Investigation of in vitro susceptibility and resistance mechanisms to amikacin among diverse carbapenemase-producing Enterobacteriaceae.

OBJECTIVE: This study aims to assess the in vitro drug susceptibility of various Carbapenemase-Producing Enterobacteriaceae (CPE) genotypes and elucidate the underlying mechanisms of amikacin resistance. METHODS: A total of 72 unique CPE strains were collected from the Second Hospital of Jiaxing between 2019 and 2022, including 51 strains of Klebsiella pneumoniae, 11 strains of Escherichia coli, 6 strains of Enterobacter cloacae, 2 strains of Klebsiella aerogenes, 1 strain of Citrobacter freundii, and 1strain of Citrobacter werkmanii. Among these strains, 24 carried blaKPC gene, 20 carried blaNDM gene, 23 carried blaOXA-48-like gene, and 5 carried both blaKPC and blaNDM. We measured the in vitro activity of amikacin and other common antibiotics. Strains carrying blaOXA-48-like gene were selected for whole genome sequencing (WGS) via next-generation sequencing to identify genes related to antimicrobial resistance (AMR) and virulence factor (VF). RESULTS: Out of the 72 CPE strains tested, 41.7% exhibited resistance to amikacin. The drug resistance rates for K. pneumoniae, E. coli, and Enterobacter spp. were 51.0%, 27.3%, and 10.0%, respectively. The majority of the CPE strains (> 90%) displayed resistance to cephalosporins and carbapenems, while most of them were sensitive to polymyxin B and tigecycline (97.2% and 94.4%). The amikacin resistance rate was 100% for strains carrying blaOXA-48, 20.8% for those with blaKPC, 5.0% for those with blaNDM, and 20.0% for those with both blaKPC and blaNDM. These differences were statistically significant (P < 0.05). Through sequencing, we detected aminoglycoside resistance genes rmtF and aac(6')-Ib, VF genes iucABCD and rmpA2 in OXA-48-producing multidrug resistance and highly virulent strains. These genes were located on a IncFIB- and IncHI1B-type plasmid, respectively. Both plasmids were highly homologous to the plasmid from OXA-232 strains in Zhejiang province and Shanghai province. Integration of these resistance genes into the IncFIB plasmid, facilitated by the IS6 and/or Tn3 transposons, resulted in OXA232-producing K. pneumoniae with amikacin resistance. CONCLUSION: This study identified significant amikacin resistance in CPE strains, particularly in those carrying the blaOXA-48 gene. Resistance genes rmtF and aac(6')-Ib were identified on plasmids. These results highlight the need for careful monitoring of amikacin resistance.

Fitness factor genes conserved within the multi-species core genome of Gram-negative Enterobacterales species contribute to bacteremia pathogenesis.

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from unregulated immune responses to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections.

Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics.

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

Whole-Genome Analysis of Extensively Drug-Resistant Enterobacter hormaechei Isolated from a Patient with Non-Hodgkin's Lymphoma.

BACKGROUND: Currently, the Enterobacteriaceae species are responsible for a variety of serious infections and are already considered a global public health problem, especially in underdeveloped countries, where surveillance and monitoring programs are still scarce and limited. Analyses were performed on the complete genome of an extensively antibiotic-resistant strain of Enterobater hormaechei, which was isolated from a patient with non-Hodgkin's lymphoma, who had been admitted to a hospital in the city of Manaus, Brazil. METHODS: Phenotypical identification and susceptibility tests were performed in automated equipment. Total DNA extraction was performed using the PureLink genomic DNA mini-Kit. The genomic DNA library was prepared with Illumina Microbial Amplicon Prep and sequenced in the MiSeq Illumina Platform. The assembly of the whole-genome and individual analyses of specific resistance genes extracted were carried out using online tools and the Geneious Prime software. RESULTS: The analyses identified an extensively resistant ST90 clone of E. hormaechei carrying different genes, including blaCTX-M-15, blaGES-2, blaTEM-1A, blaACT-15, blaOXA-1 and blaNDM-1, [aac(3)-IIa, aac(6')-Ian, ant(2″)-Ia], [aac(6')-Ib-cr, (qnrB1)], dfrA25, sul1 and sul2, catB3, fosA, and qnrB, in addition to resistance to chlorhexidine, which is widely used in patient antisepsis. CONCLUSIONS: These findings highlight the need for actions to control and monitor these pathogens in the hospital environment.

Detection of Quinolone resistance Qnr genes and its association with Extended Spectrum ß-lactamase and AmpC ß-lactamase genes in Qnr Positive Enterobacteriaceae in Bangladesh.

This cross-sectional study was conducted to explore quinolone resistant Enterobacteriaceae followed by searching the prevalence of three groups of quinolone resistance genes (QnrA, QnrB and QnrS) from January 2015 to December 2015 at Dhaka Medical College hospital, Bangladesh. Then genes for ESBL and AmpC ß-lactamase were detected among Qnr positive strains for better understanding the role of these genes for multiple drug resistance. Total 340 urines, sputum, wound swab and blood samples were collected from DMCH. Total 270(79.41%) Enterobacteriaceae were isolated from 340 samples. Out of 270 Enterobacteriaceae, 225(83.33%) were quinolone (ciprofloxacin) resistant strains. Qnr genes were detected in 141(62.67%) of the 225 quinolone resistant Enterobacteriaceae. Total 187 Qnr genes [84(59.57%) QnrS, 70(49.64%) QnrB and 33(23.40%) QnrA] were detected from 141 quinolone resistant strains. Total 48(34.04%) ESBL producers were detected by DDS test and 47(33.33%) ESBL producers were positive by PCR among 141 Qnr positive strains. QnrA was co-existed with CTX-M-15. QnrB was co-existed with TEM, CTXM-15 and OXA-1. QnrS genes were also associated with TEM, CTX-M-15 and OXA-1. Among 52 cefoxitin resistant Qnr positive strains, 22(42.31%) AmpC ß-lactamase producers were detected by Modified three-dimensional test (MTDT) and 45(86.54%) AmpC ß-lactamase producers were detected by PCR. QnrA had been identified with DHA, ACC, EBC and CIT while QnrB had been identified with DHA, ACC, EBC and CIT. QnrS had also been co-existed with DHA, ACC, EBC and CIT. The results of this study provided insights into the high proportion of Qnr genes among isolated Enterobacteriaceae. Simultaneous presence of Qnr genes and genes for extended-spectrum ß-lactamase or AmpC ß-lactamase were observed in multidrug resistant Enterobacteriaceae.

The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine.

PRMT5 is a type II arginine methyltransferase abundantly expressed in the colonic epithelium. It is up-regulated in inflammatory bowel disease and colorectal cancer. However, its role in mucosal defense against enteric infection has not been studied. Here, we report that Prmt5 in the murine colon is up-regulated in response to Citrobacter rodentium infection. Pathogen clearance in mice with haploinsufficient expression of Prmt5 is significantly delayed compared with wildtype littermate controls. Transcriptomic analyses further reveal that PRMT5 regulates the expression of canonical crypt goblet cell genes involved in mucus production, assembly, and anti-microbial responses via methyltransferase activity-dependent and -independent mechanisms. Together, these findings uncover PRMT5 as a novel regulator of mucosal defense and a potential therapeutic target for treating intestinal diseases.

Molecular Characterization of Nine TRAF Genes in Yellow Catfish (Pelteobagrus fulvidraco) and Their Expression Profiling in Response to Edwardsiella ictaluri Infection.

The yellow catfish (Pelteobagrus fulvidraco) is an economic fish with a large breeding scale, and diseases have led to huge economic losses. Tumor necrosis factor receptor-associated factors (TRAFs) are a class of intracellular signal transduction proteins that play an important role in innate and adaptive immune responses by mediating NF-κB, JNK and MAPK signaling pathways. However, there are few studies on the TRAF gene family in yellow catfish. In this study, the open reading frame (ORF) sequences of TRAF1, TRAF2a, TRAF2b, TRAF3, TRAF4a, TRAF4b, TRAF5, TRAF6 and TRAF7 genes were cloned and identified in yellow catfish. The ORF sequences of the nine TRAF genes of yellow catfish (Pf_TRAF1-7) were 1413-2025 bp in length and encoded 470-674 amino acids. The predicted protein structures of Pf_TRAFs have typically conserved domains compared to mammals. The phylogenetic relationships showed that TRAF genes are conserved during evolution. Gene structure, motifs and syntenic analyses of TRAF genes showed that the exon-intron structure and conserved motifs of TRAF genes are diverse among seven vertebrate species, and the TRAF gene family is relatively conserved evolutionarily. Among them, TRAF1 is more closely related to TRAF2a and TRAF2b, and they may have evolved from a common ancestor. TRAF7 is quite different and distantly related to other TRAFs. Real-time quantitative PCR (qRT-PCR) results showed that all nine Pf_TRAF genes were constitutively expressed in 12 tissues of healthy yellow catfish, with higher mRNA expression levels in the gonad, spleen, brain and gill. After infection with Edwardsiella ictaluri, the expression levels of nine Pf_TRAF mRNAs were significantly changed in the head kidney, spleen, gill and brain tissues of yellow catfish, of which four genes were down-regulated and one gene was up-regulated in the head kidney; four genes were up-regulated and four genes were down-regulated in the spleen; two genes were down-regulated, one gene was up-regulated, and one gene was up-regulated and then down-regulated in the gill; one gene was up-regulated, one gene was down-regulated, and four genes were down-regulated and then up-regulated in the brain. These results indicate that Pf_TRAF genes might be involved in the immune response against bacterial infection. Subcellular localization results showed that all nine Pf_TRAFs were found localized in the cytoplasm, and Pf_TRAF2a, Pf_TRAF3 and Pf_TRAF4a could also be localized in the nucleus, uncovering that the subcellular localization of TRAF protein may be closely related to its structure and function in cellular mechanism. The results of this study suggest that the Pf_TRAF gene family plays important roles in the immune response against pathogen invasion and will provide basic information to further understand the roles of TRAF gene against bacterial infection in yellow catfish.

Characterization of Japanese flounder (Paralichthys olivaceus) STAT members: An immune-related gene family involved in Edwardsiella tarda and temperature stress.

The signal transducer and activator of transcription (STAT) family members are not only the transcriptional activators, but also play important roles in regulating inflammatory response. Some members have been reported to be involved in innate bacterial and antiviral immunity in aquatic organisms. However, no systematic research on STATs has been found in teleost. In this present study, we characterized six STAT genes in Japanese flounder based on bioinformatics methods, namely PoSTAT1, PoSTAT2, PoSTAT3, PoSTAT4, PoSTAT5 and PoSTAT6. The phylogenetic analysis of STATs in fish indicated that STATs were highly conserved and revealed an absence of STAT5 in a few species. Further analysis of gene structures and motifs showed STAT proteins shared a similar structure and probably had similar functionality in Japanese flounder. The expression profiles of different development stages and tissues demonstrated that PoSTATs exhibited specificity in temporality and spatiality as well as PoSTAT4 was highly expressed in gill. The transcriptome data analysis of E. tarda and temperature stress showed that PoSTAT1 and PoSTAT2 were more respective to these two kinds of stress. In addition, the results also demonstrated that these PoSTATs might regulate immune response in different ways, manifested by up-regulation in E. tarda infection and down-regulation in temperature stress. In a word, this systematic analysis of PoSTATs would provide valuable information about the phylogenetic relationship of STATs in fish species and help understand the role of STAT genes in the immune response of Japanese flounder.

Japanese Flounder pol-miR-155 Is Involved in Edwardsiella tarda Infection via ATG3.

MicroRNAs (miRNAs) are small RNA molecules that function in the post-transcriptionally regulation of the expression of diverse genes, including those involved in immune defense. Edwardsiella tarda can infect a broad range of hosts and cause severe disease in aquatic species, including Japanese flounder (Paralichthys olivaceus). In this study, we examined the regulation mechanism of a flounder miRNA, pol-miR-155, during the infection of E. tarda. Pol-miR-155 was identified to target flounder ATG3. Overexpression of pol-miR-155 or knockdown of ATG3 expression suppressed autophagy and promoted the intracellular replication of E. tarda in flounder cells. Overexpression of pol-miR-155 activated the NF-κB signaling pathway and further promoted the expression of downstream immune related genes of interleukin (IL)-6 and IL-8. These results unraveled the regulatory effect of pol-miR-155 in autophagy and in E. tarda infection.

[The pathogenicity of Cronobacter in the light of bacterial genomics]. / La patogenicidad de Cronobacter a la luz de la genómica bacteriana.

Introduction: Cronobacter spp. is a genus of Gram-negative bacteria belonging to the family Enterobacteriaceae. Species of the genus Cronobacter, particularly C. sakazakii, are implicated in the development of severe disease in newborns, which occurs with necrotizing enterocolitis, sepsis and meningitis. The disease has been frequently associated with powdered infant formula (PIF) and can therefore occur in the form of outbreaks. The genus Cronobacter has undergone extensive diversification in the course of its evolution, with some species being clearly pathogenic to humans while the impact of other species on human health is uncertain or unknown. Whole genome sequencing is used both in population genetic studies to identify the limited number of genotypes associated with the disease and to detect genes associated with antibiotic resistance or virulence, ultimately allowing more precise epidemiological links to be established between pediatric disease and infant foods.

Introducción: Cronobacter es un género de bacterias gramnegativas perteneciente a la familia Enterobacteriaceae. Algunas especies del género Cronobacter, en particular C. sakazakii, están implicadas en el desarrollo de infecciones neonatales graves, incluyendo meningitis, sepsis y enterocolitis necrotizante. La enfermedad se ha relacionado frecuentemente con los preparados en polvo para lactantes (PPL) y se puede presentar, por tanto, en forma de brotes. El género Cronobacter ha experimentado una amplia diversificación en el curso de su evolución, siendo algunas especies claramente patógenas para los humanos mientras que el impacto de otras especies sobre la salud humana es incierto o desconocido. La secuenciación genómica se utiliza en los estudios de genética de poblaciones tanto para identificar el limitado número de genotipos asociados a la enfermedad como para detectar los genes asociados a la virulencia, la adaptación al estrés o la resistencia a antibióticos, lo que permite, en definitiva, establecer vínculos epidemiológicos más precisos entre la enfermedad pediátrica y los alimentos infantiles.

Genomic insights of Leclercia adecarboxylata strains linked to an outbreak in public hospitals in Mexico.

BACKGROUND: Leclercia adecarboxylata is a bacteria closely related to Escherichia coli according to its biochemical characteristics and is commonly considered non-pathogenic although a growing number of publications classify it as an emerging pathogen. Fosfomycin resistance is a common trait for L. adecarboxylata encoded by fosALA gene. OBJECTIVE: To analyze genomic traits of sixteen L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition. METHODS: Twenty-eight L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition were identified biochemically with a Vitek ® automated system. The strains were phenotyped by their growth on Eosin Methylene Blue agar or MacConkey agar plates. Additionally, Pulsed field gel electrophoresis (PFGE) was performed to establish the clonal relationship. The genomic DNA of sixteen strains was obtained using a Qubit ® dsDNA HS Assay Kit and sequenced on an Illumina ® MiSeq instrument. Draft genomes were assembled using PROKKA and Rast. Assemblies were submitted to Resfinder and PathogenFinder from the Center for Genomic Epidemiology in order to find resistance genes and pathogenic potential. IslandViewer4 was also used to find Pathogenicity and Phage Islands. For identification of the fosA gene, manual curation and Clustal analysis was performed. A novel FosA variant was identified. Finally, phylogenetic analysis was performed using VAMPhyRE software and Mega X. RESULTS: In this paper, we report the genomes of sixteen strains of Leclercia adecarboxylata causing an outbreak associated with parenteral nutrition in public hospitals in Mexico. The genomes were analyzed for genetic determinants of virulence and resistance. A high pathogenic potential (pathogenicity index 0.82) as well as multiple resistance genes including carbapenemics, colistin and efflux pumps were determined. Based on sequence analysis, a new variant of the fosALA gene was described. Finally, the outbreak was confirmed by establishing the clonal relationship among the sixteen genomes obtained. CONCLUSIONS: Commensal strains of L. adecarboxylata may acquire genetic determinants that provide mechanisms of host damage and go unnoticed in clinical diagnosis. L. adecarboxylata can evolve in a variety of ways including the acquisition of resistance and virulence genes representing a therapeutic challenge in patient care.

Ornithine decarboxylase supports ILC3 responses in infectious and autoimmune colitis through positive regulation of IL-22 transcription.

Group 3 innate lymphoid cells (ILC3s) are RORγT+ lymphocytes that are predominately enriched in mucosal tissues and produce IL-22 and IL-17A. They are the innate counterparts of Th17 cells. While Th17 lymphocytes utilize unique metabolic pathways in their differentiation program, it is unknown whether ILC3s make similar metabolic adaptations. We employed single-cell RNA sequencing and metabolomic profiling of intestinal ILC subsets to identify an enrichment of polyamine biosynthesis in ILC3s, converging on the rate-limiting enzyme ornithine decarboxylase (ODC1). In vitro and in vivo studies demonstrated that exogenous supplementation with the polyamine putrescine or its biosynthetic substrate, ornithine, enhanced ILC3 production of IL-22. Conditional deletion of ODC1 in ILC3s impaired mouse antibacterial defense against Citrobacter rodentium infection, which was associated with a decrease in anti-microbial peptide production by the intestinal epithelium. Furthermore, in a model of anti-CD40 colitis, deficiency of ODC1 in ILC3s markedly reduced the production of IL-22 and severity of inflammatory colitis. We conclude that ILC3-intrinsic polyamine biosynthesis facilitates efficient defense against enteric pathogens as well as exacerbates autoimmune colitis, thus representing an attractive target to modulate ILC3 function in intestinal disease.

Molecular and functional characterization of teleost-specific Interleukin-17N in yellow catfish (Pelteobagrus fulvidraco).

In mammals, six interleukin-17 (IL-17) genes, as potent pro-inflammatory cytokines, all accelerate the inflammatory responses. In teleosts, seven IL-17 genes have been found in various species, but little is known about the function of teleost-specific IL-17N. In this study, teleost IL-17N and IL-17A/F2 genes all had six conserved cysteine residues forming three intrachain disulfide bridges, the length of three exons of teleost IL-17N gene was similar to that of teleost IL-17A/F2 gene, and the neighbor-joining (NJ) phylogenetic tree showed that teleost IL-17N was clustered with vertebrate IL-17A/F, implying that teleost IL-17N gene may be a paralog of teleost IL-17A/F gene. Pelteobagrus fulvidraco (Pf) IL-17N gene was highly expressed in the blood, brain and kidney of healthy yellow catfish. Pf_IL-17N transcript and protein were notably up-regulated in the spleen, head kidney, gill and kidney detected after Edwardsiella ictaluri infection. Lipopolysaccharides (LPS), polyinosinic-polycytidylic acid (Poly I:C) and peptidoglycan (PGN) also remarkably induced the expression of Pf_IL-17N in the isolated peripheral blood leucocytes (PBLs) of yellow catfish. These results reveal that Pf_IL-17N may play important roles in preventing the invasion of pathogens. Furthermore, the recombinant (r) Pf_IL-17N protein could significantly induce the mRNA expressions of inflammatory cytokines, chemokines and antimicrobial peptide genes in yellow catfish in vivo and in vitro, and it also notably promoted the phagocytosis of myeloid cells in the PBLs and the chemotaxis of the PBLs and gill leucocytes (GLs) in yellow catfish. Besides, though the rPf_IL-17N protein could induce and aggravate inflammation infiltration in the kidney of yellow catfish, it did not effectively and notably increase the survival rate of yellow catfish after E. ictaluri infection. Furthermore, the rPf_IL-17N protein could induce the mRNA expressions of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways related genes, and the inhibitor of NF-κB and MAPK signal pathways could restrain the rPf_IL-17N protein-induced inflammatory response. This study provides crucial evidence that the Pf_IL-17N may mediate inflammatory response to eliminate invasive pathogens.

NDM-beta-lactamase-1: Where do we stand?

Multidrug-resistant (MDR) Gram-negative bacilli (GNB) have been playing havoc in the field of nosocomial as well as community-acquired infections. Of particular concern are the carbapenem-resistant GNBs, belonging to Enterobacteriaceae and encoding for New Delhi metallo-beta-lactamase-1 (NDM-1) gene. These strains spread rapidly and horizontally in the population, thus exhibiting MDR traits as these can harbour several resistance encoding genes to almost all antimicrobial groups. Several predisposing factors are responsible towards its spread, viz. excessive antibiotic usage, improper aseptic conditions by healthcare workers, lack of awareness, abruptly discontinuing medication course, alternative medications and vector-borne factors contributing to the unchecked harbouring of these super bugs in India. Thus, a bugle call has already been sounded worldwide especially in India, where the country has taken serious cognizance to build up strategy via implementation of several national programs to combat antimicrobial resistance covering human, animal, agriculture and environmental aspects. As there is an exponential rise in variants of NDM-1 harbouring strains, molecular epidemiological investigations of these strains using genotyping techniques are of paramount importance for a better understanding of this rampant spread and curbing resistance thereafter. This review explores the urgent need to develop a cost-effective, rapid molecular assay, viz. the loop-mediated isothermal amplification method for field detection of MBL harbouring bacterial strains, especially NDM-1 and its variants, thus targeting specific carbapenemase genes at a grass root level even to the remote and rural regions of the country.

Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers.

MicroRNA (miRNA) plays essential roles in post-transcriptional regulation of protein coding genes, and the quantitative real-time polymerase chain reaction (qRT-PCR) is the powerful and broadly employed tool to conduct studies of miRNA expression. Identifying appropriate references to normalize quantitative data is a prerequisite to ensure the qRT-PCR accuracy. Until now, there has been no report about miRNA reference for qRT-PCR in Japanese flounder (Paralichthys olivaceus), one important marine cultured fish along the coast of Northern Asia. In this study, combined with miRNA-Seq analysis and literature search, 10 candidates (miR-34a-5p, miR-205-5p, miR-101a-3p, miR-22-3p, miR-23a-3p, miR-210-5p, miR-30c-5p, U6, 5S rRNA, and 18S rRNA) were chosen as potential references to test their expression stability among P. olivaceus tissues, and in livers of P. olivaceus infected with Edwardsiella tarda at different time points. The expression stability of these candidates was analyzed by qRT-PCR and evaluated with Delta CT, BestKeeper, geNorm, as well as NormFinder methods, and RefFinder was employed to estimate the comprehensive ranking according to the four methods. As the result, miR-22-3p and miR-23a-3p were proved to be the suitable combination as reference miRNAs for both P. olivaceus normal tissues and livers infected with E. tarda, and they were successfully applied to normalize miR-7a and miR-221-5p expression in P. olivaceus livers in response to E. tarda infection. All these results provide valuable information for P. olivaceus miRNA quantitative expression analysis in the future.

Allelically and Differentially Expressed Genes After Infection of Edwardsiella ictaluri in Channel Catfish as Determined by Bulk Segregant RNA-Seq.

Identification of genetic markers associated with resistance against enteric septicemia of catfish (ESC) is of great interest for genetic enhancement programs of catfish. In the present study, bulk segregant RNA-Seq analysis was applied to determine differentially expressed genes and alleles after ESC infection. Here we report three genomic regions on LG1, LG12, and LG26, containing significant single-nucleotide polymorphisms (SNPs). These genomic regions aligned well with quantitative trait loci (QTL) previously identified. Within the QTL regions, eleven genes were found to be differentially regulated between phenotypic bulks. Importantly, the QTL on linkage group 1 (LG1) were found to be expressed in the liver, whereas the QTL on LG12 and LG26 were expressed in the intestine, suggesting multiple mechanisms of ESC resistance. It is apparent that apolipoproteins may be important for ESC resistance as the QTL on LG1 included the 14-kDa apolipoprotein genes that are both allelically expressed and differentially expressed between the resistant and susceptible bulks. Traf2 and NCK-interacting protein kinase (TNIK) were found in the QTL on LG12, and it was downregulated in resistant fish, suggesting the importance of NCK downregulation in ESC resistance, as previously reported. In addition, we observed divergent gene expression patterns between the liver and intestine after infection. Immune/inflammatory-related processes were overrepresented from liver DEGs, while those DEGs identified from intestine were enriched for proteolysis and wounding processes. Taken together, the BSR-Seq analysis presented here advanced the knowledge of ESC resistance, providing information of not only positions of QTL but also genes and their differential expression between resistant and susceptible fish, making it one step closer to the identification of the causal genes for ESC resistance.

Transcriptomic analysis using dual RNA sequencing revealed a Pathogen-Host interaction after Edwardsiella anguillarum infection in European eel (Anguilla anguilla).

Many studies have explored differentially expressed genes (DEGs) between some pathogens and hosts, but no study has focused on the interaction of DEGs between Edwardsiella anguillarum (Ea) and Anguilla anguilla (Aa). In this study, we examined the interactions of DEGs during Ea infection and Aa anti-infection processes by dual RNA sequencing. Total RNA from in vitro and in vivo (Aa liver) Ea culture was extracted. Using high-throughput transcriptomics, significant DEGs that were expressed between Ea cultured in vitro versus in vivo and those in the liver of the infected group versus control group were identified. Protein-protein interactions between the pathogen and host were explored using Cytoscape according to the HPIDB 3.0 interaction transcription database. The results showed that the liver in the infection group presented with severe bleeding and a large number of thrombi in the hepatic vessels. We found 490 upregulated and 398 downregulated DEGs of Ea in vivo versus Ea cultured in vitro, and 2177 upregulated and 970 downregulated genes in the liver of the infected eels. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the pathogen DEGs revealed that the upregulated genes were mainly enriched in migration, colonization, biofilm formation, and significantly enriched in ABC transport and quorum sensing; the downregulated genes were mainly involved in metabolism, information transduction, organelle formation, enzyme catalysis, molecular transport, and binding. GO of the host DEGs showed that metabolic process, catalytic activity, single organism metabolic process, small molecule binding, nucleotide binding, nucleotide phosphate binding, and anion binding were markedly enriched. Finally, we found that 79 Ea and 148 Aa proteins encoded by these DEGs were involved in an interaction network, and some pathogen (DegP, gcvP, infC, carB, rpoC, trpD, sthA, and FhuB) and host proteins (MANBA, STAT1, ETS2, ZEP1, TKT1, NMI and RBPMS) appear to play crucial roles in infection. Thus, determining the interaction networks revealed crucial molecular mechanisms underlying the process of pathogenic infection and host anti-infection.

Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease.

We report a pleiotropic disease due to loss-of-function mutations in RHBDF2, the gene encoding iRHOM2, in two kindreds with recurrent infections in different organs. One patient had recurrent pneumonia but no colon involvement, another had recurrent infectious hemorrhagic colitis but no lung involvement and the other two experienced recurrent respiratory infections. Loss of iRHOM2, a rhomboid superfamily member that regulates the ADAM17 metalloproteinase, caused defective ADAM17-dependent cleavage and release of cytokines, including tumor-necrosis factor and amphiregulin. To understand the diverse clinical phenotypes, we challenged Rhbdf2-/- mice with Pseudomonas aeruginosa by nasal gavage and observed more severe pneumonia, whereas infection with Citrobacter rodentium caused worse inflammatory colitis than in wild-type mice. The fecal microbiota in the colitis patient had characteristic oral species that can predispose to colitis. Thus, a human immunodeficiency arising from iRHOM2 deficiency causes divergent disease phenotypes that can involve the local microbial environment.

Genetic characterization of heterologous Edwardsiella piscicida isolates from diverse fish hosts and virulence assessment in a Chinook salmon Oncorhynchus tshawytscha model.

Edwardsiella piscicida is an emergent global fish pathogen with a wide host range, although host associations driving genetic diversity remain unclear. This study investigated the genetic and virulence diversity of 37 E. piscicida isolates recovered from 10 fish species in North America. Multilocus sequence analysis (MLSA) was conducted using concatenated alignments of the gyrB, pgi and phoU sequences. MLSA clustered the tested isolates into six discrete clades. In light of recent disease outbreaks in cultured salmonids, the virulence of each clade was evaluated in Chinook salmon Oncorhynchus tshawytscha fingerlings following intracoelomic challenge of ~106  CFU/fish. Challenged and control fish were monitored for 21d, and microbiological and histological examination was performed on dead and surviving fish. Peak mortality occurred 3-5 days post-challenge (dpc) regardless of isolate or genetic group. Edwardsiella piscicida was recovered from all moribund and dead animals. At 21 dpc, fish challenged with isolates from clades II, III and IV presented cumulative mortality ≥83.3%, whereas isolates from clade I, V and VI resulted in cumulative mortality ≤71.4%. This study suggests an underlying genetic basis for strain virulence and potential host associations. Further investigations using other fish models and variable challenge conditions are warranted.

RNAseq studies reveal distinct transcriptional response to vitamin A deficiency in small intestine versus colon, uncovering novel vitamin A-regulated genes.

Vitamin A (VA) deficiency remains prevalent in resource limited areas. Using Citrobacter rodentium infection in mice as a model for diarrheal diseases, previous reports showed reduced pathogen clearance and survival due to vitamin A deficient (VAD) status. To characterize the impact of preexisting VA deficiency on gene expression patterns in the intestines, and to discover novel target genes in VA-related biological pathways, VA deficiency in mice were induced by diet. Total mRNAs were extracted from small intestine (SI) and colon, and sequenced. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and co-expression network analyses were performed. DEGs compared between VAS and VAD groups detected 49 SI and 94 colon genes. By GO information, SI DEGs were significantly enriched in categories relevant to retinoid metabolic process, molecule binding, and immune function. Three co-expression modules showed significant correlation with VA status in SI; these modules contained four known retinoic acid targets. In addition, other SI genes of interest (e.g., Mbl2, Cxcl14, and Nr0b2) in these modules were suggested as new candidate genes regulated by VA. Furthermore, our analysis showed that markers of two cell types in SI, mast cells and Tuft cells, were significantly altered by VA status. In colon, "cell division" was the only enriched category and was negatively associated with VA. Thus, these data suggested that SI and colon have distinct networks under the regulation of dietary VA, and that preexisting VA deficiency could have a significant impact on the host response to a variety of disease conditions.