Characterization of the molecular role that ST3GAL1 plays in porcine susceptibility to E. coli F18 infection.

Escherichia coli F18 (E. coli F18) is the main cause of bacterial diarrhea in piglets. Previous transcriptome reported that ST3GAL1 was associated to E. coli F18 infection. However, its role in mediating the resistance to E. coli F18 remains elusive. Here, we revealed that the downregulation of ST3GAL1 expression contributed to the enhancement of E. coli F18 resistance in IPEC-J2 cells. Bisulfite sequencing identified 26 methylated CpG sites in the ST3GAL1 core promoter. Among these, the ST3GAL1 mRNA levels significantly correlated with methylation levels of the mC-8 site in the specificity protein 1 (SP1) transcription factor (P < 0.01). Interestingly, ST3GAL1 expression may enhances the immune response by activating TLRs signaling, meanwhile decreases the production of the E. coli F18 receptor by inhibiting glycosphingolipid biosynthesis signaling, thereby leading to enhance the resistance to E. coli F18 infection. Besides, low ST3GAL1 expression may increase E. coli resistance by reducing sialylation. Together, these results support the status of ST3GAL1 as a viable target for efforts to modulate E. coli F18 susceptibility, offering a theoretical foundation for the use of this gene as a key biomarker for molecular breeding to improve porcine disease resistance.

Transcriptional markers classifying Escherichia coli and Staphylococcus aureus induced sepsis in adults: A data-driven approach.

Sepsis is a life-threatening condition mainly caused by gram-negative and gram-positive bacteria. Understanding the type of causative agent in the early stages is essential for precise antibiotic therapy. This study sought to identify a host gene set capable of distinguishing between sepsis induced by gram-negative bacteria; Escherichia coli and gram-positive bacteria; Staphylococcus aureus in community-onset adult patients. In the present study, microarray expression information was used to apply the Least Absolute Shrinkage and Selection Operator (Lasso) technique to select the predictive gene set for classifying sepsis induced by E. coli or S. aureus pathogens. We identified 25 predictive genes, including LILRA5 and TNFAIP6, which had previously been associated with sepsis in other research. Using these genes, we trained a logistic regression classifier to distinguish whether a sample contains an E. coli or S. aureus infection or belongs to a healthy control group, and subsequently assessed its performance. The classifier achieved an Area Under the Curve (AUC) of 0.96 for E. coli and 0.98 for S. aureus-induced sepsis, and perfect discrimination (AUC of 1) for healthy controls from the other conditions in a 10-fold cross-validation. The genes demonstrated an AUC of 0.75 in distinguishing between sepsis patients with E. coli and S. aureus pathogens. These findings were further confirmed in two distinct independent validation datasets which gave high prediction AUC ranging from 0.72-0.87 and 0.62 in distinguishing three groups of participants and two groups of patients respectively. These genes were significantly enriched in the immune system, cytokine signaling in immune system, innate immune system, and interferon signaling. Transcriptional patterns in blood can differentiate patients with E. coli-induced sepsis from those with S. aureus-induced sepsis. These diagnostic markers, upon validation in larger trials, may serve as a foundation for a reliable differential diagnostics assay.

Multi-omics strategy reveals potential role of antimicrobial resistance and virulence factor genes responsible for Simmental diarrheic calves caused by Escherichia coli.

Escherichia coli (E. coli) is reported to be an important pathogen associated with calf diarrhea. Antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) pose a considerable threat to both animal and human health. However, little is known about the characterization of ARGs and VFGs presented in the gut microbiota of diarrheic calves caused by E. coli. In this study, we used multi-omics strategy to analyze the ARG and VFG profiles of Simmental calves with diarrhea caused by E. coli K99. We found that gut bacterial composition and their microbiome metabolic functions varied greatly in diarrheic calves compared to healthy calves. In total, 175 ARGs were identified, and diarrheal calves showed a significantly higher diversity and abundance of ARGs than healthy calves. Simmental calves with diarrhea showed higher association of VFGs with pili function, curli assembly, and ferrienterobactin transport of E. coli. Co-occurrence patterns based on Pearson correlation analysis revealed that E. coli had a highly significant (P < 0.0001) correlation coefficient (>0.8) with 16 ARGs and 7 VFGs. Metabolomics analysis showed that differentially expressed metabolites in Simmental calves with diarrhea displayed a high correlation with the aforementioned ARGs and VFGs. Phylotype analysis of E. coli genomes showed that the predominant phylogroup B1 in diarrheic Simmental calves was associated with 10 ARGs and 3 VFGs. These findings provide an overview of the diversity and abundance of the gut microbiota in diarrheic calves caused by E. coli and pave the way for further studies on the mechanisms of antibiotic resistance and virulence in the calves affected with diarrhea.IMPORTANCESimmental is a well-recognized beef cattle breed worldwide. They also suffer significant economic losses due to diarrhea. In this study, fecal metagenomic analysis was applied to characterize the antibiotic resistance gene (ARG) and virulence factor gene (VFG) profiles of diarrheic Simmental calves. We identified key ARGs and VFGs correlated with Escherichia coli isolated from Simmental calves. Additionally, metabolomics analysis showed that differentially expressed metabolites in Simmental calves with diarrhea displayed a high correlation with the aforementioned ARGs and VFGs. Our findings provide an insight into the diversity and abundance of the gut microbiota in diarrheic calves caused by Escherichia coli and pave the way for further studies on the mechanisms of antibiotic resistance and virulence in the diarrheal calves from cattle hosts.

CXCR2, as a key regulatory gene of HDP-PG-1, maintains intestinal mucosal homeostasis.

The intestine defends against pathogenic microbial invasion via the secretion of host defense peptides (HDPs). Nutritional immunomodulation can stimulate the expression of endogenous HDPs and enhance the body's immune defense, representing a novel non-antibiotic strategy for disease prevention. The project aims to explore the regulatory mechanism of protegrin-1 (PG-1) expression using sodium phenylbutyrate (PBA) by omics sequencing technology and further investigate the role of key regulatory genes on intestinal health. The results showed that PBA promoted PG-1 expression in intestinal epithelial cells based on cell density through epidermal growth factor receptor (EGFR) and G protein-coupled receptor (GPR43). Transcriptome sequencing and microRNA sequencing revealed that C-X-C motif chemokine receptor 2 (CXCR2) exhibited interactions with PG-1. Pre-treatment cells with a CXCR2 inhibitor (SB225002) effectively suppressed the induction of PG-1 by PBA. Furthermore, SB225002 significantly suppressed the gene expression of HDPs in the jejunum of mice without influencing on the morphology, number of goblet cells, and proliferation of the intestine. CXCR2 inhibition significantly reduced the expression of HDPs during E. coli infection, and resulted in the edema of jejunal epithelial cells. The 16S rDNA analysis of cecal contents showed that the E. coli and SB225002 treatments changed gut microbiota diversity and composition at different taxonomic levels. Correlation analysis suggested a potential regulatory relationship between gut microbiota and HDPs. To that end, a gene involved in the HDP expression, CXCR2, has been identified in the study, which contributes to improving intestinal immune function. PBA may be used as a functional additive to regulate intestinal mucosal function, thereby enhancing the health of the intestinal and host.

Extended-spectrum beta-lactamases in clinical isolates of Escherichia coli and Klebsiella pneumoniae recovered from patients at the Tamale Teaching Hospital, Ghana.

INTRODUCTION: Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae are pathogens of significant public health interest for which new antibiotics are urgently needed. AIM: To determine the prevalence of ESBLs in E. coli and K. pneumoniae isolates from patients attending the Tamale Teaching Hospital (TTH) in Ghana. METHODOLOGY: The study was a cross-sectional study involving convenience sampling of E. coli and K. pneumoniae isolates from consenting patients' clinical specimens, between April and June 2015. Antimicrobial susceptibility test was performed, and ESBL-producer phenotypes were further screened for BlaTEM, BlaSHV, and BlaCTX-M genes. Patients' clinical data were additionally collected using a structured questionnaire. RESULTS: Of the 150 non-duplicate E. coli and K. pneumoniae isolates identified, 140 were confirmed as E. coli (84%, n = 117) and K. pneumoniae (16%, n = 23). Of these, sixty-two (44%) [E. coli (84%; n = 52); K. pneumoniae (16%; n = 10)] phenotypically expressed ESBLs. The proportion of ESBL-producing isolates was higher in adults (15-65 years) than in neonates (< 28 days) (p = 0.14). Most of the isolates showed a high percentage resistance to ampicillin (96%) and tetracycline (89%), but a relatively lower resistance to amikacin (36%). No isolate was resistant to meropenem. More ESBL producers were multidrug resistant compared to non-ESBL-producers [23% (14/62) versus 18% (14/78); p = 0.573]. Overall, 74% (n = 46) of the ESBL genotypes expressed BlaCTX-M-1 genes, followed by 63% (n = 39) BlaTEM, and 16% (n = 10) BlaSHV. The study showed a high prevalence of ESBL-positive E. coli and K. pneumoniae, mostly CTX-M-1 producers at TTH. CONCLUSION: Routine laboratory ESBL screening is warranted to inform patient management.

Regional modulation of toll-like receptor signaling pathway genes in acute epididymitis in mice.

BACKGROUND: Region-specific immune environments in the epididymis influence the immune responses to uropathogenic Escherichia coli (UPEC) infection, a relevant cause of epididymitis in men. Toll-like receptors (TLRs) are essential to orchestrate immune responses against bacterial infections. The epididymis displays region-specific inflammatory responses to bacterial-derived TLR agonists, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), suggesting that TLR-associated signaling pathways could influence the magnitude of inflammatory responses in epididymitis. OBJECTIVES: To investigate the expression and regulation of key genes associated with TLR4 and TLR2/TLR6 signaling pathways during epididymitis induced by UPEC, LPS, and LTA in mice. MATERIAL AND METHODS: Epididymitis was induced in mice using UPEC, ultrapure LPS, or LTA, injected into the interstitial space of the initial segment or the lumen of the vas deferens close to the cauda epididymidis. Samples were harvested after 1, 5, and 10 days for UPEC-treated animals and 6 and 24 h for LPS-/LTA-treated animals. Ex vivo epididymitis was induced by incubating epididymal regions from naive mice with LPS or LTA. RT-qPCR and Western blot assays were conducted. RESULTS: UPEC infection up-regulated Tlr2, Tlr4, and Tlr6 transcripts and their associated signaling molecules Cd14, Ticam1, and Traf6 in the cauda epididymidis but not in the initial segment. In these epididymal regions, LPS and LTA differentially modulated Tlr2, Tlr4, Tlr6, Cd14, Myd88, Ticam1, Traf3, and Traf6 expression levels. NFKB and AP1 activation was required for LPS- and LTA-induced up-regulation of TLR-associated signaling transcripts in the cauda epididymidis and initial segment, respectively. CONCLUSION: The dynamic modulation of TLR4 and TLR2/TLR6 signaling pathways gene expression during epididymitis indicates bacterial-derived antigens elicit an increased tissue sensitivity to combat microbial infection in a spatial manner in the epididymis. Differential activation of TLR-associated signaling pathways may contribute to fine-tuning inflammatory responses along the epididymis.

Phylogenetic analysis, biofilm formation, antimicrobial resistance and relationship between these characteristics in Uropathogenic Escherichia coli.

BACKGROUND: In the present study, we examine the prevalence of phylogenetic groups, O-serogroups, adhesin genes, antimicrobial resistance, the level of gene expression associated with biofilm formation, and the presence of extended-spectrum beta-lactamase (ESBL) in UPEC strains isolated from both pediatric and adult patients. METHODS: In this cross-sectional study, 156 UPEC isolates were collected from UTI patients. ESBL-producing isolates were detected using the double-disc synergy (DDS) method, and biofilm formation was assessed through a microplate assay. The presence of O-serogroups, adhesion factors and resistance genes, including ESBLs and PMQR genes, was detected by PCR, and isolates were categorized into phylogenetic groups using multiplex PCR. Additionally, the quantitative real-time PCR method was also used to determine the expression level of genes related to biofilm. RESULTS: During the study period, 50.6% (79/156) of the samples were obtained from children, and 49.4% (77/156) were from adults. The highest rate of resistance was to NA (91.7%), while FM (10.9%) had the lowest rate of antibiotic resistance. In addition, 67.9% (106/156) of UPEC isolates were ESBL producers. Most of UPEC isolates belonged to phylogenetic group B2 (37.1%). This study revealed that blaCTX-M and qnrS are widely distributed among UPEC isolates. The mean expression levels of fimA genes were significantly higher in non-biofilm producers than in biofilm producers (p < 0.01). CONCLUSIONS: The high antibiotic resistance rates in this study highlight the significance of local resistance monitoring and investigating underlying mechanisms. Our findings indicate the dominance of phylogroup B2 and group D as the prevailing phylogenetic groups. Consequently, it is imperative to investigate the epidemiological aspects and characterize UPEC isolates across diverse regions and time frames.

Iron regulatory proteins 1 and 2 have opposing roles in regulating inflammation in bacterial orchitis.

Acute bacterial orchitis (AO) is a prevalent cause of intrascrotal inflammation, often resulting in sub- or infertility. A frequent cause eliciting AO is uropathogenic Escherichia coli (UPEC), a gram negative pathovar, characterized by the expression of various iron acquisition systems to survive in a low-iron environment. On the host side, iron is tightly regulated by iron regulatory proteins 1 and 2 (IRP1 and -2) and these factors are reported to play a role in testicular and immune cell function; however, their precise role remains unclear. Here, we showed in a mouse model of UPEC-induced orchitis that the absence of IRP1 results in less testicular damage and a reduced immune response. Compared with infected wild-type (WT) mice, testes of UPEC-infected Irp1-/- mice showed impaired ERK signaling. Conversely, IRP2 deletion led to a stronger inflammatory response. Notably, differences in immune cell infiltrations were observed among the different genotypes. In contrast with WT and Irp2-/- mice, no increase in monocytes and neutrophils was detected in testes of Irp1-/- mice upon UPEC infection. Interestingly, in Irp1-/- UPEC-infected testes, we observed an increase in a subpopulation of macrophages (F4/80+CD206+) associated with antiinflammatory and wound-healing activities compared with WT. These findings suggest that IRP1 deletion may protect against UPEC-induced inflammation by modulating ERK signaling and dampening the immune response.

aiGeneR 1.0: An Artificial Intelligence Technique for the Revelation of Informative and Antibiotic Resistant Genes in Escherichia coli.

BACKGROUND: There are several antibiotic resistance genes (ARG) for the Escherichia coli (E. coli) bacteria that cause urinary tract infections (UTI), and it is therefore important to identify these ARG. Artificial Intelligence (AI) has been used previously in the field of gene expression data, but never adopted for the detection and classification of bacterial ARG. We hypothesize, if the data is correctly conferred, right features are selected, and Deep Learning (DL) classification models are optimized, then (i) non-linear DL models would perform better than Machine Learning (ML) models, (ii) leads to higher accuracy, (iii) can identify the hub genes, and, (iv) can identify gene pathways accurately. We have therefore designed aiGeneR, the first of its kind system that uses DL-based models to identify ARG in E. coli in gene expression data. METHODOLOGY: The aiGeneR consists of a tandem connection of quality control embedded with feature extraction and AI-based classification of ARG. We adopted a cross-validation approach to evaluate the performance of aiGeneR using accuracy, precision, recall, and F1-score. Further, we analyzed the effect of sample size ensuring generalization of models and compare against the power analysis. The aiGeneR was validated scientifically and biologically for hub genes and pathways. We benchmarked aiGeneR against two linear and two other non-linear AI models. RESULTS: The aiGeneR identifies tetM (an ARG) and showed an accuracy of 93% with area under the curve (AUC) of 0.99 (p < 0.05). The mean accuracy of non-linear models was 22% higher compared to linear models. We scientifically and biologically validated the aiGeneR. CONCLUSIONS: aiGeneR successfully detected the E. coli genes validating our four hypotheses.

Reverse transcriptase inhibitors prevent liver abscess formation during Escherichia coli bloodstream infection.

The presence of bacteria in the bloodstream is associated with severe clinical outcomes. In mice, intravenous inoculation of Escherichia coli can lead to the formation of macroscopic abscesses in the liver. Abscesses are regions of severe necrosis and consist of millions of bacteria surrounded by inflammatory immune cells. Liver abscess susceptibility varies widely across strains of mice, but the host factors governing this variation are unknown. Here, we profiled hepatic transcriptomes in mice with varying susceptibility to liver abscess formation. We found that transcripts from endogenous retroviruses (ERVs) are robustly induced in the liver by E. coli infection and ERV expression positively correlates with the frequency of abscess formation. Hypothesizing that ERV-encoded reverse transcriptase may generate cytoplasmic DNA and heighten inflammatory responses, we tested whether nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) influence abscess formation. Strikingly, a single NRTI dose administered immediately following E. coli inoculation prevented abscess formation, leading to a concomitant 100,000-fold reduction in bacterial burden. We provide evidence that NRTIs inhibit abscess formation by preventing the tissue necrosis that facilitates bacterial replication. Together, our findings suggest that endogenous reverse transcriptases drive inflammatory responses during bacterial bloodstream infection to drive abscess formation. The high efficacy of NRTIs in preventing abscess formation suggests that the consequences of reverse transcription on inflammation should be further examined, particularly in infectious diseases where inflammation drives negative clinical outcomes, such as sepsis.

Survey on resistance occurrence for F4+ and F18+ enterotoxigenic Escherichia coli (ETEC) among pigs reared in Central Italy regions.

Porcine Post Weaning Diarrhoea (PWD) is one of the most important swine disease worldwide, caused by Enterotoxigenic Escherichia coli (ETEC) strains able to provoke management, welfare and sanitary issues. ETEC is determined by proteinaceous surface appendages. Numerous studies conducted by now in pigs have demonstrated, at the enterocytes level, that, the genes mucin 4 (MUC4) and fucosyltransferase (FUT1), coding for ETEC F4 and F18 receptors respectively, can be carriers of single nucleotide polymorphisms (SNPs) associated with natural resistance/susceptibility to PWD. The latter aspect was investigated in this study, evaluating the SNPs of the MUC4 and FUT1 genes in slaughtered pigs reared for the most in Central Italy. Genomic DNA was extracted from 362 swine diaphragmatic samples and then was subjected to the detection of known polymorphisms on MUC4 and FUT1candidate target genes by PCR-RFLP. Some of the identified SNPs were confirmed by sequencing analysis. Animals carrying the SNPs associated with resistance were 11% and 86% for the FUT1 and MUC4 genes respectively. Therefore, it can be assumed that the investigated animals may be an important resource and reservoir of favorable genetic traits for the breeding of pigs resistant to enterotoxigenic E.coli F4 variant.

Multi-drug resistance pattern and genome-wide SNP detection in levofloxacin-resistant uropathogenic Escherichia coli strains.

OBJECTIVES: Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. METHODS: Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. RESULTS: Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. CONCLUSIONS: The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics.

Parallel loss of type VI secretion systems in two multi-drug-resistant Escherichia coli lineages.

The repeated emergence of multi-drug-resistant (MDR) Escherichia coli clones is a threat to public health globally. In recent work, drug-resistant E. coli were shown to be capable of displacing commensal E. coli in the human gut. Given the rapid colonization observed in travel studies, it is possible that the presence of a type VI secretion system (T6SS) may be responsible for the rapid competitive advantage of drug-resistant E. coli clones. We employed large-scale genomic approaches to investigate this hypothesis. First, we searched for T6SS genes across a curated dataset of over 20 000 genomes representing the full phylogenetic diversity of E. coli. This revealed large, non-phylogenetic variation in the presence of T6SS genes. No association was found between T6SS gene carriage and MDR lineages. However, multiple clades containing MDR clones have lost essential structural T6SS genes. We characterized the T6SS loci of ST410 and ST131 and identified specific recombination and insertion events responsible for the parallel loss of essential T6SS genes in two MDR clones.

ArcA positively regulates the expression of virulence genes and contributes to virulence of porcine Shiga toxin-producing enterotoxigenic Escherichia coli.

IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in humans and animals, leading to death and huge economic loss worldwide. Thus, elucidation of ETEC's pathogenic mechanisms will provide powerful data for the discovery of drugs serving as prevention or therapeutics against ETEC-caused diarrheal diseases. Here, we report that ArcA plays an essential role in the pathogenicity and virulence regulation in ETEC by positively regulating the expression of several key virulence factors including F18 fimbriae, heat-labile and heat-stable toxins, Shiga toxin 2e, and hemolysin, under microaerobic conditions and in vivo. Moreover, we found that positive regulation of several virulence genes by ArcA requires a global repressor H-NS (histone-like nucleoid structuring), implying that ArcA may exert positive effects by antagonizing H-NS. Collectively, our data established a key role for ArcA in the pathogenicity of porcine ETEC and ETEC strains isolated from human infections. Moreover, our work reveals another layer of regulation in relation to oxygen control of virulence factors in ETEC.

Expression of hes, iha, and tpsA codified in locus of adhesion and autoaggregation and their involvement in the capability of shiga toxin-producing Escherichia coli strains to adhere to epithelial cells.

OBJECTIVES: Shiga toxin-producing Escherichia coli strains LAA-positive are important cause of human infection. The capability to adhere to epithelial cells is a key virulence trait, and genes codified in LAA pathogenicity island could be involved in the adhesion during the pathogenesis of LAA-positive STEC strains. Thus, our objectives were to compare hes-negative and hes-positive STEC strains in their adherence capability to epithelial cells (HEp-2) and to evaluate the expression levels of the hes, iha, and tpsA in the bacteria adhered and non-adhered to HEp-2 cells. These genes are encoded in LAA, and are virulence factors that participate in adhesion and autoaggregation. RESULTS: We could not observe differences between the adhesion of strains but also in the expression level of of hes, iha, and tpsA. Genes encoded in LAA contribute to the adhesion phenotype though the expression of STEC adhesins is a coordinated event that depends not only the strain but also on the environment as well as its genetic background. Therefore, the results of this study suggest that LAA ,the most prevalent PAI among LEE-negative STEC strains, plays a role in pathogenesis.

The bacterial genetic determinants of Escherichia coli capacity to cause bloodstream infections in humans.

Escherichia coli is both a highly prevalent commensal and a major opportunistic pathogen causing bloodstream infections (BSI). A systematic analysis characterizing the genomic determinants of extra-intestinal pathogenic vs. commensal isolates in human populations, which could inform mechanisms of pathogenesis, diagnostic, prevention and treatment is still lacking. We used a collection of 912 BSI and 370 commensal E. coli isolates collected in France over a 17-year period (2000-2017). We compared their pangenomes, genetic backgrounds (phylogroups, STs, O groups), presence of virulence-associated genes (VAGs) and antimicrobial resistance genes, finding significant differences in all comparisons between commensal and BSI isolates. A machine learning linear model trained on all the genetic variants derived from the pangenome and controlling for population structure reveals similar differences in VAGs, discovers new variants associated with pathogenicity (capacity to cause BSI), and accurately classifies BSI vs. commensal strains. Pathogenicity is a highly heritable trait, with up to 69% of the variance explained by bacterial genetic variants. Lastly, complementing our commensal collection with an older collection from 1980, we predict that pathogenicity continuously increased through 1980, 2000, to 2010. Together our findings imply that E. coli exhibit substantial genetic variation contributing to the transition between commensalism and pathogenicity and that this species evolved towards higher pathogenicity.

Transposon mutagenesis of atypical enteroaggregative Escherichia coli reveals a hemagglutinin-associated protein that mediates cell adhesion and contributes to the Galleria mellonella virulence.

Twenty-two atypical enteroaggregative Escherichia coli isolates from a previous epidemiological study harboring EAEC virulence genes were examined for their adhesion properties. Nine strains showed a typical aggregative adherence (AA) pattern, while 13 strains showed variant AA, such as AA with lined up cells characteristic of the chain-like adhesion (CLA) and AA mainly to HeLa cells characteristic of the diffuse adherence (DA). The aggregative forming pilus (AFP) genes afpA2 and afpR were detected only in strain Q015B, which exhibited an AA/DA pattern. Using Tn5-based transposon mutagenesis on Q015B strain, we identified a 5517-bp open reading frame (ORF) encoding a predicted 1838-amino-acid polypeptide that is genetically related to a putative filamentous hemagglutinin identified in E. coli strain 7-233-03_S3_C2. Therefore, the ORF was named orfHA. The regions flanking orfHA were sequenced and two ORFs were found; upstream, an ORF that encodes a 603-amino-acid polypeptide with 99% identity to hemolysin secretion/activation proteins of the ShlB/FhaC/HecB family, and downstream, another ORF, which encodes a 632-amino-acid polypeptide with 72% identity to the glycosyltransferase EtpC. An orfHA mutant (Q015BΔorfHA) was constructed from strain Q015B. Q015BΔorfHA strain did not adhere to HeLa cells, whereas Q015BΔ orfHA transformed with a pACYC184 plasmid carrying orfHA restored the AA/DA phenotype of strain Q015B. Furthermore, the Q015ΔorfHA mutant had a marked effect on the ability of strain Q015B to kill the larvae of Galleria mellonella. Our results suggest that the AA/DA pattern of strain Q015B is mediated by a hemagglutinin-associated protein which also contributes to its virulence in the G. mellonella model.

Molecular typing of mcr-1 Escherichia coli isolates from pigs and farm environment based on fumC and fimH alleles.

Background: The dissemination of polymyxin resistance represents a significant threat to public health. Materials & methods: Sequence-based typing was performed by 53 mcr-1 Escherichia coli isolates using fumC/fimH (CH) genes to characterize clones spreading from pig farming. Furthermore, 12 isolates had their whole genome sequenced for phylogenetic study. Results: The isolates were classified into 22 distinct CH types, and two novel CH types (CH41-1578 and CH4-1579) and one sequence type (ST12652) was also described. According to phylogenetic study, both multilocus sequence typing and CH methods grouped the isolates similarly. Conclusion: Our findings suggest that the dissemination of the mcr-1 gene in pig farming has occurred mainly by horizontal gene transfer, and CH typing proved to be a good tool to characterize E. coli clones.

Complete association between CHCF1 genotype and enterotoxigenic Escherichia coli F4ab-associated post-weaning diarrhea in a pig challenge trial.

Enterotoxigenic E. coli (ETEC) susceptibility in pigs is highly influenced by their genotype. The aim of this study was to determine the association between CHCF1 genotype and ETEC F4ab susceptibility in experimentally infected pigs. We investigated ETEC diarrhea development in CHCF1 heterozygous susceptible (RS) (n = 12 pigs) compared to CHCF1 homozygous resistant (RR) (n = 12 pigs) for six days after ETEC F4ab challenge. Afterwards, we genotyped with MUC4 and MUC13 markers to relate performance in identifying ETEC F4ab diarrhea susceptible pigs. In the CHCF1 RS group, 12/12 pigs developed ETEC diarrhea compared with 0/12 pigs in the CHCF1 RR group. Weight gain was lower in CHCF1 RS pigs compared with RR pigs (mean ± SD: 208 ± 323 g and 987 ± 615 g, p = 0.0007). Further, the shedding of hemolytic E. coli was significantly higher in CHCF1 RS pigs from 2 to 6 days post inoculation and they shed the challenge strain for more days (mean ± SD: 3.5 ± 1.6 days versus 0.5 ± 0.5 days, p < 0.0001). Twelve pigs with ETEC diarrhea were misclassified as resistant with the MUC4 marker and four pigs without ETEC diarrhea were misclassified as susceptible with the MUC13 marker. We found complete association between CHCF1 genotype and ETEC diarrhea development in pigs from a herd with Danbred genetics. The CHCF1 marker was more likely to determine the true host susceptibility to ETEC F4ab than the other markers. The marker shows potential for improving reliability of PWD challenge models and potentially for use in breeding for ETEC F4ab/ac resistance.

Analysis of alternative splicing in chicken macrophages infected with avian pathogenic E. coli (APEC).

Colibacillosis is a complex disease that caused by avian pathogenic Escherichia coli (APEC), resulting in huge economic loss to the global poultry industry and threatening to human health. Alternative splicing (AS) is a universal post-transcriptional regulatory mechanism, which can simultaneously produce many proteins from a single gene to involve in various diseases and individual development. Herein, we characterized genome-wide AS events in wild type macrophages (WT) and APEC infected macrophages (APEC) by high-throughput RNA sequencing technology. A total of 751 differentially expressed (DE) AS genes were identified in the comparison of APEC vs. WT, including 587 of SE, 114 of MXE, 25 of RI, 17 of A3 and 8 of A5 event. Functional analysis showed that these identified DE AS genes were involved in 'Endocytosis', 'p53 signaling pathway', 'MAPK signaling pathway', 'NOD-like receptor signaling pathway', 'Ubiquitin mediated proteolysis' and 'Focal adhesion' immune related pathways. In summary, we comprehensively investigate AS events during APEC infection. This study has expanded our understanding of the process of APEC infection and provided new insights for further treatment options for APEC infection.