Integrated Bioinformatics Exploration and Preliminary Clinical Verification for the Identification of Crucial Biomarkers in Severe Cases of COVID-19.

Background: Coronavirus disease 2019 (COVID-19) is a respiratory infectious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The objective of this study is to identify reliable and accurate biomarkers for the early stratification of disease severity, a crucial aspect that is currently lacking for the impending phases of the next COVID-19 pandemic. Methods: In this study, we identified important module and hub genes related to clinical severe COVID-19 using differentially expressed genes (DEGs) screening combing weighted gene co-expression network analysis (WGCNA) in dataset GSE213313. We further screened and confirmed these hub genes in another two new independent datasets (GSE172114 and GSE157103). In order to evaluate these key genes' stability and robustness for diagnosing or predicting the progression of illness, we used RT-PCR validation of selected genes in blood samples obtained from hospitalized COVID-19 patients. Results: A total of 968 and 52 DEGs were identified between COVID-19 patients and normal people, critical and non-critical patients, respectively. Then, using WGCNA, 10 modules were constructed. Among them, the blue module positively associated with clinic disease severity of COVID-19. From overlapped section between DEGs and blue module, 12 intersected common differential genes were obtained. Subsequently, these hub genes were validated in another two new independent datasets as well and 9 genes that overlapped showed a highly correlation with disease severity. Finally, the mRNA expression levels of these hub genes were tested in blood samples from COVID-19 patients. In severe cases, there was increased expression of MCEMP1, ANXA3, CD177, and SCN9A. In particular, MCEMP1 increased with disease severity, which suggested an unfavorable development and a frustrating prognosis. Conclusion: Using comprehensive bioinformatical analysis and the validation of clinical samples, we identified four major candidate genes, MCEMP1, ANXA3, CD177, and SCN9A, which are essential for diagnosis or development of COVID-19.

Gut microbiota composition is associated with disease severity and host immune responses in COVID-19.

Background: Human gut microbiota play a crucial role in the immune response of the host to respiratory viral infection. However, evidence regarding the association between the gut microbiome, host immune responses, and disease severity in coronavirus disease 2019 (COVID-19) remains insufficient. Methods: To better comprehend the interactions between the host and gut microbiota in COVID-19, we conducted 16S rRNA sequencing and characterized the gut microbiome compositions in stool samples from 40 COVID-19 patients and 33 non-pneumonia controls. We assessed several hematological parameters to determine the immune status. Results: We found that the gut microbial composition was significantly changed in COVID-19 patients, which was characterized by increased opportunistic pathogens and decreased commensal bacteria. The frequency of prevalent opportunistic pathogens Enterococcus and Lactobacillus increased, especially in severe patients; yet the abundance of butyrate-producing bacteria, Faecalibacterium, Roseburia, and Anaerostipes, decreased significantly, and Faecalibacterium prausnitzii might help discriminate severe patients from moderate patients and non-pneumonia people. Furthermore, we then obtained a correlation map between the clinical characteristics of COVID-19 and severity-related gut microbiota. We observed a notable correlation between the abundance of Enterococcus faecium and abnormal neutrophil or lymphocyte percentage in all COVID-19 patients. Faecalibacterium was positively correlated with lymphocyte counts, while negatively correlated with neutrophil percentage. Conclusion: These results suggested that the gut microbiome could have a potential function in regulating host immune responses and impacting the severity or consequences of diseases.

TREM-1, TREM-2 and their association with disease severity in patients with COVID-19.

BACKGROUND: Delayed diagnosis and inadequate treatment caused by limited biomarkers are associated with the outcomes of COVID-19 patients. It is necessary to identify other promising biomarkers and candidate targets for defining dysregulated inflammatory states. METHODS: The triggering receptors expressed on myeloid cell (TREM)-1 and TREM-2 expression from hospitalized COVID-19 patients were characterized using ELISA and flow cytometry, respectively. Their correlation with disease severity and contrast with the main clinical indicators were evaluated. RESULTS: Increased expression of soluble TREM-1 and TREM-2 in the plasma of COVID-19 patients was found compared to the control group. Moreover, membrane-bound TREM-1 and TREM-2 expression was upregulated on the cell surface of circulating blood T cells from COVID-19 patients. Correlation analysis showed that sTREM-2 levels were negatively correlated with PaO2/FiO2, but positively correlated with C-reactive protein (CRP), procalcitonin (PCT) and interleukin (IL)-6 levels. Receiver operating characteristic curve analysis indicated that the predictive efficacy of sTREM-1 and sTREM-2 was equivalent to CRP and IL-6, and a little better than absolute leukocyte or neutrophil count and PCT in distinguishing disease severity. CONCLUSION: TREM-2 and TREM-1 are critical host immune factors that response to SARS-COV-2 infection and could serve as potential diagnostic biomarkers and therapeutic targets for COVID-19.

The expression of soluble TREM-1 and TREM-2 in plasma and membrane-bound TREM-1 and TREM-2 on the cell surface was upregulated in COVID-19 patients.sTREM-2 level was negatively correlated with PaO₂/FiO₂, but positively correlated with CRP, PCT and IL-6 level, respectively.sTREM-1 and sTREM-2 exhibited potential predictive abilities, and their expression was equivalent to CRP and IL-6, and better than the absolute leukocytes or neutrophil counts and PCT in distinguishing disease severity.

Effect of grazing and climatic factors on biodiversity-ecosystem functioning relationships in grassland ecosystems - a case study of typical steppe in Inner Mongolia, China.

Biodiversity underpins grassland ecological functions and productive capacities. By studying the mechanisms for the maintenance of species diversity in animal communities, we can provide important theoretical guidance for the optimization of grazing management and biodiversity protection. The typical grassland of Xilingol in Inner Mongolia, China, was used as the experimental area, and a grazing intensity experiment was set up. This consisted of four gradient levels that were grazed by sheep, which were available for continuous monitoring, namely control standard sheep unit·day·hectare-1·year-1 (CK, 0 SSU·d·hm-2y-1), light grazing (LG, 170 SSU·d·hm-2·y-1), moderate grazing (MG, 340 SSU·d·hm-2·y-1), and high grazing (HG, 510 SSU·d·hm-2·y-1). Nine consecutive years of multi-indicator monitoring of vegetation was carried out from 2014-2022, using monitoring data coupled with time series and inter-annual climatic (relative moisture index, RMI) fluctuations. This was done to analyze the impacts of disturbances, such as grazing use and climatic fluctuations, on the diversity of species and above-ground productivity of the community, thereby exploring the relationship between diversity and productivity, and provide possible explanations for the emergence of a range of ecological responses. The statistical analysis methods used were One-way Analysis of Variance (ANOVA), general linear regression and mixed-effects models. The main conclusions of this study are as follows: (1) The grassland in the experimental area under CK had the highest diversity and productivity and the ecosystem was better able to buffer the negative impacts of climatic drought. Furthermore, the effect of climate on productivity and diversity weakened as the intensity of grazing increased. (2) LG to MG had a constant diversity. (3) Grazing utilization changed the relationship between community species diversity and aboveground productivity by releasing spatial community resources, altering the structure of plant communities, weakening competitive exclusion, and strengthening complementary effects. However, under all of the conditions there is a brief stage in the time series when diversity is stimulated to increase, and the higher the grazing intensity, the earlier this occurs.

Cycling and activated CD8+ T lymphocytes and their association with disease severity in influenza patients.

BACKGROUND: T cell lymphopenia was a significant characteristic of severe influenza infection and it was associated with the functional changes of T cells. It is necessary to clarify the T cells characteristics of kinetic changes and their correlation with disease severity. METHODS: In a cohort of hospitalized influenza patients with varying degrees of severity, we characterized lymphocyte populations using flow cytometry. RESULTS: The numbers of cycling (Ki67+) T cells at the acute phase of severe influenza were higher, especially in the memory (CD45RO+) T cell subsets. T cells from hospitalized influenza patients also had significantly higher levels of the exhausted marker PD-1. Cycling status of T cells was associated with T cell activation during the acute phase of influenza infection. The recruitment of cycling and activated (CD38+HLA-DR+) CD8+ T cells subset is delayed in severe influenza patients. CONCLUSIONS: The increased numbers of cycling memory (Ki67+CD45RO+) T cells subsets and delayed kinetics of activated (CD38+HLA-DR+) CD8+ T cells, could serve as possible biological markers for disease severity.

High Prevalence and Persistence of Escherichia coli Strains Producing Shiga Toxin Subtype 2k in Goat Herds.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a zoonotic pathogen with the ability to cause severe diseases like hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is the key virulence factor in STEC and can be classified into two types, Stx1 and Stx2, and different subtypes. Stx2k is a newly reported Stx2 subtype in E. coli strains from diarrheal patients, animals, and raw meats exclusively in China so far. To understand the reservoir of Stx2k-producing E. coli (Stx2k-STEC), we investigated Stx2k-STEC strains in goat herds and examined their genetic characteristics using whole-genome sequencing. A total of 448 STEC strains were recovered from 2,896 goat fecal samples, and 37.95% (170/448) were Stx2k-STEC. Stx2k-STEC strains of serotype O93:H28 and sequence type 4038 (ST4038) were the most predominant and were detected over several years. Notably, 55% of Stx2k-STEC strains carried the heat-labile toxin (LT)-encoding gene (elt) defining enterotoxigenic E. coli (ETEC), thereby exhibiting the hybrid STEC/ETEC pathotype. Stx2k-converting prophage genomes clustered into four groups and exhibited high similarity within each group. Strains from patients, raw meat, sheep, and goats were intermixed distributed in the phylogenetic tree, indicating the risk for cross-species spread of Stx2k-STEC and pathogenic potential for humans. Further studies are required to investigate the Stx2k-STEC strains in other reservoirs and to understand the mechanism of persistence in these hosts. IMPORTANCE Strains of the recently reported Stx2k-STEC have been circulating in a variety of sources over time in China. Here, we show a high prevalence of Stx2k-STEC in goat herds. More than half of the strains were of the hybrid STEC/ETEC pathotype. Stx2k-STEC strains of predominant serotypes have been widespread in the goat herds over several years. Stx2k-converting prophages have exhibited a high level of similarity across geographical regions and time and might be maintained and transmitted horizontally. Given that goat-derived Stx2k-STEC strains share similar genetic backbones with patient-derived strains, the high prevalence of Stx2k-STEC in goats suggests that there is a risk of cross-species spread and that these strains may pose pathogenetic potential to humans. Our study thus highlights the need to monitor human Stx2k-STEC infections in this region and, by extension, in other geographic locations.

Geographic distribution of the cagA, vacA, iceA, oipA and dupA genes of Helicobacter pylori strains isolated in China.

BACKGROUND: There are geographic variations in the genotypes of Helicobacter pylori (H. pylori) cagA, vacA, iceA, oipA and dupA. The aim of the study was to investigate the distribution of these genotypes among H. pylori strains from five regions of China and their association with clinical outcomes. MATERIALS AND METHODS: Gastric biopsy specimens were obtained from 348 patients with different gastrointestinal diseases in the five regions of China. The regional distribution was 89 patients from Shandong, 91 from Guangxi, 57 from Hunan, 58 from Qinghai and 53 from Heilongjiang. The presence of cagA, vacA, iceA, oipA and dupA genotypes was determined by polymerase chain reaction (PCR) from H. pylori DNA. RESULTS: A total of 269 H. pylori isolates were obtained, of which 74 isolates were from Shandong, 78 from Guangxi, 46 from Hunan, 33 from Qinghai and 38 from Heilongjiang. The cagA-positive status was predominant in the five regions. The predominant vacA genotypes were s1c (73.4%), m2 (70.6%) and i1 (92.9%). In strains from Shandong, s1a and m1 were dominant. By contrast, s1c was dominant in Guangxi and i1 was dominant in Hunan and Heilongjiang. The prevalence of m2 subtype in Qinghai (78.8%) was significantly higher than that in other regions (P < 0.05). The predominant iceA genotype was iceA1 and the frequency of iceA1 was significantly more prevalent in Hunan than in other regions (P < 0.05). The oipA status "on" gene was more frequent in Shandong (91.9%) and Guangxi (91%) than in Heilongjiang (71.7%) (P < 0.05). Conversely, the dupA-positive status was less than half in Shandong (31.1%) and Guangxi (15.4%), whereas it was 73.9% in Hunan and 81.8% in Qinghai (P < 0.001). There were no significant associations between the cagA, vacA, iceA, oipA genotypes and clinical outcomes. The dupA-positive strains were more common in peptic ulcer disease (PUD) patients than in non-ulcer dyspepsia (NUD) patients in Shandong and Guangxi (P < 0.05), but the association was not observed in other geographic regions. CONCLUSIONS: There was significant geographic diversity of H. pylori genotypes in different regions of China and the presence of dupA gene can be considered as a marker for the development of gastroduodenal diseases. However, the cagA, iceA, vacA and oipA genes cannot be regarded for prediction of the clinical presentation of H. pylori infection in China.

Genetic and Transcriptomic Variations for Amoxicillin Resistance in Helicobacter pylori under Cryopreservation.

Some amoxicillin-resistant strains of H. pylori show a sharp decrease in amoxicillin resistance after freezing. In China, most clinical gastric mucosal specimens are frozen and transported for isolation and drug susceptibility testing for H. pylori, which may lead to an underestimation of the amoxicillin resistance. The objective of this study is to investigated reasons for the decreased amoxicillin resistance after cryopreservation. A high-level amoxicillin-resistant clone (NX24r) was obtained through amoxicillin pressure screening. After cryopreservation at -80 °C for 3 months, the minimum inhibitory concentration (MIC) of NX24r was reduced sharply. Mutations and changes of transcriptome were analyzed after amoxicillin screening and cryopreservation. Mutations in PBP1 (I370T, E428K, T556S) and HefC (M337K, L378F, D976V) were detected in NX24r, which may be the main reason for the induced amoxicillin resistance. No mutations were found in PBP1 or HefC after cryopreservation. However, transcriptome analysis showed that down-regulated genes in the cryopreserved clone were significantly enriched in plasma membrane (GO:0005886), including lepB, secD, gluP, hp0871 and hp1071. These plasma membrane genes are involved in the biosynthesis and transport function of the membrane. The decreased amoxicillin resistance after cryopreservation may be related to the down-regulation of genes involved in membrane structure and transport function.

Diversity of 3' variable region of cagA gene in Helicobacter pylori strains isolated from Chinese population.

BACKGROUND: The cytotoxin-associated gene A (cagA) is one of the most important virulence factors of Helicobacter pylori (H. pylori). There is a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the C-terminal of CagA protein. This repeat region is thought to play an important role in the pathogenesis of gastrointestinal diseases. The aim of this study was to investigate the diversity of cagA 3' variable region and the amino acid polymorphisms in the EPIYA segments of the CagA C-terminal region of H. pylori, and their association with gastroduodenal diseases. METHODS: A total of 515 H. pylori strains from patients in 14 different geographical regions of China were collected. The genomic DNA from each strain was extracted and the cagA 3' variable region was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and analyzed using MEGA 7.0 software. RESULTS: A total of 503 (97.7%) H. pylori strains were cagA-positive and 1,587 EPIYA motifs were identified, including 12 types of EPIYA or EPIYA-like sequences. In addition to the four reported major segments, several rare segments (e.g., B', B″ and D') were defined and 20 different sequence types (e.g., ABD, ABC) were found in our study. A total of 481 (95.6%) strains carried the East Asian type CagA, and the ABD subtypes were most prevalent (82.1%). Only 22 strains carried the Western type CagA, which included AC, ABC, ABCC and ABCCCC subtypes. The CagA-ABD subtype had statistical difference in different geographical regions (P = 0.006). There were seven amino acid polymorphisms in the sequences surrounding the EPIYA motifs, among which amino acids 893 and 894 had a statistical difference with gastric cancer (P = 0.004). CONCLUSIONS: In this study, 503 CagA sequences were studied and analyzed in depth. In Chinese population, most H. pylori strains were of the CagA-ABD subtype and its presence was associated with gastroduodenal diseases. Amino acid polymorphisms at residues 893 and 894 flanking the EPIYA motifs had a statistically significant association with gastric cancer.

Alterations of Fucosyltransferase Genes and Fucosylated Glycans in Gastric Epithelial Cells Infected with Helicobacter pylori.

Helicobacter pylori (H. pylori) adhesion to human gastric epithelial cells is closely linked with fucosylated glycans. Therefore, investigation of fucosylation in the interaction of gastric epithelial cells with H. pylori is critical. In this study we used lectin microarrays to detect the expression of fucosylated glycans in gastric epithelial cells (GES-1) infected with H. pylori strains isolated from patients with different diseases including chronic gastritis, duodenal ulcers, and gastric cancer (each containing two strains) at 4 h. In addition, we investigated the time-course expression of fucosyltransferase (FUT) 1-6 genes in GES-1 cells stimulated with H. pylori strains at 0.5-8 h. At 4 h post-infection, Lotus, AAA, BC2LCN, PA-IIL, CNL and ACG lectins had increased signals in H. pylori-infected GES-1 cells compared to uninfected cells. Higher expression of FUT1 and FUT2 was detected in all H. pylori-infected GES-1 cells within 2 h, regardless of the H. pylori strain. In particular, the expression of FUT2 was higher in H. pylori-infected GES-1 cells with a higher fold change in levels of BC2LCN lectin specific to α1-2 linked fucose (Fuc) at 4 h. The results suggest that the high levels of α1, 2-linked Fuc synthesized by FUT1/2, might play a role in the preliminary stage of H. pylori infection. This provides us with pivotal information to understand the adhesion of H. pylori to human gastric epithelial cells.

Identification of Functional Interactome of Gastric Cancer Cells with Helicobacter pylori Outer Membrane Protein HpaA by HPLC-MS/MS.

HpaA as an outer membrane protein of Helicobacter pylori (H. pylori) plays a significant role in the adhesion to the human stomach, but the functional relation between HpaA and gastric epithelial cells is still not clear. To screen the interaction between HpaA and cellular proteins in gastric epithelial cells, the HpaA protein from H. pylori 26695 fused with a tag (6× His) was expressed and purified successfully, the secondary structure was estimated by the Circular Dichroism (CD) spectrum, and the purified recombinant protein was used to perform the pull-down assays with gastric cancer cell lines (AGS and SGC-7901) lysates, respectively. The pull-down proteins were identified by high-performance liquid chromatography tandem mass spectrometry system (HPLC-MS/MS). A total of 9 and 13 proteins related were analyzed from AGS and SGC-7901 cell lysates, respectively. ANXA2 was considered as putative HpaA functional partner discovered from lysates of both cell lines with high score and coverage. It is hypothesized that HpaA may be involved in the biological process of regulation of transcription and nucleic acid metabolism during the adhesion of H. pylori to human gastric epithelial cells, and HpaA-binding proteins also be used as targets for the development of antiadhesion drugs against H. pylori.

Genetic diversity of the intimin gene (eae) in non-O157 Shiga toxin-producing Escherichia coli strains in China.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. The increasing incidence of non-O157 STEC has posed a great risk to public health. Besides the Shiga toxin (Stx), the adherence factor, intimin, coded by eae gene plays a critical role in STEC pathogenesis. In this study, we investigated the prevalence and polymorphisms of eae gene in non-O157 STEC strains isolated from different sources in China. Among 735 non-O157 STEC strains, eae was present in 70 (9.5%) strains. Eighteen different eae genotypes were identified in 62 eae-positive STEC strains with the nucleotide identities ranging from 86.01% to 99.97%. Among which, seven genotypes were newly identified in this study. The eighteen eae genotypes can be categorized into five eae subtypes, namely ß1, γ1, ε1, ζ3 and θ. Associations between eae subtypes/genotypes and serotypes as well as origins of strains were observed in this study. Strains belonging to serotypes O26:H11, O103:H2, O111:H8 are associated with particular eae subtypes, i.e., ß1, ε1, θ, respectively. Most strains from diarrheal patients (7/9, 77.8%) carried eae-ß1 subtype, while most isolates from cattle (23/26, 88.5%) carried eae-ζ3 subtype. This study demonstrated a genetic diversity of eae gene in non-O157 STEC strains from different sources in China.

Escherichia coli strains producing a novel Shiga toxin 2 subtype circulate in China.

Shiga toxin (Stx) is the key virulence factor in Shiga toxin producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with life-threatening complications. Stx comprises two toxin types, Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli, which are variable in sequences, toxicity and host specificity. Here, we report the identification of a novel Stx2 subtype, designated Stx2k, in E. coli strains widely detected from diarrheal patients, animals, and raw meats in China over time. Stx2k exhibits varied cytotoxicity in vitro among individual strains. The Stx2k converting prophages displayed considerable heterogeneity in terms of insertion site, genetic content and structure. Whole genome analysis revealed that the stx2k-containing strains were genetically heterogeneous with diverse serotypes, sequence types, and virulence gene profiles. The nine stx2k-containing strains formed two major phylogenetic clusters closely with strains belonging to STEC, enterotoxigenic E. coli (ETEC), and STEC/ETEC hybrid. One stx2k-containing strain harbored one plasmid-encoded heat-stable enterotoxin sta gene and two identical copies of chromosome-encoded stb gene, exhibiting STEC/ETEC hybrid pathotype. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of STEC strains. Given the wide distribution of the Stx2k-producing strains in diverse sources and their pathogenic potential, Stx2k should be taken into account in epidemiological surveillance of STEC infections and clinical diagnosis.

High prevalence of non-O157 Shiga toxin-producing Escherichia coli in beef cattle detected by combining four selective agars.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are emerging foodborne pathogens that are public health concern. Cattle have been identified as the major STEC reservoir. In the present study, we investigated the prevalence and characteristics of STEC strains in beef cattle from a commercial farm in Sichuan province, China. RESULTS: Among 120 beef cattle fecal samples, stx genes were positive in 90% of samples, as assessed using TaqMan real-time PCR, and 87 (72.5%) samples were confirmed to yield at least one STEC isolate by culture using four selective agars, MacConkey, CHROMagar™ ECC, modified Rainbow® Agar O157, and CHROMagar™ STEC, from which 31, 32, 91, and 73 STEC strains were recovered, respectively. A total of 126 STEC isolates were selected and further characterized. Seventeen different O:H serotypes were identified, all of which belonged to the non-O157 serotypes. One stx1 subtype (stx1a) and three stx2 subtypes (stx2a, stx2c, and stx2d) were present among these isolates. The intimin encoding gene eae, and other adherence-associated genes (iha, saa, and paa) were present in 37, 125, 74, and 30 STEC isolates, respectively. Twenty-three isolates carried the virulence gene subA, and only one harbored both cnf1 and cnf2 genes. Three plasmid-origin virulence genes (ehxA, espP, and katP) were present in 111, 111, and 7 isolates, respectively. The 126 STEC isolates were divided into 49 pulsed-field gel electrophoresis (PFGE) patterns. CONCLUSIONS: Our study showed that the joint use of the selective MacConkey and modified Rainbow® Agar O157 agars increased the recovery frequency of non-O157 STEC strains in animal feces, which could be applied to other samples and in regular STEC surveillance. Moreover, the results revealed high genetic diversity of non-O157 STEC strains in beef cattle, some of which might have the potential to cause human diseases.

Occurrence of multidrug-resistant and ESBL-producing atypical enteropathogenic Escherichia coli in China.

BACKGROUND: Atypical enteropathogenic Escherichia coli (aEPEC) is regarded as a globally emerging enteropathogen. aEPECs exhibit various level of resistance to a range of antibiotics, which is increasing alarmingly. The present study investigated the antimicrobial resistance of aEPEC isolates recovered from diarrheal patients, healthy carriers, animals, and raw meats. RESULTS: Among 267 aEPEC isolates, 146 (54.7%) were resistant to tetracycline, followed by ampicillin (49.4%), streptomycin (46.1%), and piperacillin (41.2%). Multidrug resistance (MDR) was detected in 128 (47.9%) isolates, and 40 MDR isolates were resistant to ≥ 10 antimicrobial agents. A total of 47 (17.6%) aEPEC isolates were identified as extended-spectrum ß-lactamase (ESBL)-producers. The blaCTX-M-14 and blaCTX-M-15 genes were predominant among ESBL-producing isolates. CONCLUSIONS: This investigation depicted the occurrence of multidrug-resistant and ESBL-producing aEPEC isolates in China. The results suggested that it is necessary to continuously monitor the emergence and spread of MDR aEPEC.

Identification and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype.

Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and seven Stx2 subtypes have been described in E. coli, which differed in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated Stx2h in E. coli strains isolated from wild marmots in the Qinghai-Tibetan plateau, China. Stx2h shares 91.9% nucleic acid sequence identity and 92.9% amino acid identity to the nearest Stx2 subtype. The expression of Stx2h in type strain STEC299 was inducible by mitomycin C, and culture supernatant from STEC299 was cytotoxic to Vero cells. The Stx2h converting prophage was unique in terms of insertion site and genetic composition. Whole genome-based phylo- and patho-genomic analysis revealed STEC299 was closer to other pathotypes of E. coli than STEC, and possesses virulence factors from other pathotypes. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of E. coli strains. As the emergence of new Shiga toxin genotypes and new Stx-producing pathotypes pose a great threat to the public health, Stx2h should be further included in E. coli molecular typing, and in epidemiological surveillance of E. coli infections.

Multidrug-Resistant Escherichia albertii: Co-occurrence of ß-Lactamase and MCR-1 Encoding Genes.

Escherichia albertii is an emerging member of the Enterobacteriaceae causing human and animal enteric infections. Antimicrobial resistance among enteropathogens has been reported to be increasing in the past years. The purpose of this study was to investigate antibiotic resistance and resistance genes in E. albertii isolated from Zigong city, Sichuan province, China. The susceptibility to 21 antimicrobial agents was determined by Kirby-Bauer disk diffusion method. The highest prevalence was tetracycline resistance with a rate of 62.7%, followed by resistance to nalidixic acid and streptomycin with a rate of 56.9 and 51.0%, respectively. All isolates were sensitive or intermediate susceptible to imipenem, meropenem, amoxicillin-clavulanic acid, and levofloxacin. Among 51 E. albertii isolates, 15 were extended-spectrum ß-lactamase-producing as confirmed by the double disk test. The main ß-lactamase gene groups, i.e., blaTEM, blaSHV, and blaCTX-M, were detected in17, 20, and 22 isolates, respectively. Furthermore, four colistin-resistant isolates with minimum inhibitory concentrations of 8 mg/L were identified. The colistin-resistant isolates all harbored mcr-1 and blaCTX-M-55. Genome sequencing showed that E. albertii strain SP140150 carried mcr-1 and blaCTX-M-55 in two different plasmids. This study provided significant information regarding antibiotic resistance profiles and identified the co-occurrence of ß-lactamase and MCR-1 encoding genes in E. albertii isolates.

Genetic diversity of the enterohaemolysin gene (ehxA) in non-O157 Shiga toxin-producing Escherichia coli strains in China.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) is increasingly recognized as an important enteric foodborne pathogen. The hallmark of the disease is the production of Shiga toxins; however, there are other virulence factors that contribute to the pathogenesis of STEC. This study aimed to investigate the prevalence and genetic diversity of the enterohaemolysin gene, ehxA, among non-O157 STEC strains from human, animal, and food sources. The ehxA gene was amplified from 138 (31.8%) of 434 non-O157 STEC strains, among which 36 unique ehxA sequences were identified. Based on ehxA sequence analysis, three phylogenetic ehxA groups (I II, and III) were determined. Correlations between ehxA groups and sources, serotypes, and virulent gene profiles were observed. The ehxA group II strains were mostly diarrhoeal patient-derived and may demonstrate higher pathogenic potential compared with the ehxA group I and group III strains. Five types of replicons (I1-Ig, FIB, K, F, and B/O) were identified in the 138 ehxA-positive strains, and 3.6%, 5.8%, and 52.2% of the strains harboured toxB, katP and espP genes, respectively, implying marked genetic diversity of ehxA containing plasmids in non-O157 STEC strains. Sequence-based ehxA genotyping might be important in modern strain typing and in epidemiological surveillance of non-O157 STEC infections.

Tellurite resistance profiles and performance of different chromogenic agars for detection of non-O157 Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) are globally important food-borne pathogens. The isolation of non-O157 STEC is a significant public health challenge due to the dramatic diversity of their phenotypes and genotypes. In the present study, 476 non-O157 STEC strains representing 95 different O-serogroups were used to evaluate tellurite resistance and the performance of 12 different chromogenic agars. Of 476 strains, only 108 (22.7%) strains showed the minimal inhibitory concentration (MIC) values for potassium tellurite being higher than 4µg/ml, and 96 (20.2%) strains harbored intact ter genes cluster. The presence of ter genes was significantly correlated with tellurite resistance. Six commercial chromogenic agars (TBX, MAC, SMAC, Rainbow® Agar O157, CHROMagar™ ECC, and Fluorocult O157) supported the growth of all strains. However, CT-SMAC, CHROMagar™ O157, and CHROMagar™ STEC agars exhibited 12.2%, 31.1%, and 38.0% of growth inhibition, respectively. Furthermore, 4.6%, 33.2%, and 45.0% of strains were inhibited on RBA-USDA, RBA-NT, and BCM O157 agar media. Variations in tellurite resistance and colony appearance might result in discrepant performance of non-O157 STEC recovery from different chromogenic agars. Using inclusive agars or less selective agar in combination with highly selective agar should be suggested to recover most non-O157 STEC strains, which would increase the probability of recovering STECs from complex background microflora.

High Prevalence of Virulence Genes in Specific Genotypes of Atypical Enteropathogenic Escherichia coli.

Atypical enteropathogenic Escherichia coli (aEPEC) strains are emerging enteropathogens that have been detected worldwide. A collection of 228 aEPEC strains (121 from diarrheal patients, 27 from healthy carriers, 47 from animals and 33 from raw meats) were investigated for serotypes, virulence gene profiles and phylogenetic relationships. Sixty-six O serogroups were identified. Serogroup O51 was the most prevalent, followed by O119, O26 and O76. For the 20 virulence genes detected, statistically significant differences were observed in the overall prevalence of efa1 (lifA), nleB, nleE, set/ent, paa, and ehxA genes among strains from diarrheal patients, healthy carriers, animals and raw meats, respectively. Strains from diarrheal patients had significantly higher levels of efa1 (lifA) (29.8 vs. 0%, P = 0.0002), nleB (41.3 vs. 7.4%, P = 0.0004), nleE (43.8 vs. 7.4%, P = 0.0002) and set/ent (41.3 vs. 7.4%, P = 0.0004) genes than strains obtained from healthy carriers. The paa gene was identified more often in isolates from raw meats (63.6 vs. 14.8%, P < 0.0001), animals (42.6 vs. 14.8%, P < 0.0122), and diarrheal patients (36.4 vs. 14.8%, P < 0.0225) than in strains obtained from healthy carriers. The ehxA gene was detected more frequently in strains from raw meats than in strains from diarrheal patients (27.3 vs. 2.5%, P = 0.0000) and healthy carriers (27.3 vs. 7.4%, P = 0.0474). The phylogenetic marker, yjaA, was more frequently observed in strains among healthy carriers than in diarrheal patient strains. Among the 228 aEPEC strains, 79 sequence types (STs) were identified. The prominent STs, which comprised strains carrying the four OI-122 genes and lpfA, were ST40, ST328, and ST29. Overall, the results indicate that aEPEC strains isolated in China are highly heterogeneous. aEPEC strains that are potentially more pathogenic appear to be related to specific STs or clonal complexes and serotypes. The high prevalence of diarrhea-associated genes in animal or raw meat strains suggests a zoonotic transmission pathway for potentially human pathogenic aEPEC.