Isolation, antimicrobial resistance and virulence characterization of Salmonella spp. from fresh foods in retail markets in Hangzhou, China.

Salmonella can cause severe foodborne diseases. This study investigated the prevalence of Salmonella spp. in fresh foods in Hangzhou market and their harborage of antibiotic resistance and virulence genes, antibiotic susceptibility, and pathogenicity. A total of 500 samples (pork, n = 140; chicken, n = 128; vegetable, n = 232) were collected over a one-year period. Salmonella was found in 4.2% (21) of samples with the detection rate in pork, chicken and vegetables as 4.3% (6), 6.3% (8), and 3% (7), respectively. One Salmonella strain was recovered from each positive sample. The isolates were identified as six serotypes, of which S. Enteritidis (n = 7) and S. Typhimurium (n = 6) were the most predominant serotypes. The majority of isolates showed resistance to tetracycline (85.7%) and/or ciprofloxacin (71.4%). Tetracycline resistance genes showed the highest prevalence (90.5%). The occurrence of resistance genes for ß-lactams (blaTEM-1, 66.7%; and blaSHV, 9.5%) and aminoglycosides (aadA1, 47.6%; Aac(3)-Ia, 19%) was higher than sulfonamides (sul1, 42.9%) and quinolones (parC, 38.1%). The virulence gene fimA was detected in 57.1% of isolates. Gene co-occurrence analysis implied that resistance genes were associated with virulence genes. Furthermore, selected S. Typhimurium isolates (n = 4) carrying different resistance and virulence genes up-regulated the secretions of cytokines IL-6 and IL-8 by Caco-2 cells in different degrees, suggesting that virulence genes may play a role in inflammatory transcription. In in vivo virulence test, microbiological counts in mouse feces and tissues showed that all included S. Typhimurium were able to infect mice, with one strain showing significantly higher virulence than others. In conclusion, this study indicates Salmonella contamination in fresh foods in Hangzhou market poses a risk to public health and it should be closely monitored to prevent and control foodborne diseases.

Characterization and fitness cost analysis of two plasmids carrying different subtypes of blaNDM in aquaculture farming.

In recent years, the blaNDM gene, which mediate resistance to carbapenems, has disseminated all over the world, and has also been detected in animals. Understanding the dissemination and accumulation of antibiotic resistance genes (ARGs) in a human-impacted environment is essential to solve the food safety problems caused by antibiotics. In this study, two strains of carbapenem bacteria carrying blaNDM were screened from 244 strains isolated from two T. sinensis farms in Zhejiang province, China. After their plasmids were isolated and sequenced, their structure and gene environment were analyzed and the mechanism of blaNDM gene transfer was explored. The study measured the fitness cost of plasmids carrying different blaNDM subtypes by four biological characteristics experiments. The results showed that the fitness cost of IncC plasmid carrying blaNDM-1 was higher than that of IncX3 plasmid carrying blaNDM-5. Furthermore, the real-time PCR showed that the decrease of transcription level of fitness-related genes lead to the different fitness cost of plasmids carrying different blaNDM subtypes. Fitness of many blaNDM-harboring plasmids enhanced the further dissemination of this gene and increase the risk of blaNDM gene spreading in aquatic environment, and thus further investigation of carbapenem-resistant bacterias among food animals are in urgent need.

Encapsulation of Salmonella phage SL01 in alginate/carrageenan microcapsules as a delivery system and its application in vitro.

Phages can be used successfully to treat pathogenic bacteria including zoonotic pathogens that colonize the intestines of animals and humans. However, low pH and digestive enzyme activity under harsh gastric conditions affect phage viability, thereby reducing their effectiveness. In this study, alginate (ALG)/κ-carrageenan (CG) microcapsules were developed to encapsulate and release phage under simulated gastrointestinal conditions. The effects of ALG and CG concentrations on the encapsulation and loading efficiency of microcapsules, as well as the release behavior and antibacterial effects of microcapsules in simulating human intestinal pH and temperature, were investigated. Based on various indicators, when the concentration of ALG and CG were 2.0 and 0.3%, respectively, the obtained microcapsules have high encapsulation efficiency, strong protection, and high release efficiency in simulated intestinal fluid. This effect is attributed to the formation of a more tightly packed biopolymer network within the composite microcapsules based on the measurements of their microstructure properties. Bead-encapsulation is a promising, reliable, and cost-effective method for the functional delivery of phage targeting intestinal bacteria.

The diversity in antimicrobial resistance of MDR Enterobacteriaceae among Chinese broiler and laying farms and two mcr-1 positive plasmids revealed their resistance-transmission risk.

This research aimed to investigate the microbial composition and diversity of antimicrobial resistance genes (ARGs) found in Chinese broiler and layer family poultry farms. We focused on the differences in resistance phenotypes and genotypes of multidrug-resistant Enterobacteriaceae (MDRE) isolated from the two farming environments and the existence and transmissibility of colistin resistance gene mcr-1. Metagenomic analysis showed that Firmicutes and Bacteroides were the dominant bacteria in broiler and layer farms. Many aminoglycoside and tetracycline resistance genes were accumulated in these environments, and their absolute abundance was higher in broiler than in layer farms. A total of 526 MDRE were isolated with a similar distribution in both farms. The results of the K-B test showed that the resistance rate to seven antimicrobials including polymyxin B and meropenem in broiler poultry farms was significantly higher than that in layer poultry farms (P ≤ 0.05). PCR screening results revealed that the detection rates of mcr-1, aph(3')Ia, aadA2, bla oxa-1 , bla CTX-M , fosB, qnrD, sul1, tetA, and catA1 in broiler source MDRE were significantly higher than those in layers (P ≤0.05). A chimeric plasmid p20432-mcr which carried the novel integron In1866 was isolated from broiler source MDRE. The high frequency of conjugation (10-1 to 10-3) and a wide range of hosts made p20432-mcr likely to play an essential role in the high detection rate of mcr-1, aph(3')-Ia, and aadA2 in broiler farms. These findings will help optimize disinfection and improve antimicrobial-resistant bacteria surveillance programs in poultry farms, especially broilers.

Perceived Stigma and Self-Efficacy of Patients With Inflammatory Bowel Disease-Related Stoma in China: A Cross-Sectional Study.

BACKGROUND: Patients with inflammatory bowel disease (IBD)-related stoma face physical, psychological, and social adjustment challenges. However, knowledge about stigmatization and self-management, which is important for clinical care and patient education strategies, is lacking. OBJECTIVE: To evaluate the level of stigma and self-management ability of Chinese patients with IBD-related stoma using an online questionnaire. METHODS: Participants were recruited from two general hospitals in mainland China and the internet platforms of the China Crohn's and Colitis Foundation (CCCF). Participants completed a questionnaire, which gathered data on sociodemographic, clinical, and experience in online groups, self-efficacy scale, and social impact scale. The influencing factors of self-efficacy and perceived stigma were assessed by stepwise multivariate regression analyses. RESULTS: One hundred and seventy-six respondents were included. Most of the respondents (78/176, 44.32%) spent between 500 and 999 RMB ($77-153) on ostomy care accessories monthly. Three patients reported using homemade ostomy products. The average score on the self-efficacy scale was 75.79 ± 23.91, which reflected a moderate level of self-efficacy, and 69 (39.2%) respondents had low-level self-efficacy. The average social impact scale score was 62.76 ± 12.69, which reflected a moderate level of perceived stigma. Forty-three (24.43%) patients experienced severe levels of perceived stigma. Stepwise multivariate regression analysis revealed that self-efficacy was associated with educational level (P = 0.007), whereas stigma was associated with nursing privacy (P = 0.021) and acceptance by the closest person (P = 0.005). A total of 91% of respondents who participated in online peer support groups believed the groups were helpful for disease management. CONCLUSIONS: Chinese patients with IBD-related stoma reported a moderate degree of perceived stigma; their level of self-efficacy was low to moderate. High educational level was associated with high self-efficacy. Notably, acceptance of the stoma by the closest person was an influencing factor protecting patients from perceived stigma. Interventions aimed at improving patient education, reducing economic burden, and strengthening social support should be considered to help improve the living conditions of patients with IBD-related stoma.

Patients with IBD have a more cautious attitude towards COVID-19 vaccination.

Background: To understand the awareness of COVID-19 vaccine, the willingness to vaccinate and the influencing factors of willingness to vaccinate in inflammatory bowel disease (IBD) patients. Methods: The online questionnaire was distributed to conduct a survey to analyze and evaluate the willingness, awareness and trust in vaccines of IBD patients. Bivariate analyses and logistic regression models were used to analysis influencing factors. Results: We sent the questionnaire to the WeChat group for patient management and 304 patients responded, out of which 16 respondents had to be excluded and 288 respondents were included for the analysis. Among them, 209 patients vaccinated with COVID-19 vaccine. Among the non-vaccinated 79 patients, the main reasons for their concerns were afraid of vaccination aggravating IBD and fear of adverse effects. Our results showed that IBD patients with long disease duration were more willing to receive COVID-19 vaccination (P<0.05). We also observed that a high perception of benefits and cues to action to receive the vaccine were the two most important constructs affecting a definite intention for COVID-19 vaccination (P<0.05). Conclusions: Patients with IBD have a more cautious attitude towards COVID-19 vaccination, which may lead to a higher rate of vaccine hesitancy. Further efforts should be made to protect patients with IBD from COVID-19 infections and achieve adequate vaccination coverage.

Patients' views of shared decision making in inflammatory bowel disease: a survey in China.

BACKGROUND: Recently, decision-making process has become increasingly complex. But there is limited information on Chinese patients' views of shared decision making (SDM) in inflammatory bowel disease (IBD). This questionnaire investigation aimed to understand Chinese patients' perspectives and expectations of SDM in IBD and analyze the possible factors that influence their views. METHODS: An online survey was conducted from July 19th to 24th, 2020. A total of 1118 patients completed the survey. RESULTS: One-third of patients were dissatisfied with the current decision-making model, and the satisfaction of inpatients was lower than that of outpatients. 84% of patients preferred to participate in SDM, who were young and had a high education level, high income, commercial insurance, strong learning ability and knowledge of SDM. Most of those who did not want to participate (72%) were worried about the cost. The kind of medicine (948, 84.8%), surgical indications (505, 45.2%) and operation methods (482, 43.1%) were the topics that patients thought most require SDM. Side effects of medicine (837, 74.9%), costs of therapy (675, 60.4%), and surgical risks (563, 50.4%) were considered to be the most influential factors for SDM. 52.7% of all patients hoped experts in different disciplines would participate in SDM. The most desirable amount of time for discussion was 30 to 60 min (562/1118, 50.3%), that were associated with the cost of SDM. CONCLUSION: We can meet the needs of patients by reducing costs and strengthening online patient education and exploring a model suitable for Chinese IBD patients.

The Clustered Regularly Interspaced Short Palindromic Repeats-Associated System and Its Relationship With Mobile Genetic Elements in Klebsiella.

Microorganisms have developed many strategies in the process of long-term defense against external attacks, one of which is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. In this study, the whole genome of 300 strains of Klebsiella was collected, the CRISPR-Cas system in the strains was statistically analyzed, and the types and structures of CRISPR system in Klebsiella were explored, as well as the correlation between CRISPR and mobile genetic elements (MGEs). Through principal component analysis (PCA), we found that Cas gene, plasmids, integron, IS1, IS609, and enzymes of DNA metabolism were closely related to CRISPR-Cas. Compared the structural characteristics of plasmids, the DinG family helicases, Cas6, Csf2, and IS5 were observed near the CRISPR loci in plasmid, which is also confirmed by the results of PCA that they may be important factors affecting the plasmid with CRISPR.

Internet and WeChat used by patients with Crohn's disease in China: a multi-center questionnaire survey.

BACKGROUND: Currently, WeChat is widely used in disease education for patients with Crohn's disease (CD) in China. It is beneficial for the patients to actively engage in their disease management. METHODS: In this study, we examined the source and expectations of disease information for Chinese CD patients, analysing the content of popular WeChat public accounts and their potential association with medication adherence. RESULTS: Between November 24th, 2017 and April 10th, 2018, online questionnaires were sent to CD patients from eight different large urban hospitals in China. In all, 436 patients with CD were surveyed, and 342 patients responded. Patients most frequently visited Baidu (65%), WeChat (61%) and medical websites such as Haodaifu (35%) when searching for IBD-related information. Among ten WeChat IBD public accounts, the China Crohn's and Colitis Foundation (CCCF) (73%), "IBD Academic Officer" (21%) and "IBD in love" (21%) were the most popular. CD patients were most interested in information from the internet about diet and day-to-day health-related living with IBD (83%), an introduction to the disease (80%), and medication advances and side effects (80%). The correlation between the information provided by the top five WeChat public accounts and patients' expectations was low. Additionally, most patients (64%) had greater confidence in overcoming the disease after learning about CD through their internet searches. Medical adherence was also related to internet access and income (p < 0.05). CONCLUSIONS: WeChat has become a major source of information for IBD education in China, but the content of WeChat didn't fully meet patients' expectations. Therefore, future initiatives should aim to provide high-quality information that based on patients' demands.

Efficacy predictors of a 2-month exclusive enteral nutrition for inducing remission of active Crohn's disease.

In order to identify the factors that could predict the efficacy of exclusive enteral nutrition (EEN) in inducing remission of active CD. Baseline clinical and laboratory covariates were correlated with the outcome (clinical remission) of EEN in active CD (CDAI ≥150) by both univariable and multivariable analyses. A total of 67 from a consecutive of 136 active CD patients were enrolled. L4b (from treitz ligament to distal 1/3 ileum) involvement was negatively and high serum albumin (≥35 g/L) was positively associated with clinical remission (OR: 0.14, OR: 14.71). In conclusion, L4b sparing and high serum albumin might predict a favorable outcome of EEN in active CD (ClinicalTrials.gov ID: NCT 02942511).

Expression of IRAK­3 is associated with colitis­associated tumorigenesis in mice.

Interleukin­1 receptor­associated kinase­3 (IRAK­3) is a negative regulator in Toll­like receptor (TLR) pathways. The present study investigated the importance of IRAK­3 in a mouse model of chemically­induced colitis­associated tumorigenesis. The colitis­associated tumorigenesis was induced in ICR mice by the administration of 1,2­dimethyl hydrazine (DMH) and dextran sodium sulfate (DSS), termed the DMH + DSS group. In the DSS group, mice were administered with DSS; in the DMH group, mice were injected with DMH; in the control group, mice were injected with physiological saline. The clinical signs were examined for 20 weeks; tissue samples were analyzed at week 4, 9, 13 and 20. At week 20, the levels of IRAK­3 were analyzed using immunohistochemistry, western blot analysis, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis, and methylation­specific PCR. At week 20, the DMH + DSS group mice exhibited a decrease in total body weight and had developed canalicular adenoma or adenocarcinoma. The mice in the DSS group and DMH group presented with significant colitis at week 20. The mice with colitis­associated tumorigenesis were found to have decreased levels of IRAK­3, compared with the mice in the other groups, as evidenced by the results of the immunohistochemistry (P=0.002), RT­qPCR analysis (P<0.001) and western blot analysis (P<0.001). IRAK­3 methylation was observed in all experimental groups. Taken together, DMH + DSS induction in colitis led to increased inflammation and risk of tumorigenesis. IRAK­3 methylation may be a predictive factor in the transition from colitis to cancer.

Up-regulated NRIP2 in colorectal cancer initiating cells modulates the Wnt pathway by targeting RORß.

BACKGROUND: Colorectal cancer remains one of the most common malignant tumors worldwide. Colorectal cancer initiating cells (CCICs) are a small subpopulation responsible for malignant behaviors of colorectal cancer. Aberrant activation of the Wnt pathways regulates the self-renewal of CCIC. However, the underlying mechanism(s) remain poorly understood. METHODS: Via retroviral library screening, we identified Nuclear Receptor-Interacting Protein 2 (NRIP2) as a novel interactor of the Wnt pathway from enriched colorectal cancer colosphere cells. The expression levels of NRIP2 and retinoic acid-related orphan receptor ß (RORß) were further examined by FISH, qRT-PCR, IHC and Western blot. NRIP2 overexpressed and knockdown colorectal cancer cells were produced to study the role of NRIP2 in Wnt pathway. We also verified the binding between NRIP2 and RORß and investigated the effect of RORß on CCICs both in vitro and in vivo. Genechip-scanning speculated downstream target HBP1. Western blot, ChIP and luciferase reporter were carried to investigate the interaction between NRIP2, RORß, and HBP1. RESULTS: NRIP2 was significantly up-regulated in CCICs from both cell lines and primary colorectal cancer tissues. Reinforced expression of NRIP2 increased Wnt activity, while silencing of NRIP2 attenuated Wnt activity. The transcription factor RORß was a key target through which NRIP2 regulated Wnt pathway activity. RORß was a transcriptional enhancer of inhibitor HBP1 of the Wnt pathway. NRIP2 prevented RORß to bind with downstream HBP1 promoter regions and reduced the transcription of HBP1. This, in turn, attenuated the HBP1-dependent inhibition of TCF4-mediated transcription. CONCLUSIONS: NRIP2 is a novel interactor of the Wnt pathway in colorectal cancer initiating cells. interactions between NRIP2, RORß, and HBP1 mediate a new mechanism for CCIC self-renewal via the Wnt activity.

Promyelocytic leukemia protein enhances apoptosis of gastric cancer cells through Yes-associated protein.

It has been shown that Yes-associated protein (YAP) acts as a transcriptional co-activator to regulate p73-dependent apoptosis in response to DNA damage in some cell types, and promyelocytic leukemia (PML) protein is involved in the regulation loop through stabilization of YAP through sumoylation. Although YAP has been shown to be significantly upregulated in gastric cancer, whether the YAP/PML/p73 regulation loop also functions in gastric cancer is unknown. Here, we show significantly higher levels of YAP and significantly lower levels of PML in the gastric cancer specimen. Overexpression of YAP in gastric cancer cells significantly increased cell growth, but did not affect apoptosis. However, overexpression of PML in gastric cancer cells significantly increased cell apoptosis, resulting in decreases in cell growth, which seemed to require the presence of YAP. The effect of PML on apoptosis appeared to be conducted through p73-mediated modulation of apoptosis-associated genes, Bcl-2, Bak, and caspase9. Thus, our study suggests the presence of a YAP/PML/p73 regulatory loop in gastric cancer, and highlights PML as a promising tumor suppressor in gastric cancer through YAP-coordinated cancer cell apoptosis.

Relationship among symptoms, mucosal injury, and acid exposure in gastroesophageal reflux disease.

BACKGROUND: Symptoms, endoscopy, and pH monitoring form the basis of diagnosis of gastroesophageal reflux disease (GERD). Their relationship was meaningful for primary care physicians, but still unclear. Our research aimed to compare questionnaire, endoscopy, and pH monitoring and to analyze their correlations. METHODS: Three hundred patients who underwent the Reflux Disease Questionnaire (RDQ), endoscopy, and esophageal 24-hour pH monitoring from March 2007 to December 2010 in Peking University People's Hospital were enrolled. We analyzed the characteristics of different investigations and their relationships. RESULTS: Male (OR for mild reflux esophagitis (RE) = 2.433, severe RE = 8.386), body mass index (BMI) (OR for mild RE = 1.222, severe RE = 1.297), and hernia (OR for mild RE = 6.059, severe RE = 17.547), were found to be the risk factors for RE; age (OR = 1.074) was correlated with severe RE. The consistency of questionnaire, endoscopy, and pH monitoring was poor: RDQ did not agree well with pH monitoring (κ = 0.061), nor with endoscopy (κ = 0.044); pH monitoring did not agree well with endoscopy (κ = 0.316). However, the severity of mucosa injury in RE was associated with pathological acid exposure (PAE): reflux episodes of >5 minutes (P = 0.035), the percentage time pH <4 (P = 0.017), and the DeMeester score (P = 0.016) increased significantly in patients with severe RE. Chest pain had poor relationship with RE or PAE. CONCLUSIONS: Male, age, BMI, and hernia were probably risk factors for esophagitis. RDQ, endoscopy, and pH monitoring have their own focus and reinforce each other in diagnosis. Of the GERD symptoms, chest pain had negative correlation with RE or PAE.

[Influences of diagnostic modes on an early diagnosis of colorectal cancer].

OBJECTIVE: To explore the influences of diagnostic modes of colorectal cancer (CRC) on an early diagnosis of CRC. METHODS: A total of 405 cases were randomly collected from 1798 CRC patients registered at our hospital from January 2000 to December 2010. A retrospective chart review was undertaken for all identified cases. Besides the demographics and tumor features, TNM stage was obtained from medical records and pathological forms. Other collected data were as follows: (1) Type of clinical examinations leading to diagnosis. (2) Diagnostic duration: including patient duration (period from initial symptoms to consulting a doctor and hospitalization duration (period from patient first seeing a doctor to a confirmed CRC case). (3) Diagnostic delay and its rate: diagnostic delay was identified as the diagnostic duration of over 30 days. We compared the different delay periods of 31 - 60, 61 - 90, 91 - 150 and > 150 days and its corresponding tumor stages at diagnosis. (4) Misdiagnosis and its rate: that CRC subjects were diagnosed and treated as other diseases for at least 30 days was identified as misdiagnosis. The t and Mann-Whitney U tests were performed for the quantitative data and χ(2) test for the qualitative data. RESULTS: Among the study subjects, 67.2% (270/402) CRC cases were examined by colonoscopy, 17.4 % (70/402) cases by CT scan or B ultrasound. The median diagnostic duration of CRC was 90 days (25% percentile: 40 days, 75% percentile: 210 days). The median patient duration was 30 days and median hospital duration 10 days. The overall misdiagnostic rate of CRC was 27.9% (112/401), higher (39.7%, 48/121) in right sited CRC than in left sited CRC (22.9%, 63/275) (χ(2) = 11.7, P = 0.00). 77.7% (313/403) cases had > 30 days diagnostic delay, 50.8% (156/307) delay attributable to the patients, while 29.0% (89/307) attributable to hospitals and 20.2% (62/307) attributable to both. The diagnostic duration of early-stage CRC and advanced-stage CRC appeared to be in the same length. On the other hand, no difference of TNM stage at the initial diagnosis was found in groups with different diagnostic durations of 31 - 60, 61 - 90, 91 - 150 and > 150 days (all P > 0.05). CONCLUSION: Colonoscopy is the most effective in the diagnosis of CRC. The median diagnostic time of CRC is 90 days. The phenomenon of its diagnostic delay often occurs. However, the delay is not a major cause for its advanced stage and a poor outcome. Its inherent biological characteristics may be more important.