Comparison of different hypervariable regions of 16S rRNA for taxonomic profiling of vaginal microbiota using next-generation sequencing.

The exploration of vaginal microbiota by using next-generation sequencing (NGS) of 16S ribosomal RNA (rRNA) gene is widely used. Up to now, different hypervariable regions have been selected to study vaginal microbiota by NGS and there is no standard method for analysis. The study aimed to characterize vaginal microbiota from clinical samples using NGS targeting the 16S rRNA gene and to determine the performance of individual and concatenated hypervariable region sequences to generate the taxonomic profiles of the vaginal microbiota. Fifty-one vaginal DNA samples were subjected to 16S rRNA gene NGS based on the Ion Torrent PGM platform with the use of two primer sets spanning seven hypervariable regions of the 16S rRNA gene. Our analysis revealed that the predominant bacterial genera were Lactobacillus, Gardnerella and Atopobium, which accounted for 78%, 14% and 2%, respectively, of sequences from all vaginal bacterial genera. At the species level, Lactobacillus iners, Gardnerella vaginalis and Atopobium vaginae accounted for 72%, 10% and 6%, respectively, of the bacterial cells present. Analyses using the V3 region generally indicated the highest bacterial diversity followed by the V6-V7 and V4 regions, while the V9 region gave the lowest bacterial resolution. NGS based on the 16S rRNA gene can give comprehensive estimates of the diversity of vaginal bacterial communities. Selection of sequences from appropriate hypervariable regions is necessary to provide reliable information on bacterial community diversity.

Novel mutations detected from drug resistant Mycobacterium tuberculosis isolated from North East of Thailand.

The emergence of drug-resistant tuberculosis is a major global public health threat. Thailand is one of the top 14 countries with high tuberculosis and multi-drug resistant tuberculosis rates. Immediate detection of drug-resistant tuberculosis is necessary to reduce mortality and morbidity by effectively providing treatment to ameliorate the formation of resistant strains. Limited data exist of mutation profiles in Northeastern Thailand. Here, 65 drug-resistant Mycobacterium tuberculosis isolates were used to detect mutations by polymerase chain reaction (PCR) and DNA sequencing. In the katG gene, mutations were occurred in 47 (79.7%) among 59 isoniazid resistant samples. For rpoB gene, 31 (96.9%) were observed as mutations in 32 rifampicin resistant isolates. Of 47 katG mutation samples, 45 (95.7%) had mutations in katG315 codon and 2 (4.3%) showed novel mutations at katG365 with amino acid substitution of CCG-CGG (Pro-Arg). Moreover, out of 31 rpoB mutation isolates, the codon positions rpoB516, rpoB526, rpoB531 and rpoB533 were 3 (9.7%), 8 (25.8%), 11 (35.5%) and 1 (3.2%), respectively. Seven isolates of double point mutation were found [rpoB516, 526; 1 (3.2%) and rpoB516, 531; 6 (19.4%)]. In addition, 1 (3.2%) sample had triple point mutation at codon positions rpoB516, 526 and 531. Common and novel mutation codons of the rpoB and katG genes were generated. Although DNA sequencing showed high accuracy, conventional PCR could be applied as an initial marker for screening drug-resistant Mycobacterium tuberculosis isolates in limit resources region. Mutations reported here should be considered when developing new molecular diagnostic methods for implementation in Northeastern Thailand.

Detection and genotyping of Helicobacter pylori in saliva versus stool samples from asymptomatic individuals in Northeastern Thailand reveals intra-host tissue-specific H. pylori subtypes.

BACKGROUND: Two-thirds of the world's population is thought to be infected by Helicobacter pylori. Although most people infected with H. pylori are asymptomatic, this pathogen is associated with several gastric pathologies including cancer. The risk factors for colonization are still unclear and the genetic diversity within individual hosts has never been clearly investigated. RESULT: This study determined the prevalence of, and explored risk factors for, H. pylori infection directly from paired saliva (n = 110) and stool (n = 110) samples from asymptomatic persons in Northeast Thailand. Samples were subjected to indirect immunofluorescence assay (IFA), 16S rRNA-based real-time PCR and vacA-based semi-nested PCR. Partial vacA gene sequences of H. pylori were compared between saliva and stool samples. The overall prevalence of H. pylori infection in our asymptomatic study population was 64%. Age, gender, occupation and frequency of brushing teeth were not found to be associated with H. pylori colonization. The vacA gene was successfully sequenced from both saliva and stool samples of 12 individuals. For seven of these individuals, saliva and stool sequences fell into different clusters on a phylogenetic tree, indicating intra-host genetic variation of H. pylori. CONCLUSION: This study reports a high prevalence of H. pylori infection in asymptomatic persons in this region of Thailand and demonstrates that genotypes (vacA gene sequences) of H. pylori may differ between the oral cavity and intestinal tract.

DETECTION OF HELICOBACTER PYLORI AND VIRULENCE-ASSOCIATED GENES IN SALIVA SAMPLES OF ASYMPTOMATIC PERSONS IN NORTHEAST THAILAND.

The aims of the study were to develop nested-PCR (targeting vacA and cagA), SYBR green quantitative PCR (targeting 16S rDNA) tests and compared them with indirect fluorescent-monoclonal antibody (IFA) method for determination of the prevalence of Helicobacter pylori in 118 saliva samples from asymptomatic individuals in Khon Kaen, Thailand. Detection limit of both PCR-based assays was one cell. Prevalence of H. pylori in saliva samples was 55% based on the criterion of positivity of IFA test and one of the PCR-based methods or positivity of both PCR assays. Forty-nine percent of H. pylori detected carried cagA, encoding a cytotoxin associated with severe clinical outcomes. These results imply that the mouth may be an important reservoir for H. pylori, with nearly 50% of the virulent type that could possibly lead to gastroduodenal disease.

PHENOTYPIC AND GENOTYPIC DETECTION OF ENTEROTOXINS, TOXIC SHOCK SYNDROME TOXIN-1 AND OF METHICILLIN RESISTANCE IN STAPHYLOCOCCUS AUREUS ISOLATED FROM RETAIL READY-TO-EAT FOODS IN NORTHEASTERN THAILAND.

Toxigenic Staphylococcus aureus contamination of ready-to-eat (RTE) foods is a leading cause of foodborne illness in Thailand. From 151 RTE food samples randomly collected from food vendors and food shops in Khon Kaen municipality, Thailand and after culture-based identification of S. aureus isolates, pentaplex PCR was used for simultaneous detection of super-antigenic toxin (SE) genes (sea, seb, sec, sed and tst-1) and presence of their toxins by reversed passive latex agglutination assay. S. aureus was identified in 57 isolates, of which 60% and 25% was positive for presence of super-antigenic toxin genes and toxins, respectively; and among the former isolates sea was the most common (46%), as well as its product (SEA) (14%) among the latter group. Methicillin resistance S. aureus mecA was not found in any of the isolates using both PCR and disk diffusion methods. These results showed that pentaplex PCR is a useful tool for detection of SE-encoding genes in S. aureus isolates from RTE food.

DOUBLE-STEP MULTIPLEX REAL TIME PCR WITH MELTING CURVE ANALYSIS FOR DETECTION AND DIFFERENTIATION OF MYCOBACTERIA IN SPUTUM.

Mycobacterium tuberculosis (M. tb) is a causative agent of tuberculosis, a worldwide public health problem. In recent years, the incidence of human mycobacterial infection due to species other than M. tb has increased. However, the lack of specific, rapid, and inexpensive methods for identification of mycobacterial species remains a pressing problem. A diagnostic test was developed for mycobacterial strain differentiation utilizing a double-step multiplex real time PCR together with melting curve analysis for identifying and distinguishing among M. tb, M. bovis BCG, other members of M. tb. complex, M. avium, and non-tuberculosis mycobacteria. The assay was tested using 167 clinical sputum samples in comparison with acid-fast staining and culturing. Using only the first step (step A) the assay achieved sensitivity and specificity of 81% and 95%, respectively. The detection limit was equivalent to 50 genome copies.

Development of single-tube multiplex PCR for classification of Mycobacterium tuberculosis lineages based on large sequence polymorphisms.

Large sequence polymorphisms (LSPs) or regions of differences (RDs) are molecular epidemiological and evolutionary markers used to classify Mycobacterium tuberculosis (MTB) into East Asian (Beijing), Indo-Oceanic (IO), Euro-American (EuA) and East African Indian (EAI) lineages. The most used method is separate PCR and sequencing for each RD. We developed a single-tube multiplex PCR using four primer pairs specific to the four MTB lineages and a primer pair for species-specific RD9 with genomic DNA extracted from isolated colonies. The single-tube multiplex PCR produced lineage-specific amplicon patterns capable of differentiating the four MTB lineages. Sensitivity and specificity of the assay were 100% when differentiating MTB lineages from other species and strains of bacteria. The limit of detection of genomic MTB DNA was 12.5 ng. This single-tube multiplex PCR method offers a simple, rapid and reliable method for classification of MTB lineages based on LSPs.

Cloning, expression and characterization of Mycobacterium tuberculosis sirR.

Identification of new drug targets is important for the improvement of chemotherapy for tuberculosis treatment. Metal-associated gene products are candidates for novel drug development. A Mycobacterium tuberculosis (MTB) sirR-encoded protein has been proposed, but the function of MTB SirR has not yet been elucidated. Bioinformatics analysis revealed that MTB SirR contains iron binding domains with 34%-59% similarity to previously described metal-dependent gene regulators and that the gene lies in Rv2787-sirR operon. RT-PCR revealed that the Rv2787-sirR operon is transcribed a single bicistronic mRNA. Heterologous expression, purification and characterization of recombinant MTB His-tagged SirR demonstrated a 25 kDa protein (by SDS-PAGE and immunoblotting) that exists as a dimer (native PAGE). Based on electrophoretic mobility shift assay, MTB SirR bound a cis element located at -85 bp upstream of its operon. As Rv2787-sirR operon is unique only to MTB (and M. bovis), further studies on its regulation and other functions of the encoded proteins should provide leads towards the discovery of novel anti-TB drugs.

Characterization of a novel two-component system response regulator involved in biofilm formation and a low-iron response of Burkholderia pseudomallei.

Two-component systems (TCSs) regulate an adaptive response to environmental conditions, leading to changes in bacterial cellular processes. In this study, we identified a novel TCS response regulator gene, designated as bfmR (biofilm formation-associated regulator) that regulates biofilm formation by Burkholderia pseudomallei (Bp). An insertion mutant of the Bp bfmR gene resulted in a significant decrease in expression of fimbriae chaperone-usher assembly genes (BPSL2O28 and BPSL22 7), leading to suppression of assembly of fimbriae on the cell surface and reduced biofilm formation. The defective phenotypes of the mutant strain were restored by introducing a complementing plasmid having an intact bfmR gene. Using RT-PCR analyses, we found that bfmR gene expression was upregulated under low-iron growth conditions. In addition, the bfmR mutant strain showed retarded growth in low-iron medium and in phagocytic cells compared to the wild-type strain. These results indicate that bfmR is a novel positive regulator for controlling assembly of fimbriae and biofilm formation, and is upregulated under low-iron conditions.

Comparison of stress adaptation and survival rate between Burkholderia pseudomallei with mutant and wild type bfmR.

Burkholderia pseudomallei (Bp) is highly adaptable to a wide range of environmental changes for survival and pathogenesis. However, the underlying mechanisms of such adaptability are still unclear. Two-component system (TCS) is a common signal transduction used by bacteria in response to environmental changes. A gene designated as bfmR (locus tag of BPSL2024) has been proposed to encode a response regulator, a member of the TCS, and was studied by mutagenesis and comparison of its phenotypic changes compared with those of the wild type. The growth rates of the mutant Bp at temperatures of 37 degrees-39 degrees C and pH 5-8 were significantly lower than the wild type strain (p < 0.05), especially at 39 degrees C (p = 0.01) and pH 7 (p = 0.01). The survival rate of the mice infected with the mutant strain is not significantly different from mice infected with wild type strain. The defective phenotypes were recovered in the complemented strain. These results indicated that bfmR is involved in adaptation of Bp to thermal- and pH-induced stress conditions.

Evaluation of an immunochromatographic test kit for detecting Mycobacterium tuberculosis complex in sputum samples and on solid and in liquid cultures.

A rapid, cheap and effective method for diagnosing tuberculosis (TB) is essential for TB control. We evaluated the performance of an immunochromatographic assay (ICA) kit (SD Bioline TB Ag MPT64 rapid test) designed for detecting Mycobacterium tuberculosis complex (MTC) in liquid and solid cultures to determine its ability to detect and differentiate MTC directly in sputum samples. We attempted to optimize antigen extraction using several sputum solvents under various conditions. Adding the sputum solvent prior to using the ICA kit gave a sensitivity of 71.7% (43/60) for all acid-fast bacillus (AFB) stain positive specimens and a 100% specificity (20/20) among AFB negative specimens. Without sputum solvent, the ICA kit had 0% sensitivity for detecting MTC in sputum. We also evaluated the ICA test kit for its designed purpose of detecting MTC in 80 solid and liquid culture specimens positive for MTC using the niacin accumulation test or polymerase chain reaction (PCR) (16s-23s ITS). The ICA kit gave 100% sensitivity and specificity. We also evaluated the ICA test kit on 3 reference specimens of MTC, 15 nontuberculous mycobacteria (NTM) species, 7 bacterial species and 5 Candida albicans specimens. The tested ICA kit gave 100% specificity. The tested ICA kit was useful for detecting and differentiating MTC in solid and liquid cultures, but not useful for detecting MTC in sputum samples even treated with sputum solvent. The tested ICA kit should be used only for liquid and solid culture specimens. Therefore, the tested kit is inappropriate for use in evaluating sputum samples.

Application of tetraplex PCR for detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle.

A tetraplex PCR method was developed for simultaneous detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle samples in comparison with conventional culture method. Specific primers targeting ompW of V. cholerae, tl of V. parahaemolyticus, hsp60 of V. vulnificus and sodB of V. mimicus were employed in the same PCR. Detection limit of the tetraplex PCR assay was 104 cfu/ml (400 cfu/PCR reaction) for pure cultures of all four species of Vibrio. In Vibrio spiked cockle samples, the limit of detection after 6 hours enrichment in alkaline peptone water was 1 cfu/10 g of cockle tissue for three Vibrio spp, except for V. mimicus that was 102 cfu/10 g of cockle tissue. When the tetraplex PCR and culture methods were applied to 100 cockle samples, V. parahaemolyticus, V. vulnificus, V. cholerae and V. mimicus were detected in 100, 98, 80 and 9% of the samples by tetraplex PCR and in 76, 42, 0 and 0% by the culture method, respectively. This developed tetraplex PCR method should be suitable for simultaneous and rapid detection of Vibrio species in food samples and for food safety assessment.

Detection of Mycobacterium tuberculosis Complex in Sputum Samples Using Droplet Digital PCR Targeting mpt64.

Tuberculosis (TB) is one of the top 10 causes of death worldwide. It is challenging to find methods of diagnosis of active pulmonary TB that are sensitive enough to detect cases for proper treatment before unintentional transmission. Droplet digital PCR (ddPCR) is a highly sensitive method to detect genetic material of pathogens, but it has rarely been used for diagnosis of TB. This study compared the sensitivity of ddPCR with that of GeneXpert and AFB smear microscopy in 180 leftover sputum samples from patients suspected of having TB on the basis of clinical symptoms and radiography. Absolute quantification of copy numbers of MTB-specific genes was possible using ddPCR targeting the mpt64 gene. Among the 180 samples, 41.1% were diagnosed as having TB using ddPCR. The sensitivities of AFB smear microscopy, GeneXpert and ddPCR were 41.9%, 82.4% and 100%, respectively. AFB smear microscopy and GeneXpert both had a specificity of 100%, and the specificity of ddPCR was 95.3%. The accuracy of ddPCR (97.2%) is higher than that of GeneXpert (92.7%). This robust ddPCR system could potentially be used as a method for early diagnosis of TB.

Heteroresistance of Mycobacterium tuberculosis in the Sputum Detected by Droplet Digital PCR.

Heteroresistance in MTB refers to the presence of distinct subpopulations of bacteria with varying levels of antibiotic susceptibility within a population. Multidrug-resistant and rifampicin-resistant TB are serious global health concerns. In this study, we aimed to determine the prevalence of heteroresistance in MTB from sputum samples of new TB cases using Droplet Digital PCR mutation detection assays for katG and rpoB genes, which are commonly associated with resistance to isoniazid and rifampicin, respectively. We found that out of 79 samples, 9 (11.4%) exhibited mutations in katG and rpoB genes. INH mono-resistant TB, RIF mono-resistant TB, and MDR-TB samples constituted 1.3%, 6.3%, and 3.8% of new TB cases, respectively. Heteroresistance in katG, rpoB, and both genes were found in 2.5%, 5%, and 2.5% of total cases, respectively. Our results suggest that these mutations may have arisen spontaneously, as the patients had not yet received anti-TB drugs. ddPCR is a valuable tool for the early detection and management of DR-TB, as it can detect both mutant and wild-type strains in a population, enabling the detection of heteroresistance and MDR-TB. Overall, our findings highlight the importance of early detection and management of DR-TB for effective TB control (in katG, rpoB, and katG/rpoB).

Relationship between Helicobacter Pylori Infection and the Risk of Esophageal Cancer in Thailand.

OBJECTIVE: Esophageal cancer (EC) is a multifactorial disease and a leading cause of mortality. Epidemiological and molecular studies have provided evidence that Helicobacter pylori (H. pylori) infection is an important cause of gastric carcinogenesis and thus, may be related to EC. However, esophagus H. pylori infection in Thai patients with newly diagnosed EC has not been reported. Moreover, the evidence of the association with H. pylori to EC is controversial. This study investigated the possible association between H. pylori infection with a virulence gene and EC in Thailand. METHODS: A case-control study was conducted that involved 105 newly diagnosed EC patients and 108 healthy controls. The prevalence of H. pylori infection detected in formalin-fixed, paraffin-embedded EC tissue in esophageal biopsy specimens from the subjects was measured using real-time PCR. All the data were collected in face to face interviews using a structured questionnaire. Multivariable unconditional logistic regression was used to calculate and analyses the odds ratios (ORs) of the data. RESULTS: A significant association was found between H. pylori infection and EC (p < 0.001, 95% CI:3.11-10.48). H. pylori-positive subjects had a 2.76 times higher risk of developing ESCC. Moreover, the H. pylori-positive subjects who were CagA-positive had slightly higher ORs and statistically significant risk factors. CONCLUSIONS: H. pylori infection was found to be associated with a risk of EC in Thailand, and among the H. pylori-positive subjects who were CagA-positive had a higher risk factor of ESCC but not of EAC.

Sequencing and analysis of three plasmids from Lactobacillus casei TISTR1341 and development of plasmid-derived Escherichia coli-L. casei shuttle vectors.

Pyrosequencing followed by conventional PCR and sequencing was used to determine the complete nucleotide sequence of three plasmids (pRCEID2.9, pRCEID3.2, and pRCEID13.9) from the Lactobacillus casei strain TISTR1341. The plasmid sequences were found to be almost identical, respectively, to those of pLA106, pLA105, and pLA103 from Lactobacillus acidophilus strain TK8912, suggesting that these strains may be related. Sequence analysis and comparison indicated that pRCEID2.9 replicates by a rolling circle (RC) mechanism, while pRCEID3.2 and pRCEID13.9 probably follow a theta-type mode of replication. Replicons of pRCEID2.9 and pRCEID13.9 were used to develop Escherichia coli/L. casei compatible shuttle vectors, which were stably maintained in different genetic backgrounds. Real-time quantitative PCR analysis showed copy numbers of around 4 and 15, respectively, for the pRCEID13.9- and pRCEID2.9-derived shuttle vectors per chromosome equivalent. The functionality of vector pRCEID-LC13.9 was proved by cloning and expressing in L. casei of a green fluorescent protein gene variant from Aequorea victoria under the control of the promoter from a homologous lactate dehydrogenase gene. The new vectors might complement those currently in use for the exploitation of L. casei as a cellular factory and in other biotechnological applications.

PCR-RFLP and antimicrobial susceptibility profiles of Helicobacter pylori isolated from antrum and corpus of dyspeptic patients in Thailand.

The study determined the genetic heterogeneity of Helicobacter pylori isolates from antrum and corpus of the same dyspeptic patients in a Thai population and determined the relationship between the antimicrobial susceptibility (AS) profile (antibiogram) and PCR-restriction fragment length polymorphism (PCR-RFLP) pattern. One hundred and nineteen H. pylori isolates comprising 7 single and 56 paired antrum and corpus isolates obtained by gastric biopsy from 160 dyspeptic patients were analyzed. For PCR-RFLP, the 820 bp amplicon of ureC was digested with Sau3AI and HhaI, which revealed 16 (A-Q) and 19 (a- s) different PCR-RFLP patterns after Sau3AI and HhaI digestion, respectively. Combination of the restriction enzyme digestion patterns resulted in 35 distinct RFLP types. Among the 56 paired isolates, 47 were infected with H. pylori having the same AS and PCR-RFLP profiles, 7 with different AS profiles but the same PCR-RFLP profiles and 2 with different PCR-RFLP profiles but the same AS profiles. No patient was infected with H. pylori different in both PCR-RFLP and AS profiles. The results indicate that the majority of the paired H. pylori isolates displayed identical AS profile and PCR-RFLP patterns suggesting that most patients were infected with a single strain. Some patients could have been infected with single strains that were different in the AS profiles.

Prevalence of cagA EPIYA motifs in Helicobacter pylori among dyspeptic patients in northeast Thailand.

The aims of this study were to determine the prevalence of cagA type in Helicobacter pylori isolated from dyspeptic patients in northeastern Thailand and to determine whether the pattern of cagA EPIYA motifs were associated with clinical outcomes. One hundred and forty-seven H. pylori-infected dyspeptic patients were enrolled, of whom 68 had non-ulcer dyspepsia (NUD), 57 peptic ulcer disease (PUD), 18 gastric cancer (GCA), and 4 other gastroduodenal diseases. PCR and DNA sequence analysis were used to determine the cagA genotype and the pattern of EPIYA motifs. cagA-positive H. pylori were identified in 138 (94%) of H. pylori-infected dyspeptic patients of whom 75 (54%) were of the Western-type, 44 (32%) the East Asian type and 19 (14%) of the other types. The Western type is significantly found in PUD patients (p = 0.0175). The majority of cagA EPIYA was EPIYA-ABC (43%) and EPIYA-ABD (28%). There is no significant correlation between the increase in number of EPIYA-C motifs and clinical outcomes. Thus, the most frequent cagA type found among northeastern Thai dyspeptic patients was the Western cagA type, which is significantly associated with PUD indicating a possible predictive parameter for clinical outcome.

Molecular analysis of Helicobacter pylori virulent-associated genes in hepatobiliary patients.

OBJECTIVES: The Helicobacter pylori virulence-associated genes in hepatobiliary patients, including vacA, iceA, babA2, cagA and cagE, have not been reported. The aim of this study was to investigate these genes and the association of those and the clinical outcomes in hepatobiliary diseases. METHODS: Eighty H. pylori-PCR-positive cases were obtained from hepatobiliary patients, representing both cholangiocarcinoma (CCA) (n= 58) and cholelithiasis (n= 22). The diversity of virulence genes was examined by polymerase chain reaction and DNA sequencing. Phylogenetic analysis of cagA was determined using the maximum parsimony method. RESULTS: The vacAs1a + c/m1, iceA1 and babA2 genes were the most predominant genotypes in both CCA and cholelithiasis patients. The cagA and cagE genes were found significantly more frequently in patients with CCA than those with cholelithiasis (P < 0.05). The cagA positive samples were the Western-type cagA and showed that almost all of the detected sequences in Thai hepatobiliary and Thai gastric cancer patients were classified in the same cluster but separated from the cluster of Japan and other countries. CONCLUSIONS: The cagA and cagE genes may be associated in the pathogenesis of hepatobiliary diseases, especially of CCA. Besides the bacterial variation, other host factors may be involved in the pathogenesis of hepatobiliary cancer.

The relationship between P16INK4A and TP53 promoter methylation and the risk and prognosis in patients with oesophageal cancer in Thailand.

DNA methylation can regulate the expression of tumour suppressor genes P16 and TP53, environmental factors, which are both important factors related to an increased risk and prognosis of oesophageal cancer (EC). However, the association between these two genes methylation status, as well as the effects of gene-environment interactions, EC risk remains unclear. A Hospital-based case-control study data were collected from 105 new EC cases and 108 controls. Promoter methylation status was investigated for P16 and TP53 genes using methylation-specific polymerase (MSP) chain reaction methods with SYBR green. Logistic and Cox regression models were used to analyse the association of P16 and TP53 promotor methylation status with EC risk and prognosis, respectively. Our results suggest P16, TP53 methylation significantly increased the risk of EC (OR = 5.24, 95% CI: 2.57-10.66, P < 0.001; OR = 3.38, 95% CI: 1.17-6.67, P < 0.001, respectively). In addition, P16 and TP53 promoter methylation status and the combined effects between environmental factors and its methylations in tissue were correlated with the EC risk and prognosis of EC patients. As a new biomarker, the methylation of P16 and TP53 can serve as a potential predictive biomarker of EC.