Despite of the widespread use of hydrogen and methane breath test, the variability in testing protocols, gas measurement techniques, and interpretation criteria continues to challenge the reproducibility and comparability across centers, especially in the Asia-Pacific region. The Asian Neurogastroenterology and Motility Association hence presents the first Asian monograph guiding application and interpretation of breath test. The monograph was formulated according to the framework of indications, preparatory process, performance, and interpretation of results, as well as future direction for research.

Objective:To explore the data distribution characteristics of hydrogen and methane breath test (HMBT) in Chinese population and to evaluate its applicability for diagnosing small intestinal bacterial overgrowth (SIBO) in Chinese population.Methods:HMBT data of 18 634 individuals who underwent health check-up nationwide from March 2019 to september 2022 were retrospectively collected, which included the levels of hydrogen and methane at 0, 30, 60, and 90 min. After quality control and data cleaning, the final valid sample size was 12 654 cases, comprising 7 146 SIBO-negative cases and 5 508 SIBO-positive cases. In order to exclude confounding factors such as oral hygiene, the 12 654 cases were divided into D0 and D1 dataset, when the 0 min-value of hydrogen and methane were both lower than the 30 min-value, the 0 min-value was taken as the baseline, and induded into the D0 dataset (5 556 cases), and other situations were induded into the D1 dataset (7 098 cases). There were 2 879 SIBO-negative cases and 2 659 SIBO-positive cases in D0 dataset, and 4 249 SIBO-negative cases and 2 849 SIBO-positive cases in D1 dataset. The hydrogen and methane level at each testing time point in the SIBO-negative and SIBO-positive individuals, the difference between the peak gas level at 90 min and the baseline, and the distribution of time points at which peak level occurred were analyzed. Independent-sample t test and Mann-Whitney U test were used for statistical analysis. Results:The overall SIBO positive rate was 43.53% (5 508/12 654). In SIBO-positive cases the hydrogen level at 0, 30, 60, and 90 min were 9.41×10 -6 (5.01×10 -6, 21.90×10 -6), 11.34×10 -6 (6.13×10 -6, 22.94×10 -6), 18.16×10 -6 (11.03×10 -6, 29.37×10 -6) and 29.59×10 -6 (20.12×10 -6, 43.36×10 -6), respectively, and methane level were 9.13×10 -6 (7.12×10 -6, 12.03×10 -6), 9.23×10 -6 (8.07×10 -6, 12.03×10 -6), 10.21×10 -6 (9.02×10 -6, 13.01×10 -6), and 12.03×10 -6 (10.01×10 -6, 14.11×10 -6), respectively, which were higher than those of SIBO-negative cases (6.04×10 -6 (3.10×10 -6, 11.08×10 -6), 6.04×10 -6 (3.21×10 -6, 10.06×10 -6), 6.95×10 -6 (4.03×10 -6, 11.01×10 -6), 8.96×10 -6 (5.01×10 -6, 13.91×10 -6); 8.04×10 -6 (7.02×10 -6, 10.00×10 -6), 8.03×10 -6 (7.03×10 -6, 9.95×10 -6), 8.04×10 -6 (7.03×10 -6, 10.00×10 -6) 8.98×10 -6 (7.12×10 -6, 10.03×10 -6)], and the differences were statistically significant ( U=1.41×10 7, 1.09×10 7, 6.66×10 6, 4.14×10 6, 1.51×10 7, 1.23×10 7, 1.02×10 7, 8.86×10 6; all P<0.001). In both D0 and D1 datasets, the increase in hydrogen and methane of SIBO positive subgroup were higher than those of SIBO negative subgroup (22.39×10 -6(14.82×10 -6, 33.37×10 -6) vs. 4.82×10 -6(1.96×10 -6, 7.85×10 -6), 20.61×10 -6(7.87×10 -6, 31.44×10 -6) vs. 3.25×10 -6(0.79×10 -6, 7.88×10 -6); 3.98×10 -6(2.87×10 -6, 6.87×10 -6) vs. 1.95×10 -6(0.98×10 -6, 2.99×10 -6), 2.95×10 -6(0.98×10 -6, 4.93×10 -6) vs. 0.98×10 -6(0.00×10 -6, 1.99×10 -6)), and the differences were statistically significant( U=7.24×10 6, 9.72×10 6, 5.74×10 6, 8.27×10 6; all P<0.001). In both D0 and D1 datasets, hydrogen and methane concentrations peaked at 90 min. Conclusion:HMBT can be used for non-invasive diagnosis of SIBO in Chinese population, and the differences in hydrogen and methane concentrations at 90 min of the test have critical value for SIBO diagnosis.

Objective:To analyze the drug resistance and virulence characteristics of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae(CRKP). Methods:A total of 26 non-repeating CRKP strains clinically isolated from a Class Ⅲ hospital in Kunming from August 2021 to March 2022 were collected. Mass spectrometry and the VITEK 2 Compact system were used to identify the bacteria and perform drug susceptibility tests. PCR was used to amplify the drug resistance and virulence genes carried by the strains. These CRKP strains were divided into a hypervirulent CRKP(CR-hvKP) group and a CR-non-hvKP group according to the characteristic virulence genes of hypervirulent Klebsiella pneumoniae. The virulence phenotypes of CRKP were investigated by wire drawing test, serum resistance test and siderophore qualitative and quantitative tests. The whole genomes of CRKP-67 (a CR-hvKP strain) and CRKP-94 (a CR-non-hvKP strain) were sequenced by the Illumina high-throughput sequencing platform, to further analyze the drug resistance genes, virulence genes, and virulence plasmidds carried by the strains. Results:The drug sensitivity results indicated that all 26 strains were resistant to carbapenem, cephalosporins, fluoroquinolones and β-lactam/β-lactam inhibitor complexes. The resistance rates to amicacin, cotrimoxazole and gentamicin were 61.54%(6/26), 57.69%(15/26) and 73.08%(9/26), respectively. Regarding the drug resistance gene amplification results, the carrying rates of blaKPC-2, blaNDM-1, blaOXA-48, blaVIM, blaIMP, blaSME, blaSHV, blaCTX-M and blaTEM were 100.00%(26/26), 0, 0, 0, 0, 100.00%(26/26), 100.00%(26/26), 15.38% (4/26) and 73.08%(19/26), respectively. In the 26 strains, the carrying rates of toxic genes entB, entC, ureA, uge, wabG, ycf, irp1, irp2, mrkD, fimH and ybtS were 100.00%(26/26), while the carrying rates of virulence genes kfuB, iroN, aero, magA and alls were 0. The positive rate of string test was 66.7%(6/9) in the CR-hvKP group and 0 in the CR-non-hvKP group. The serum killing test showed a high sensitivity rate of 77.78%(7/9), a low sensitivity rate of 11.11%(1/9), and a serum resistance rate of 11.11%(1/9) in the CR-hvKP group. In the CR-non-hvKP group, the high sensitivity rate was 29.41%(5/17); the low sensitivity rate was 17.65%(3/17), and the serum resistance rate was 52.94%(9/17). There was no statistical significance between the two groups( P>0.05). The qualitative results of siderophore showed that all strains produced yellow chelating circles with slightly different color depth and size. The quantitative results of siderophore experiment showed that the average siderophore production level of CR-hvKP group was 40.74%, and that of CR-non-hvKP group was 28.21%. The level was higher in the CR-hvKP group than in the CR-non-hvKP group, and the difference was statistically significant( P<0.05). Whole-genome sequencing results showed that CRKP-67 was ST11 type and contained 3 plasmids. Among them, plasmid pCRKP-67-A carried a series of virulence genes, including iucABCD, iutA, rmpA, rmpA2, iroB and peg344, which were highly virulent characteristic genes. Plasmid pCRKP-67-B carried blaKPC-2, blaCTX-M, blaSHV, blaTEM and other drug-resistant genes. Plasmid pCRKP-67-C carried sul2, tetR, tetA and other drug-resistant genes. The CRKP-94 was of ST340 type and contained a drug-resistant plasmid carrying blaKPC-2, blaCTX-M, blaSHV, blaTEM and other drug-resistant genes. Conclusions:CRKP strains are highly resistant, and are only sensitive to a few antibiotics, and carry a variety of drug resistance genes. The main resistance mechanism to carbapenem antibiotics is the presence of the blaKPC-2 gene, which is located on the plasmids, which results in the spread of carbapenem resistance. The types and quantity of virulence genes carried by the CR-hvKP strain are more and greater respectively than those carried by the CR-non-hvKP strain. The co-existence of drug-resistant and virulence plasmids in CR-hvKP strains may lead to the co-transmission of high drug resistance and hypervirulence, which should be highly valued by relevant departments.

Objective:To explore the data distribution characteristics of hydrogen and methane breath test (HMBT) in Chinese population and to evaluate its applicability for diagnosing small intestinal bacterial overgrowth (SIBO) in Chinese population.Methods:HMBT data of 18 634 individuals who underwent health check-up nationwide from March 2019 to september 2022 were retrospectively collected, which included the levels of hydrogen and methane at 0, 30, 60, and 90 min. After quality control and data cleaning, the final valid sample size was 12 654 cases, comprising 7 146 SIBO-negative cases and 5 508 SIBO-positive cases. In order to exclude confounding factors such as oral hygiene, the 12 654 cases were divided into D0 and D1 dataset, when the 0 min-value of hydrogen and methane were both lower than the 30 min-value, the 0 min-value was taken as the baseline, and induded into the D0 dataset (5 556 cases), and other situations were induded into the D1 dataset (7 098 cases). There were 2 879 SIBO-negative cases and 2 659 SIBO-positive cases in D0 dataset, and 4 249 SIBO-negative cases and 2 849 SIBO-positive cases in D1 dataset. The hydrogen and methane level at each testing time point in the SIBO-negative and SIBO-positive individuals, the difference between the peak gas level at 90 min and the baseline, and the distribution of time points at which peak level occurred were analyzed. Independent-sample t test and Mann-Whitney U test were used for statistical analysis. Results:The overall SIBO positive rate was 43.53% (5 508/12 654). In SIBO-positive cases the hydrogen level at 0, 30, 60, and 90 min were 9.41×10 -6 (5.01×10 -6, 21.90×10 -6), 11.34×10 -6 (6.13×10 -6, 22.94×10 -6), 18.16×10 -6 (11.03×10 -6, 29.37×10 -6) and 29.59×10 -6 (20.12×10 -6, 43.36×10 -6), respectively, and methane level were 9.13×10 -6 (7.12×10 -6, 12.03×10 -6), 9.23×10 -6 (8.07×10 -6, 12.03×10 -6), 10.21×10 -6 (9.02×10 -6, 13.01×10 -6), and 12.03×10 -6 (10.01×10 -6, 14.11×10 -6), respectively, which were higher than those of SIBO-negative cases (6.04×10 -6 (3.10×10 -6, 11.08×10 -6), 6.04×10 -6 (3.21×10 -6, 10.06×10 -6), 6.95×10 -6 (4.03×10 -6, 11.01×10 -6), 8.96×10 -6 (5.01×10 -6, 13.91×10 -6); 8.04×10 -6 (7.02×10 -6, 10.00×10 -6), 8.03×10 -6 (7.03×10 -6, 9.95×10 -6), 8.04×10 -6 (7.03×10 -6, 10.00×10 -6) 8.98×10 -6 (7.12×10 -6, 10.03×10 -6)], and the differences were statistically significant ( U=1.41×10 7, 1.09×10 7, 6.66×10 6, 4.14×10 6, 1.51×10 7, 1.23×10 7, 1.02×10 7, 8.86×10 6; all P<0.001). In both D0 and D1 datasets, the increase in hydrogen and methane of SIBO positive subgroup were higher than those of SIBO negative subgroup (22.39×10 -6(14.82×10 -6, 33.37×10 -6) vs. 4.82×10 -6(1.96×10 -6, 7.85×10 -6), 20.61×10 -6(7.87×10 -6, 31.44×10 -6) vs. 3.25×10 -6(0.79×10 -6, 7.88×10 -6); 3.98×10 -6(2.87×10 -6, 6.87×10 -6) vs. 1.95×10 -6(0.98×10 -6, 2.99×10 -6), 2.95×10 -6(0.98×10 -6, 4.93×10 -6) vs. 0.98×10 -6(0.00×10 -6, 1.99×10 -6)), and the differences were statistically significant( U=7.24×10 6, 9.72×10 6, 5.74×10 6, 8.27×10 6; all P<0.001). In both D0 and D1 datasets, hydrogen and methane concentrations peaked at 90 min. Conclusion:HMBT can be used for non-invasive diagnosis of SIBO in Chinese population, and the differences in hydrogen and methane concentrations at 90 min of the test have critical value for SIBO diagnosis.

Objective:To analyze the drug resistance and virulence characteristics of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae(CRKP). Methods:A total of 26 non-repeating CRKP strains clinically isolated from a Class Ⅲ hospital in Kunming from August 2021 to March 2022 were collected. Mass spectrometry and the VITEK 2 Compact system were used to identify the bacteria and perform drug susceptibility tests. PCR was used to amplify the drug resistance and virulence genes carried by the strains. These CRKP strains were divided into a hypervirulent CRKP(CR-hvKP) group and a CR-non-hvKP group according to the characteristic virulence genes of hypervirulent Klebsiella pneumoniae. The virulence phenotypes of CRKP were investigated by wire drawing test, serum resistance test and siderophore qualitative and quantitative tests. The whole genomes of CRKP-67 (a CR-hvKP strain) and CRKP-94 (a CR-non-hvKP strain) were sequenced by the Illumina high-throughput sequencing platform, to further analyze the drug resistance genes, virulence genes, and virulence plasmidds carried by the strains. Results:The drug sensitivity results indicated that all 26 strains were resistant to carbapenem, cephalosporins, fluoroquinolones and β-lactam/β-lactam inhibitor complexes. The resistance rates to amicacin, cotrimoxazole and gentamicin were 61.54%(6/26), 57.69%(15/26) and 73.08%(9/26), respectively. Regarding the drug resistance gene amplification results, the carrying rates of blaKPC-2, blaNDM-1, blaOXA-48, blaVIM, blaIMP, blaSME, blaSHV, blaCTX-M and blaTEM were 100.00%(26/26), 0, 0, 0, 0, 100.00%(26/26), 100.00%(26/26), 15.38% (4/26) and 73.08%(19/26), respectively. In the 26 strains, the carrying rates of toxic genes entB, entC, ureA, uge, wabG, ycf, irp1, irp2, mrkD, fimH and ybtS were 100.00%(26/26), while the carrying rates of virulence genes kfuB, iroN, aero, magA and alls were 0. The positive rate of string test was 66.7%(6/9) in the CR-hvKP group and 0 in the CR-non-hvKP group. The serum killing test showed a high sensitivity rate of 77.78%(7/9), a low sensitivity rate of 11.11%(1/9), and a serum resistance rate of 11.11%(1/9) in the CR-hvKP group. In the CR-non-hvKP group, the high sensitivity rate was 29.41%(5/17); the low sensitivity rate was 17.65%(3/17), and the serum resistance rate was 52.94%(9/17). There was no statistical significance between the two groups( P>0.05). The qualitative results of siderophore showed that all strains produced yellow chelating circles with slightly different color depth and size. The quantitative results of siderophore experiment showed that the average siderophore production level of CR-hvKP group was 40.74%, and that of CR-non-hvKP group was 28.21%. The level was higher in the CR-hvKP group than in the CR-non-hvKP group, and the difference was statistically significant( P<0.05). Whole-genome sequencing results showed that CRKP-67 was ST11 type and contained 3 plasmids. Among them, plasmid pCRKP-67-A carried a series of virulence genes, including iucABCD, iutA, rmpA, rmpA2, iroB and peg344, which were highly virulent characteristic genes. Plasmid pCRKP-67-B carried blaKPC-2, blaCTX-M, blaSHV, blaTEM and other drug-resistant genes. Plasmid pCRKP-67-C carried sul2, tetR, tetA and other drug-resistant genes. The CRKP-94 was of ST340 type and contained a drug-resistant plasmid carrying blaKPC-2, blaCTX-M, blaSHV, blaTEM and other drug-resistant genes. Conclusions:CRKP strains are highly resistant, and are only sensitive to a few antibiotics, and carry a variety of drug resistance genes. The main resistance mechanism to carbapenem antibiotics is the presence of the blaKPC-2 gene, which is located on the plasmids, which results in the spread of carbapenem resistance. The types and quantity of virulence genes carried by the CR-hvKP strain are more and greater respectively than those carried by the CR-non-hvKP strain. The co-existence of drug-resistant and virulence plasmids in CR-hvKP strains may lead to the co-transmission of high drug resistance and hypervirulence, which should be highly valued by relevant departments.

Objective:To investigate the mechanism by which non-enterotoxin-producing Bacteroides fragilis (NTBF) inhibits TNF-α-induced inflammatory responses in human normal colonic epithelial cells hcoEPIC, and explore new probiotic therapies for the prevention and treatment of colitis. Methods:The co-culture system of NTBF and hcoEPIC cells was established, and the adhesion and invasion ability of NTBF were detected, respectively. TNF-α was added to induce cellular inflammation after 4 h of co-culture of NTBF and hcoEPIC cells, and cell survival and apoptosis were detected by the CCK-8 assay and the AnnexinⅤ-FITC/PI assay respectively after 24 h. Key proteins of the NF-κB signalling pathway in hcoEPIC cells in different treatment groups were detected by Western blot and RT-qPCR, and the expression of downstream cytokines of this pathway incluing IL-1β, TNF-α and IL-10 were detected by ELISA. The effect of NTBF intervention on dextran sodium sulfate(DSS)-induced colitis mice was assessed by in vivo animal experiments. Results:NTBF adhered to hcoEPIC cells, and was non-toxic to the cells. Compared with control group, NTBF treatment alone did not affect cell survival and apoptosis of hcoEPIC cells ( P>0.05), but significantly reduced cell damage and apoptosis induced by TNF-α ( P<0.05); Compared with the TNF-α treatment alone group, the expression levels of p-NF-κB p65 and p-IκBα protein as well as NF-κB and IκBα mRNA were significantly reduced ( P<0.05); the production of IL-1β and TNF-α in the cell supernatant was reduced and the release of IL-10 was increased ( P<0.05). Animal experiments demonstrated that NTBF was indeed effective in alleviating DSS-induced colitis in ulcerative colitis model mice, which was mainly manifested by inhibiting weight loss, lowering DAI scores, improving colonic shortening, and attenuating colonic pathological damage in colitis-induced mice. Conclusions:NTBF may inhibit TNF-α-induced inflammatory responses in colonic epithelial cells by down-regulating the NF-κB pathway.

Ureaplasma is a common pathogen in the human reproductive tract and consists of two distinct biotypes: biotype 1 and biotype 2. In 2002, based on the differences between biotypes, biotype 1 was further classified to a separate species named Ureaplasma parvum (Up), whereas biotype 2 is referred to as Ureaplasma urealyticum (Uu). Uu infection is associated with various urogenital diseases including infertility, preterm birth, and urethritis, while the pathogenicity of Up remains controversial. Researches have shown that different serotypes showed distinct pathogenicity and drug resistance in different diseases and populations, highlighting the importance of clinical tests of serotype and biotype for Ureaplasma. This article reviews the factors that may be associated with Ureaplasma infection, and the current status of the diagnosis and treatment in clinical practice, aiming to provide insights into the clinical significance and necessity of biotypes and serotype tests for Ureaplasma-positive cases and to serve as a reference for the clinical diagnosis and treatment of related diseases.

BACKGROUND: Colorectal carcinogenesis and progression are related to the gut microbiota and the tumor immune microenvironment. Our previous clinical trial demonstrated that berberine (BBR) hydrochloride might reduce the recurrence and canceration of colorectal adenoma (CRA). The present study aimed to further explore the mechanism of BBR in preventing colorectal cancer (CRC). METHODS: We performed metagenomics sequencing on fecal specimens obtained from the BBR intervention trial, and the differential bacteria before and after medication were validated using quantitative polymerase chain reaction. We further performed ApcMin/+ animal intervention tests, RNA sequencing, flow cytometry, immunohistochemistry, and enzyme-linked immunosorbent assays. RESULTS: The abundance of fecal Veillonella parvula ( V . parvula ) decreased significantly after BBR administration ( P = 0.0016) and increased through the development from CRA to CRC. Patients with CRC with a higher V. parvula abundance had worse tumor staging and a higher lymph node metastasis rate. The intestinal immune pathway of Immunoglobulin A production was activated, and the expression of TNFSF13B (Tumor necrosis factor superfamily 13b, encoding B lymphocyte stimulator [BLyS]), the representative gene of this pathway, and the genes encoding its receptors (interleukin-10 and transforming growth factor beta) were significantly upregulated. Animal experiments revealed that V. parvula promoted colorectal carcinogenesis and increased BLyS levels, while BBR reversed this effect. CONCLUSION: BBR might inhibit V. parvula and further weaken the immunomodulatory effect of B cells induced by V. parvula , thereby blocking the development of colorectal tumors. TRIAL REGISTRAION ClinicalTrials.gov, No. NCT02226185.
Animals ; Humans ; Berberine/therapeutic use* ; Carcinogenesis ; Veillonella ; Colorectal Neoplasms/genetics* ; Tumor Microenvironment

ObjectiveTo investigate the structural features of vaginal microbiota in the early threatened abortion patients with the syndrome of kidney deficiency. MethodThirty-one patients with early threatened abortion of kidney deficiency syndrome （DK-TA group） and 116 women with normal early pregnancy （NP group） attending the First Affiliated Hospital of Guangzhou University of Chinese Medicine from May 2018 to December 2020 were selected. The vaginal secretions were collected for 16S rRNA sequencing， which can reveal the vaginal microbiota composition and differential bacteria between the two groups. ResultThe DK-TA group had higher abundance and diversity of vaginal microbiota than the NP group. The Binary jaccard and unweighted_unifrac distance matrix analysis showed that the similarity， dispersion， abundance， and phylogenetic relationship of vaginal microbiota were significantly different between the two groups. At the phylum level， the DK-TA group had lower relative abundance of Bacteroidetes and Fusobacteria and higher relative abundance of Proteobacteria and Gemmatimonadetes than the NP group. At the genus level， the DK-TA group had lower relative abundance of Sneathia and Bifidobacterium and higher relative abundance of Escherichia-Shigella and Shuttleworthia than the NP group. Linear discriminant analysis Effect Size（LEfSe）revealed that Bacteroidetes， Fusobacteria， and Bifidobacteria were dominant in the NP group and Proteobacteria and Firmicutes in the DK-TA group. The function prediction found that DK-TA was closely associated with 38 functional pathways， including cyclic adenosine monophosphate signaling pathway and regulation of tryptophan channels by inflammatory mediators. In addition， the vaginal differential bacteria between the two groups had significant positive or negative association with the differential metabolic pathways. ConclusionProteobacteria， Gemmatimonadetes， and Bacteroidetes in the vaginal microbiota may be biomarkers for threatened abortion of kidney deficiency syndrome.