA gene was amplified and the sequencing was performed on the Illumina MiniSeq platform.Data from 16S rRNA gene sequencing was processed using QIIME(version 2.0)to evaluate differences in alpha diversity,beta diversity and taxonomic relative abundances between different specimen types.ResultsSimilar alpha diversity was observed for swab specimens compared to biopsies.For ESCC carcinoma,paired specimens had similar observed operational taxonomic units(OTUs)(swab=69,biopsy=64,p=0.46)and Shannon index(swab=4.5,biopsy=4.3,p=0.41)values.Similar to carcinoma paired specimens,observed OTUs(swab=56,biopsy=61,p=0.46)and the Shannon diversity index(swab=4.2,biopsy=4.0,p=0.56)of ESCC adjacent normal specimens showed no differences in alpha diversity between the swabs and the biopsies.Alpha diversity of paired specimens was correlated for biopsies and mucosal swabs(rho>0.60,p<0.001).For beta diversity,distinct clustering by sampling method was not observed for adjacent normal or tumor tissue from ESCC.The Mantel test showed a high correlation for both weighted UniFrac distance and Bray-Curtis distance in paired specimens(rho>0.60,p=0.001).The ten dominant bacterial genera were similar between swab and biopsy specimens.However,higher levels of relative abundance of Veillonella(p=0.0002)were detected in ESCC adjacent normal swabs compared to the biopsies.ConclusionsIn conclusion,mucosal swab specimens from ESCC participants appear to yield similar microbial profiles as tissue biopsies.The collection method for a new study should be determined based on feasibility and sampling invasiveness.Additional studies of samples collected during an upper endoscopy from precancerous lesions of ESCC are needed to confirm our findings.