Diarrhea is the leading infectious cause of childhood morbidity and mortality. Among bacterial agents, diarrheagenic Escherichia coli (DEC) is the major causal agent of childhood diarrhea in developing countries, particularly in children under the age of 5 years. Here, we performed a hospital-based prospective study to explore the pathotype distribution, epidemiological characteristics and antibiotic resistance patterns of DEC from < 5-year-old diarrheal children. Between August 2015 and September 2016, 684 stool samples were collected from children (< 5 years old) with acute diarrhea. All samples were cultured and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and biochemical tests. PCR was used for subtyping, and enteropathogenic E. coli (EPEC) isolates were identified simultaneously with serology. Furthermore, antimicrobial sensitivity tests and sequencing of antibiotic resistance-related genes were conducted. DEC strains were identified in 7.9% of the 684 stool samples. Among them, the most commonly detected pathotype was EPEC (50.0% of DEC), of which 77.8% were classified as atypical EPEC (aEPEC). Age and seasonal distribution revealed that DEC tended to infect younger children and to occur in summer/autumn periods. Multidrug-resistant DEC isolates were 66.7%; resistance rates to ampicillin, co-trimoxazole, cefazolin, cefuroxime, cefotaxime, and ciprofloxacin were ≥ 50%. Among 5 carbapenem-resistant DEC, 60.0% were positive for carbapenemase genes (2 blaNDM-1 and 1 blaKPC-2). Among 30 cephalosporin-resistant DEC, 93.3% were positive for extended-spectrum β-lactamase (ESBL) genes, with blaTEM-1 and blaCTX-M-55 being the most common types. However, no gyrA or gyrB genes were detected in 16 quinolone-resistant isolates. Notably, aEPEC, which has not received much attention before, also exhibited high rates of drug resistance (81.0%, 66.7%, and 14.3% for ampicillin, co-trimoxazole , and carbapenem resistance, respectively). EPEC was the most frequent DEC pathotype in acute diarrheal children, with aEPEC emerging as a dominant diarrheal agent in central China. Most DEC strains were multidrug-resistant, making even ciprofloxacin unsuitable for empiric treatment against DEC infection. Among carbapenem-resistant DEC strains, those harboring blaNDM-1 and blaKPC-2 were the main causal agents. blaTEM-1 and blaCTX-M-55 were the major genetic determinants associated with high levels of cephalosporin resistance.

Clostridium difficile infection (CDI) is an emerging healthcare problem in the world. The purpose of this study was to perform a systematic epidemiological research of CDI in Tongji hospital, the central of China. Stool samples from hospitalized adults suspected of CDI were enrolled. The diagnosis of CDI were based on the combination of clinical symptoms and laboratory results. Clinical features of CDI and non-CDI patients were compared by appropriate statistical tests to determine the risk factors of CDI. Multilocus sequence typing (MLST) was employed for molecular epidemiological analysis. Susceptibility testing and relevant antimicrobial agent resistance genes were performed as well. From June 2016 to September 2017, 839 hospitalized adults were enrolled. Among them, 107 (12.8%, 107/839) patients were C. difficile culture positive, and 73 (8.7%, 73/839) were infected with toxigenic C. difficile (TCD), with tcdA + tcdB+ strains accounting for 90.4% (66/73) and tcdA-tcdB+ for 9.6% (7/73). Meanwhile, two TCD strains were binary toxin positive and one of them was finally identified as CD027. Severe symptoms were observed in these two cases. Multivariate analysis indicated antibiotic exposure (p = 0.001, OR = 5.035) and kidney disease (p = 0.015, OR = 8.329) significantly increased the risk of CDI. Phylogenetic tree analysis demonstrated 21 different STs, including one new ST (ST467); and the most dominant type was ST54 (35.6%, 26/73). Multidrug-resistant (MDR) TCD were 53.4% (39/73); resistance to ciprofloxacin, erythromycin, and clindamycin were > 50%. Other antibiotics showed relative efficiency and all strains were susceptible to metronidazole and vancomycin. All moxifloxacin-resistant isolates carried a mutation in GyrA (Thr82 → Ile), with one both having mutation in GyrB (Ser366 → Ala). Knowledge of epidemiological information for CDI is limited in China. Our finding indicated tcdA + tcdB+ C. difficile strains were the dominant for CDI in our hospital. Significant risk factors for CDI in our setting appeared to be antibiotic exposure and kidney disease. Metronidazole and vancomycin were still effective for CDI. Although no outbreak was observed, the first isolation of CD027 in center China implied the potential spread of this hypervirulent clone. Further studies are needed to enhance our understanding of the epidemiology of CDI in China.

Tuberculosis (TB) is a leading global public health problem; however, the mechanisms underlying the immunopathology of TB progression are not well understood. It is currently believed that Mycobacterium tuberculosis (Mtb) infection can modify natural killer (NK) cell phenotypic signatures. Hence, our study was designed to investigate the diversity of circulating NK cells in patients with different TB infection status. NK subsets, as well as their expression of activating and inhibitory receptors between active TB (ATB) and latent TB infection (LTBI) were evaluated. There were significant differences in NK cell phenotypes between ATB, LTBI and healthy controls. Notably, the proportion of KLRG1 in NK cells (P = 0.036), as well as in their subsets CD56^DimCD16+ (P = 0.046) and CD27⁺ (P = 0.027) NK cells, increased significantly in LTBI group than in ATB group; while Mtb specific IFN-γ+CD56^BrightCD16^Dim NK cells expressed higher KLRG1 in ATB than in LTBI (P = 0.027). However, the expression of activating receptor NKG2D in NK subsets showed no significant difference among the study groups. Our results suggest that different TB infection status are coupled with the diversity of NK cell compartments, and the expression of KLRG1 in NK cells may be a specific phenotype that modulates the progression of TB from latent to active.

Human T cells play an important role in immunity against tuberculosis (TB) infection. Activating receptor HLA-DR and inhibitory receptor KLRG1 are critical regulators of T cell function during viral infection and tumorigenesis, but they have been less studied in TB infection. In this study, we explored the relationship between CD3⁺ T cell expression of HLA-DR and KLRG1 receptors and function against TB infection. Flow cytometry was conducted to assess the immunomodulatory effects of HLA-DR and KLRG1 receptors on CD3⁺ T cells in patients with different TB infection status. We found activating receptors HLA-DR, NKG2C, CD57 and NKP46, and inhibitory receptors KLRG1 and KIR on CD3⁺ T cells in different TB infection status showed different distribution patterns; the cytotoxic potential and cytokine secretion capacity of CD3⁺ T cells after Mtb-specific antigen stimulation were significantly enhanced in TB infection groups. Further studies revealed HLA-DR⁺ T and KLRG1⁺ T cells expressed higher activating and inhibitory receptors than the negative population. In addition, the expression of cytotoxic potential and cytokine secretion capacity of HLA-DR⁺ T and KLRG1⁺ T cells was significantly higher than that of HLA-DR^- T and KLRG1^- T cells. Expression of HLA-DR and KLRG1 enhances the cytotoxic potential and cytokine secretion capacity of CD3⁺ T cells in TB patients, suggesting CD3⁺ T cells expressing HLA-DR and KLRG1 are important effector cell phenotypes involved in the host anti-TB infection. HLA-DR and KLRG1 expressed by CD3⁺ T cells may be potential predictive markers of TB disease progression and clinical immune assessment.

Interferon gamma release assays (IGRAs) for tuberculosis (TB) remain limited in their ability to discriminate between active TB (ATB) and latent TB infection (LTBI). The objective of our study was to evaluate the value of additional cytokines/chemokines other than interferon gamma (IFN-γ) as biomarkers to identify different TB infection status. A total of 128 subjects were enrolled to detect the quantification of IL-2, IP-10, MCP-1 and RANTES in the supernatants of QuantiFERON®-TB (QFT-TB). Area under the curve (AUC) was used to evaluate the diagnostic efficiency. Notably, Mycobacterium tuberculosis (Mtb) induced cytokines/chemokines of ATB patients were significantly higher than those of the LTBI, other lung related diseases (ORD) and healthy controls (HC). Moreover, ROC analysis indicated that all cytokine/chemokine parameters detected were more capable of distinguishing ATB from LTBI than IFN-γ, especially IL-2. The diagnostic model including TB specific IL-2 and RANTES improved the performance in distinguishing ATB from LTBI, which was superior to single cytokines/chemokines in QFT-TB supernatants. Our results suggest that the combination of Mtb specific cytokines/chemokines has great prospects in the diagnosis of ATB, and the diagnostic model based on IL-2 and RANTES can be used as an alternative to distinguish ATB from LTBI.