The ROK kinase N-acetylglucosamine kinase uses a sequential random enzyme mechanism with successive conformational changes upon each substrate binding.

N-acetyl-d-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolize environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate. In bacteria, the ROK family kinase N-acetylglucosamine kinase (NagK) performs this activity. Although ROK kinases have been studied extensively, no ternary complex showing the two substrates has yet been observed. Here, we solved the structure of NagK from the human pathogen Plesiomonas shigelloides in complex with GlcNAc and the ATP analog AMP-PNP. Surprisingly, PsNagK showed distinct conformational changes associated with the binding of each substrate. Consistent with this, the enzyme showed a sequential random enzyme mechanism. This indicates that the enzyme acts as a coordinated unit responding to each interaction. Our molecular dynamics modeling of catalytic ion binding confirmed the location of the essential catalytic metal. Additionally, site-directed mutagenesis confirmed the catalytic base and that the metal-coordinating residue is essential. Together, this study provides the most comprehensive insight into the activity of a ROK kinase.

Transcriptome Analysis Reveals Cross-Talk between the Flagellar Transcriptional Hierarchy and Secretion System in Plesiomonas shigelloides.

Plesiomonas shigelloides, a Gram-negative bacillus, is the only member of the Enterobacteriaceae family able to produce polar and lateral flagella and cause gastrointestinal and extraintestinal illnesses in humans. The flagellar transcriptional hierarchy of P. shigelloides is currently unknown. In this study, we identified FlaK, FlaM, FliA, and FliAL as the four regulators responsible for polar and lateral flagellar regulation in P. shigelloides. To determine the flagellar transcription hierarchy of P. shigelloides, the transcriptomes of the WT and ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were carried out for comparison in this study. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and luminescence screening assays were used to validate the RNA-seq results, and the Electrophoretic Mobility Shift Assay (EMSA) results revealed that FlaK can directly bind to the promoters of fliK, fliE, flhA, and cheY, while the FlaM protein can bind directly to the promoters of flgO, flgT, and flgA. Meanwhile, we also observed type VI secretion system (T6SS) and type II secretion system 2 (T2SS-2) genes downregulated in the transcriptome profiles, and the killing assay revealed lower killing abilities for ΔflaK, ΔflaM, ΔfliA, and ΔfliAL compared to the WT, indicating that there was a cross-talk between the flagellar hierarchy system and bacterial secretion system. Invasion assays also showed that ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were less effective in infecting Caco-2 cells than the WT. Additionally, we also found that the loss of flagellar regulators causes the differential expression of some of the physiological metabolic genes of P. shigelloides. Overall, this study aims to reveal the transcriptional hierarchy that controls flagellar gene expression in P. shigelloides, as well as the cross-talk between motility, virulence, and physiological and metabolic activity, laying the groundwork for future research into P. shigelloides' coordinated survival in the natural environment and the mechanisms that infect the host.

Pathogenicity of Plesiomonas shigelloides causing mass mortalities of largemouth bass (Micropterus salmoides) and its induced host immune response.

The outbreak of mass mortality of M. salmoides occurred in an aquaculture farm in Jiangsu province of China, showing signs of skin ulceration and haemorrhages. The bacteria were isolated from diseased largemouth bass, and identified as Plesiomonas shigelloides based on morphological, physiological and biochemical features, as well as 16S rRNA gene sequence analysis. The pathogenicity of P. shigelloides was determined by challenge experiments, and the median lethal dosage (LD50) of the isolate NJS1 for M. salmoides was calculated as 1.6 × 105 CFU/mL at 7 d post-infection. Histopathological analysis revealed that extensive necrosis, vacuolization and inflammation were presented in the kidney, liver and gill of the diseased fish. Detection of virulence-related genes showed that P. shigelloides NJS1 was positive for astA, astB, astD, astE, actP and 6 ahpA. Additionally, the host defensive response of M. salmoides infected by P. shigelloides was analyzed by quantitive real-time PCR (qRT-PCR), and the results showed that the expression levels of Cas3, Hep1, HIF, IgM, IL15 and TGF were significantly up-regulated in head kidney, liver and spleen in different hours post-infection, which revealed varying expression profiles and clear transcriptional activation of immune related genes. The results suggested that P. shigelloides was an etiological element in the mass mortalities of M. salmoides and this study provided deeper insights for the pathogenesis and host defensive system in P. shigelloides invasion.

Transcriptome Analysis Reveals the Effect of PdhR in Plesiomonas shigelloides.

The pyruvate dehydrogenase complex regulator (PdhR) was originally identified as a repressor of the pdhR-aceEF-lpd operon, which encodes the pyruvate dehydrogenase complex (PDHc) and PdhR itself. According to previous reports, PdhR plays a regulatory role in the physiological and metabolic pathways of bacteria. At present, the function of PdhR in Plesiomonas shigelloides is still poorly understood. In this study, RNA sequencing (RNA-Seq) of the wild-type strain and the ΔpdhR mutant strains was performed for comparison to identify the PdhR-controlled pathways, revealing that PdhR regulates ~7.38% of the P. shigelloides transcriptome. We found that the deletion of pdhR resulted in the downregulation of practically all polar and lateral flagella genes in P. shigelloides; meanwhile, motility assay and transmission electron microscopy (TEM) confirmed that the ΔpdhR mutant was non-motile and lacked flagella. Moreover, the results of RNA-seq and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) showed that PdhR positively regulated the expression of the T3SS cluster, and the ΔpdhR mutant significantly reduced the ability of P. shigelloides to infect Caco-2 cells compared with the WT. Consistent with previous research, pyruvate-sensing PdhR directly binds to its promoter and inhibits pdhR-aceEF-lpd operon expression. In addition, we identified two additional downstream genes, metR and nuoA, that are directly negatively regulated by PdhR. Furthermore, we also demonstrated that ArcA was identified as being located upstream of pdhR and lpdA and directly negatively regulating their expression. Overall, we revealed the function and regulatory pathway of PdhR, which will allow for a more in-depth investigation into P. shigelloides pathogenicity as well as the complex regulatory network.

RpoN is required for the motility and contributes to the killing ability of Plesiomonas shigelloides.

BACKGROUND: RpoN, also known as σ54, first reported in Escherichia coli, is a subunit of RNA polymerase that strictly controls the expression of different genes by identifying specific promoter elements. RpoN has an important regulatory function in carbon and nitrogen metabolism and participates in the regulation of flagellar synthesis, bacterial motility and virulence. However, little is known about the effect of RpoN in Plesiomonas shigelloides. RESULTS: To identify pathways controlled by RpoN, RNA sequencing (RNA-Seq) of the WT and the rpoN deletion strain was carried out for comparison. The RNA-seq results showed that RpoN regulates ~ 13.2% of the P. shigelloides transcriptome, involves amino acid transport and metabolism, glycerophospholipid metabolism, pantothenate and CoA biosynthesis, ribosome biosynthesis, flagellar assembly and bacterial secretion system. Furthermore, we verified the results of RNA-seq using quantitative real-time reverse transcription PCR, which indicated that the absence of rpoN caused downregulation of more than half of the polar and lateral flagella genes in P. shigelloides, and the ΔrpoN mutant was also non-motile and lacked flagella. In the present study, the ability of the ΔrpoN mutant to kill E. coli MG1655 was reduced by 54.6% compared with that of the WT, which was consistent with results in RNA-seq, which showed that the type II secretion system (T2SS-2) genes and the type VI secretion system (T6SS) genes were repressed. By contrast, the expression of type III secretion system genes was largely unchanged in the ΔrpoN mutant transcriptome and the ability of the ΔrpoN mutant to infect Caco-2 cells was also not significantly different compared with the WT. CONCLUSIONS: We showed that RpoN is required for the motility and contributes to the killing ability of P. shigelloides and positively regulates the T6SS and T2SS-2 genes.

Pathogenicity of Plesiomonas shigelloides and Citrobacter freundii isolated from the endangered Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon (Acipenser sinensis) is an indigenous species of China and is listed as a critically endangered species. Recently, second filial generations of Chinese sturgeon in the Yangtze River Fisheries Research Institute suffered from a severe disease. In this study, two kinds of pathogenic bacteria were isolated from diseased sturgeon and identified as Plesiomonas shigelloides and Citrobacter freundii, based on 16S rDNA gene sequence alignment analysis. Antimicrobial susceptibility testing showed that P. shigelloides was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to tocefatriaxone, piperacillin, cefoperazone, cefazolin, and ciprofloxacin. C. freundii was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to chloramphenicol, cefuroxime, norfloxacin, ciprofloxacin, and ceftazidime. The median lethal dose (LD50) values of P. shigelloides and C. freundii were 4.50 × 103 colony forming units (CFU)/g and 3.20 × 103 CFU/g, respectively. Clinical symptoms of challenged sturgeons were the same as those of naturally infected sturgeons. Histopathological examination disclosed severe damage in the viscera of P. shigelloides and C. freundii-infected sturgeons. This is the first report suggesting that P. shigelloides infection is associated with mortality of Chinese sturgeon. The results of this study revealed the pathogenesis and severe pathogenicity of P. shigelloides and C. freundii in cultured Chinese sturgeon, and offer insights into the prevention and treatment of bacterial infection caused by P. shigelloides and C. freundii in cultured sturgeons.

γ-proteobacteria eject their polar flagella under nutrient depletion, retaining flagellar motor relic structures.

Bacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large molecular machine remains unclear. Here, we describe the previously unreported ejection of polar motors by γ-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane; furthermore, we show that ejection is triggered only under nutritional depletion and is independent of the filament as a possible mechanosensor. We show that filament ejection is a widespread phenomenon demonstrated by the appearance of relic structures in diverse γ-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens, and Pseudomonas aeruginosa. While the molecular details remain to be determined, our results demonstrate a novel mechanism for bacteria to halt costly motility when nutrients become scarce.

Bacterial and parasite co-infection in Mexican golden trout (Oncorhynchus chrysogaster) by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator.

BACKGROUND: Bacterial infections are responsible of high economic losses in aquaculture. Mexican golden trout (Oncorhynchus chrysogaster) is a threatened native trout species that has been introduced in aquaculture both for species conservation and breeding for production and for which no studies of bacterial infections have been reported. CASE PRESENTATION: Fish from juvenile stages of Mexican golden trout showed an infectious outbreak in a farm in co-culture with rainbow trout (Oncorhynchus mykiss), showing external puntiform red lesions around the mouth and caudal pedunculus resembling furuncles by Aeromonas spp. and causing an accumulated mortality of 91%. Isolation and molecular identification of bacteria from lesions and internal organs showed the presence of Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator isolated from a single individual. All bacterial isolates were resistant to amoxicillin-clavulanic acid and cefazoline. P. shigelloides was resistant to third generation ß-lactamics. CONCLUSIONS: This is the first report of coinfection by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator in an individual of Mexican golden trout in co-culture with rainbow trout. Resistance to ß-lactams suggests the acquisition of genetic determinants from water contamination by human- or livestock-associated activities.

Clinical and microbiological features of intratumor abscess with bloodstream infection caused by Plesiomonas shigelloides, Citrobacter freundii, Streptococcus mitis/oralis, Clostridium perfringens, and Candida albicans in a patient with cholangiocarcinoma: A case report.

Plesiomonas shigelloides is a gram-negative facultative anaerobic bacillus, usually found in soil and freshwater, which causes self-limited diarrhea, although reports of bacteremia are rare. Here, we report the first case of an intratumoral abscess with mixed bacteremia caused by P. shigelloides, Citrobacter freundii, Streptococcus mitis/oralis, Clostridium perfringens, and Candida albicans in a patient with recurrent postoperative cholangiocarcinoma. A 77-year-old man with hilar cholangiocarcinoma and hypertension was admitted to our hospital with fever and abdominal pain. He had visited Vietnam for 3 years, 20 years ago. Abdominal computed tomography showed air within the recurrent tumor at the left liver lobectomy resection margin site, which was diagnosed as an intratumor abscess perforating the intestinal tract. P. shigelloides, C. freundii, S. mitis/oralis, C. perfringens, and C. albicans were isolated in blood culture. P. shigelloides was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and 16S ribosomal RNA (16S rRNA) sequencing. Piperacillin-tazobactam was administered for almost a week, ampicillin-sulbactam and levofloxacin for almost 3 weeks, and antifungal agents for almost 2 weeks, and the patient was discharged thereafter. Although bloodstream infections caused by P. shigelloides in patients with cancer are extremely rare, long-term colonization and the potential for future intra-abdominal infections were implicated.

The effect of ArcA on the growth, motility, biofilm formation, and virulence of Plesiomonas shigelloides.

BACKGROUND: The anoxic redox control binary system plays an important role in the response to oxygen as a signal in the environment. In particular, phosphorylated ArcA, as a global transcription factor, binds to the promoter regions of its target genes to regulate the expression of aerobic and anaerobic metabolism genes. However, the function of ArcA in Plesiomonas shigelloides is unknown. RESULTS: In the present study, P. shigelloides was used as the research object. The differences in growth, motility, biofilm formation, and virulence between the WT strain and the ΔarcA isogenic deletion mutant strain were compared. The data showed that the absence of arcA not only caused growth retardation of P. shigelloides in the log phase, but also greatly reduced the glucose utilization in M9 medium before the stationary phase. The motility of the ΔarcA mutant strain was either greatly reduced when grown in swim agar, or basically lost when grown in swarm agar. The electrophoretic mobility shift assay results showed that ArcA bound to the promoter regions of the flaK, rpoN, and cheV genes, indicating that ArcA directly regulates the expression of these three motility-related genes in P. shigelloides. Meanwhile, the ability of the ΔarcA strain to infect Caco-2 cells was reduced by 40%; on the contrary, its biofilm formation was enhanced. Furthermore, the complementation of the WT arcA gene from pBAD33-arcA+ was constructed and all of the above features of the pBAD33-arcA+ complemented strain were restored to the WT level. CONCLUSIONS: We showed the effect of ArcA on the growth, motility, biofilm formation, and virulence of Plesiomonas shigelloides, and demonstrated that ArcA functions as a positive regulator controls the motility of P. shigelloides by directly regulating the expression of flaK, rpoN and cheV genes.

High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris.

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

A multiplex PCR assay for the detection of five human pathogenic Vibrio species and Plesiomonas.

A multiplex PCR (mPCR) assay was established to detect five pathogenic Vibrio species and Plesiomonas shigelloides. Twelve genes were included: ompW, ctxA, rfbN, and wbfR from V. cholerae; tl, tdh, and trh from V. parahaemolyticus; toxR and vmhA from V. mimicus; toxR from V. fluvialis; vvhA from V. vulnificus; and the 23S rRNA gene from P. shigelloides. The specificity of the mPCR assay was 100% for the detection of 136 strains and the limits of detection (LoD) were 12.5-50 pg/reaction. The assay exhibited higher sensitivity than cultivation methods in the detection of APW cultures of 113 diarrhea samples. In the analysis of 369 suspected Vibrio populations from estuarine water samples, the specificity of the mPCR for V. cholerae and V. parahaemolyticus was 100% for both, while the sensitivities were 100% and 96.1%, respectively. The assay can be applied to screen enrichment cultures and suspected colonies from environmental and clinical samples.

Plesiomonas: A Review on Food Safety, Fish-Borne Diseases, and Tilapia.

Fish and fish products are considered a fundamental part of the human diet due to their high nutritional value. Food-borne diseases are considered a major public health challenge worldwide due to their incidence, associated mortality, and negative economic repercussions. Food safety is the guarantee that foods will not cause harm to the health of those who consume them, and it is a fundamental property of food quality. Food safety can be at risk of being lost at any stage of the food chain if the food is contaminated by pathogenic microorganisms. Many diverse bacteria are present in the environment and as part of the microbiota of food that can be transmitted to humans during the handling and consumption of food. Plesiomonas shigelloides has been mainly associated with outbreaks of gastrointestinal diseases due to the consumption of fish. This bacterium inhabits the environment and aquatic animals and is associated with the microbiota of fish such as tilapia, a fish of importance in fishing, aquaculture, commercialization, and consumption worldwide. The purpose of this document is to provide, through a bibliographic review of databases (Scopus, Web of Science, and Google Scholar, among others), a general informative perspective on food-borne diseases and, in particular, the consumption of fish and tilapia. Diseases derived from contamination by Plesiomonas shigelloides are included, and control and prevention actions and sanitary regulations for fishery products established in several countries around the world are discussed to promote the safety of foods of aquatic origin intended for human consumption and to protect public health.

Structural Studies of the Lipopolysaccharide Isolated from Plesiomonas shigelloides O22:H3 (CNCTC 90/89).

Plesiomonas shigelloides is a Gram-negative, rod-shaped bacterium which causes foodborne intestinal infections, including gastroenteritis. It is one of the most frequent causes of travellers' diarrhoea. Lipopolysaccharide (LPS, endotoxin), an important virulence factor of the species, is in most cases characterised by a smooth character, demonstrated by the presence of all regions, such as lipid A, core oligosaccharide, and O-specific polysaccharide, where the latter part determines O-serotype. P. shigelloides LPS is still a poorly characterised virulence factor considering a "translation" of the particular O-serotype into chemical structure. To date, LPS structure has only been elucidated for 15 strains out of 102 O-serotypes. Structures of the new O-specific polysaccharide and core oligosaccharide of P. shigelloides from the Czechoslovak National Collection of Type Cultures CNCTC 90/89 LPS (O22), investigated by chemical analysis, mass spectrometry, and 1H,13C nuclear magnetic resonance (NMR) spectroscopy, have now been reported. The pentasaccharide repeating unit of the O-specific polysaccharide is built of one d-QuipNAc and is rich in four d-GalpNAcAN residues. Moreover, the new core oligosaccharide shares common features of other P. shigelloides endotoxins, i.e., the lack of phosphate groups and the presence of uronic acids.

Effects of dietary blackberry syrup supplement on growth performance, antioxidant, and immunological responses, and resistance of Nile tilapia, Oreochromis niloticus to Plesiomonas shigelloides.

The present study investigated the effects of dietary blackberry syrup on growth performance, haematological, non-specific immune and spleen gene expression responses of Nile tilapia, Oreochromis niloticus. Five experimental groups of fish with mean weights of 26.75 ±â€¯2.67 g were used in the study; three of them were fed with blackberry syrup incorporated diets (7.5 g kg-1- BBRY7.5, 15 g kg-1- BBRY15, 30 g kg-1- BBRY30), whereas an additive free basal diet served as the control. Additionally, the fifth group was an antibiotic medicated diet (0.02 g kg-1- ABTC), prepared with the florfenicol. Dietary blackberry syrup especially at 15 g kg-1 significantly increased growth performance, respiratory burst activity, potential killing activity, phagocytic activity, phagocytic index, lysozyme activity, myeloperoxidase activity, total immunoglobulin levels, serum SOD activity and serum CAT activity (p < 0.05). Furthermore, dietary blackberry syrup increased the expression levels of immune [heat shock protein 70 (HSP70), interleukin 1, beta (IL-1ß), tumor necrosis factor (TNF-α), interferon gamma (IFN-γ), immunoglobulin M (IgM)] and antioxidant [glutathione peroxidase (GPx)] related genes in the spleen of fish fed with especially 15 g kg-1 blackberry syrup (p < 0.05). At the end of the 20-day challenge period the survival rates were significantly higher in the BBRY15 and ABTC groups compared to all other treatment groups (p < 0.05). As a result, feeding Nile tilapia with a diet containing 15 g kg-1 blackberry syrup over a period of 90 days might be adequate to improve growth performance, fish immune parameters, antioxidant status, as well as survival rate against P. shigelloides, similar to antibiotic treatment. Hence, blackberry syrup can be used as an antibiotics replacer for controlling P. shigelloides in tilapia feed.

Putative virulence factors of Plesiomonas shigelloides.

Plesiomonas shigelloides is a Gram-negative rod-shaped bacterium which has been isolated from humans, animals and the environment. It has been associated with diarrhoeal disease in humans and various epizootic diseases in animals. In this study P. shigelloides strains were isolated from the faecal material of a captive Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) living in semi-natural conditions in China. Plesiomonas shigelloides strain EE2 was subjected to whole genome sequencing. The draft genome was then compared to the genome sequences of ten other P. shigelloides isolates using the Pathosystems Resource Integration Center pipeline. In addition to several virulence factors which have been previously reported, we are proposing new candidate virulence factors such as a repeats-in-toxin protein, lysophospholipase, a twin-arginine translocation system and the type VI secretion effector Phospholipase A1.

Emerging agents of gastroenteritis: Aeromonas, Plesiomonas, and the diarrheagenic pathotypes of Escherichia coli.

Knowledge of the pathogenic roles of certain bacterial agents in gastroenteritis has been growing over the past few decades. With the increasing use of multiplex molecular-based syndromic stool pathogen panels, the roles of Plesiomonas shigelloides and some of the diarrheagenic pathotypes of Escherichia coli (enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], enteroinvasive E. coli [EIEC], and enteroaggregative E. coli [EAEC]) have been better understood. Although not currently targeted on Food and Drug Administration (FDA)-cleared commercial multiplex stool panels, Aeromonas has also emerged as a possible cause of bacterial gastroenteritis. The clinical presentation, pathophysiology, and diagnostic approaches to these pathogens in stool specimens are reviewed. Variability in inclusion of these pathogens on multiplex molecular panels and difficulties in detection by stool culture techniques utilized by clinical microbiology laboratories have contributed to an unclear understanding of the pathogenic role of several of these pathogens. Nonetheless, most evidence points towards a clear pathogenic role for P. shigelloides and ETEC, and possibly EPEC and EIEC. The contribution of Aeromonas spp. and EAEC to bacterial gastroenteritis has not been fully established. Further studies of pathogenicity of these pathogens are needed.

Visual and rapid detection of Plesiomonas shigelloides using loop-mediated isothermal amplification method.

Plesiomonas shigelloides is a common pathogen of aquatic animals and can pose a certain hazard to aquaculture. Here, we aimed to develop a loop-mediated isothermal amplification (LAMP) method for the visual detection of P. shigelloides to aid the diagnosis of infections caused by this pathogen in aquatic animals. We used LAMP to amplify P. shigelloides DNA and combined it with calcein or nucleic acid dipstick assay (NADA) to visualize the amplified products. The optimal LAMP amplification temperature was 64°C, and the reaction lasted for 50 min. The limit of detection of recombinant plasmids containing the target gene using the LAMP method was 2·0 × 102 copies per µl, which is ten times higher than that using conventional polymerase chain reaction (PCR). LAMP products could be visualized without agarose gel electrophoresis. We tested 85 fish specimens using the established LAMP method and conventional PCR. The detection rate was 42·4% using the LAMP method and 34·1% using conventional PCR. Based on our results, the LAMP method combined with calcein or NADA is a rapid, specific, sensitive and accurate method for visual detection of fish-derived P. shigelloides and can be used for the laboratory diagnosis of infections caused by it. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of loop-mediated isothermal amplification (LAMP) and calcein and nucleic acid dipstick assay (NADA) provided a rapid, specific and sensitive method for detecting Plesiomonas shigelloides, which is an important pathogen that causes diseases in aquatic animals worldwide. In the present study, the LAMP method showed a higher detection rate than conventional PCR for P. shigelloides using templates from 85 fish specimens. Thus, the LAMP method could be a reliable and convenient tool for diagnosing diseases in aquatic animals in the laboratory.

Total Synthesis of a Densely Functionalized Plesiomonas shigelloides Serotype 51 Aminoglycoside Trisaccharide Antigen.

Plesiomonas shigelloides, a pathogen responsible for frequent outbreaks of severe travelers' diarrhea, causes grave extraintestinal infections. Sepsis and meningitis due to P. shigelloides are associated with a high mortality rate as antibiotic resistance increases and vaccines are not available. Carbohydrate antigens expressed by pathogens are often structurally unique and are targets for developing vaccines and diagnostics. Here, we report a total synthesis of the highly functionalized trisaccharide repeating unit 2 from P. shigelloides serotype 51 from three monosaccharides. A judicious choice of building blocks and reaction conditions allowed for the four amino groups adorning the sugar rings to be installed with two N-acetyl (Ac) groups, rare acetamidino (Am), and d-3-hydroxybutyryl (Hb) groups. The strategy for the differentiation of amino groups in trisaccharide 2 will serve well for the syntheses of other complex glycans.

Enteric Infections Are Common in Patients with Flares of Inflammatory Bowel Disease.

OBJECTIVES: Few studies have examined the role of non-Clostridium difficile enteric infections in flares of inflammatory bowel disease (IBD). Our objective was to investigate enteric infection detected by multiplex PCR stool testing in patients with IBD. METHODS: We performed a cross-sectional analysis of 9403 patients who underwent 13,231 stool tests with a gastrointestinal pathogen PCR panel during a diarrheal illness from March 2015 to May 2017. Our primary outcome was the presence of an infection. Secondary outcomes included endoscopic and histologic predictors of infection, and IBD outcomes following testing. RESULTS: A total of 277 patients with Crohn's disease (CD), 300 patients with ulcerative colitis (UC), and 8826 patients without IBD underwent 454, 503, and 12,275 tests, respectively. Compared to patients without IBD, patients with IBD were less likely to test positive (CD 18.1%, UC 16.1%, no IBD 26.6%, p < 0.001). Compared to patients without IBD, CD had a higher prevalence of norovirus (p = 0.05) and Campylobacter (p = 0.043), whereas UC had a lower prevalence of norovirus (p = 0.001) and a higher prevalence of Campylobacter (p = 0.013), Plesiomonas (p = 0.049), and Escherichia coli species (p < 0.001). Of 77 patients who underwent endoscopy, there were no major endoscopic or histologic predictors of a positive test. Patients who tested negative were more likely to have IBD therapy escalated (p = 0.004). Enteric infection did not impact IBD outcomes following testing (log-rank 0.224). CONCLUSIONS: Non-Clostridium difficile enteric infections were identified in 17% of symptomatic patients with IBD. Endoscopic and histologic findings may not differentiate flare from infection. Norovirus and E.coli may play an important role in flare of IBD.