RESUMO
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.
Assuntos
Proteínas de Bactérias , Flagelos , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Plesiomonas , Flagelos/metabolismo , Flagelos/genética , Plesiomonas/genética , Plesiomonas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Transcriptoma , Regiões Promotoras Genéticas , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Transcrição Gênica , HumanosRESUMO
This study aimed to investigate the cause of a foodborne disease outbreak in Huzhou on August 14, 2023. Multiple enteropathogens were detected using FilmArray, and the pathogen was subsequently isolated and cultured from anal swabs of the cases and stream water. The isolated strains were identified using VITEK MS, and antimicrobial susceptibility test, pulsed field gel electrophoresis (PFGE) molecular typing, and whole genome sequencing (WGS) were performed on the isolates of Plesiomonas shigelloides. Gene annotation and sequence alignment were used to analyze the virulence genes and drug resistance genes of the strains. A phylogenetic tree was constructed based on single nucleotide polymorphism (SNP), and homology analysis was conducted to trace the origin of P. shigelloides. A total of 7 strains of P.shigelloides were isolated, with 3 from stream water and 4 from anal swabs. All 7 strains exhibited the same PFGE pattern and showed resistance to amikacin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, cefazolin, streptomycin, and florfenicol. The isolated strains carried the same resistance genes and virulence factors. In the sequences of the isolated strains from this outbreak, 11 mutation sites were detected. The phylogenetic tree based on SNP sites showed that these strains were homologous. This foodborne disease outbreak caused by P.shigelloides was the first reported in Huzhou. WGS can be used as a complementary method to PFGE for epidemiological investigations of disease outbreaks.
Assuntos
Doenças Transmitidas por Alimentos , Plesiomonas , Humanos , Plesiomonas/genética , Rios , Filogenia , Diarreia , ÁguaRESUMO
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.
Assuntos
Proteínas de Escherichia coli , Plesiomonas , Humanos , Complexo Piruvato Desidrogenase/metabolismo , Proteínas de Escherichia coli/metabolismo , Plesiomonas/genética , Escherichia coli/metabolismo , Proteínas Repressoras/genética , Células CACO-2 , Perfilação da Expressão GênicaRESUMO
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.
Assuntos
Bass , Plesiomonas , Animais , Plesiomonas/genética , Virulência , RNA Ribossômico 16S/genética , ImunidadeRESUMO
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.
Assuntos
Regulação Bacteriana da Expressão Gênica , Plesiomonas , Humanos , RNA Polimerase Sigma 54/genética , Plesiomonas/genética , Plesiomonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células CACO-2RESUMO
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.
Assuntos
Plesiomonas , Animais , Plesiomonas/genética , Citrobacter freundii/genética , Virulência , Clindamicina , Peixes/genética , Oxacilina , Ampicilina , CiprofloxacinaRESUMO
Various bacterial diseases have caused great economic losses to the high-density and intensive aquaculture industry; however, the pathogenic mechanism underlying the large-scale challenged to caused by many bacteria remain unclear, making the prevention and treatment of these diseases difficult. In the present study, we isolated a bacterial strain from Cyprinus carpio having a typical bacterial disease and named it Cc2021. Through subsequent morphological observations, a regression challenge, biochemical identification, and 16S rRNA gene sequence analysis, we determined Cc2021 to be Plesiomonas shigelloides. Subsequently, we comprehensively investigated the pathogenicity of P. shigelloides in C. carpio through a regression challenge and assessed the underlying the pathogenic mechanism. Mortality results revealed that P. shigelloides is highly pathogenic and infects various tissues throughout the body, resulting in edema of the liver, spleen, and body and head kidneys. Histopathological analysis revealed obvious inflammation, bleeding, and necrosis in the intestine, spleen, and head kidney. The body's immune tissues actively produce complement C3, superoxide dismutase, and lysozyme after a challenge to resist bacterial invasion. With regard to the underlying pathogenesis of P. shigelloides, comparative transcriptome analysis revealed 876 upregulated genes and 828 downregulated genes in the intestine of C. carpio after the challenge. Analysis of differentially expressed unigenes revealed the involvement of major immune pathways, particularly the TNF signaling pathway, interleukin (IL)-17 signaling pathway, and Toll-like receptor signaling pathway. The present study provides new valuable information on the immune system and defense mechanisms of P. shigelloides.
Assuntos
Carpas , Plesiomonas , Animais , Plesiomonas/genética , RNA Ribossômico 16S/genética , Transcriptoma , VirulênciaRESUMO
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.
Assuntos
Aeromonas , Coinfecção , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Oncorhynchus mykiss , Oncorhynchus , Parasitos , Plesiomonas , Aeromonas/genética , Animais , Coinfecção/veterinária , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Necator , Plesiomonas/genéticaRESUMO
The genus Plesiomonas, represented by a single species, Plesiomonas shigelloides, is a gram-negative bacillus associated with gastrointestinal and extraintestinal diseases in humans. In this study, 44 clinical isolates (gastrointestinal, n = 41; extraintestinal, n = 3) were genetically confirmed to be P. shigelloides using the hug gene. All 20 virulence genes were detected in the gastrointestinal isolates, ranging from 7.7% to 100%; however, only 12 genes were detected in the extra-gastrointestinal isolates, ranging from 33.3% to 100%. The phlA gene was significantly associated with the gastrointestinal isolates (P = 0.0216). The results of this study suggest that phlA may play a role in gastrointestinal infections. However, pilF, tolC, and fur were detected in both gastrointestinal and extraintestinal clinical isolates, and further investigations are warranted to elucidate their role in the pathogenesis of P. shigelloides.
Assuntos
Infecções por Bactérias Gram-Negativas , Plesiomonas , Humanos , Plesiomonas/genética , Virulência/genéticaRESUMO
About 10% of bacteria have a multichromosome genome with a primary replicon of bacterial origin, called the chromosome, and other replicons of plasmid origin, the chromids. Studies on multichromosome bacteria revealed potential points of coordination between the replication/segregation of chromids and the progression of the cell cycle. For example, replication of the chromid of Vibrionales (called Chr2) is initiated upon duplication of a sequence carried by the primary chromosome (called Chr1), in such a way that replication of both replicons is completed synchronously. Also, Chr2 uses the Chr1 as a scaffold for its partition in the daughter cells. How many of the features detected so far are required for the proper integration of a secondary chromosome in the cell cycle? How many more features remain to be discovered? We hypothesized that critical features for the integration of the replication/segregation of a given chromid within the cell cycle program would be conserved independently of the species in which the chromid has settled. Hence, we searched for a chromid related to that found in Vibrionales outside of this order. We identified one in Plesiomonas shigelloides, an aquatic and pathogenic enterobacterium that diverged early within the clade of Enterobacterales. Our results suggest that the chromids present in P. shigelloides and Vibrionales derive from a common ancestor. We initiated in silico genomic and proteomic comparative analyses of P. shigelloides, Vibrionales, and Enterobacterales that enabled us to establish a list of features likely involved in the maintenance of the chromid within the host cell cycle.
Assuntos
Plesiomonas , Vibrio , Cromossomos Bacterianos/genética , Genoma Bacteriano , Plesiomonas/genética , Proteômica , Vibrio/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Plesiomonas/genética , Plesiomonas/patogenicidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genéticaRESUMO
Plesiomonas shigelloides is an important fish pathogen that causes significant losses in aquaculture. Phage therapy is a new approach to overcome the problem of multidrug-resistant bacteria. Herein, a virulent phage of P. shigelloides was isolated from the intestines of grass carp. This phage belongs to the Siphoviridae family and was designated PSP01. The optimal multiplicity of infection of PSP01 was 1 with a latent period of 30 min and a lytic period of 140 min. Good activity was observed over a wide range of temperatures (-20⯰C-50⯰C), pH values (3-12), and NaCl concentrations (0.1-3.5%). The phage PSP01 lysis cassette is composed of 3 genes, HolPSP, LysPSP-1 and LysPSP-2. Expression of HolPSP or LysPSP-2 in Escherichia coli resulted in bacterial lysis, and a synergistic effect was observed when the HolPSP and LysPSP-1 proteins were co-expressed. In-frame deletion uncovered an important role of the transmembrane domain (TMD) in HolPSP and the signal peptide (SP) in LysPSP-2 for bacterial lysis function. The protective effects of phage PSP01 were investigated by intraperitoneal injection into grass carp infected with P. shigelloides, showing a 33.3% increase in the survival rate of the infected grass carp. Pathological analysis of the spleens from the infected grass carp revealed alleviation of the pathological symptoms. In conclusion, isolation and bacterial lysis investigations of phage PSP01 provide a new tool for the control of fish pathogens and possesses potential for aquaculture applications.
Assuntos
Bacteriófagos , Carpas , Plesiomonas , Animais , Aquicultura , Bacteriófagos/genética , Escherichia coli , Plesiomonas/genéticaRESUMO
Plesiomonas shigelloides, a member of the family Vibrionaceae, is a gram-negative, rod-shaped, facultative anaerobic bacterium with flagella. P. shigelloides has been isolated from such sources as freshwater, surface water, and many wild and domestic animals. P. shigelloides contains 102 Oantigens and 51 H-antigens. The diversity of O-antigen gene clusters is relatively poorly understood. In addition to O1 and O17 reported by other laboratories, and the 12 O serogroups (O2, O10, O12, O23, O25, O26, O32, O33, O34, O66, O75, and O76) reported previously by us, in the present study, nine new P. shigelloides serogroups (O8, O17, O18, O37, O38, O39, O44, O45, and O61) were sequenced and annotated. The genes for the O-antigens of these nine groups are clustered together in the chromosome between rep and aqpZ. Only O38 possesses the wzm and wzt genes for the synthesis and translocation of O-antigens via the ATP-binding cassette (ABC) transporter pathway; the other eight use the Wzx/Wzy pathway. Phylogenetic analysis using wzx and wzy showed that both genes are diversified. Among the nine new P. shigelloides serogroups, eight use wzx/wzy genes as targets. In addition, we developed an O-antigen-specific PCR assay to detect these nine distinct serogroups with no cross reactions among them.
Assuntos
Família Multigênica , Antígenos O/genética , Plesiomonas/classificação , Sorotipagem , Filogenia , Plesiomonas/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
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.
Assuntos
Proteínas de Bactérias , Enzimas Imobilizadas , Expressão Gênica , Monoéster Fosfórico Hidrolases , Plesiomonas/genética , Saccharomycetales , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Enzimas Imobilizadas/biossíntese , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Plesiomonas/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/genética , Saccharomycetales/metabolismoRESUMO
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.
Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Plesiomonas/genética , Plesiomonas/isolamento & purificação , Vibrio/genética , Vibrio/isolamento & purificação , Eletroforese Capilar , Estuários , Humanos , Sensibilidade e Especificidade , Microbiologia da ÁguaRESUMO
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.
Assuntos
DNA Bacteriano/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Plesiomonas/isolamento & purificação , Animais , Aquicultura , Peixes/microbiologia , Plesiomonas/genética , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Plesiomonas/genética , Plesiomonas/isolamento & purificação , Toninhas/microbiologia , Fatores de Virulência/genética , Animais , China , Fezes/microbiologia , Genoma Bacteriano , Sequenciamento Completo do GenomaRESUMO
Plesiomonas shigelloides is widely associated with human diarrheal disease. Research on this pathogen has been hampered by the absence of an effective genetic manipulation system. In the present study, an efficient and precise conjugation transfer procedure, mediated by suicide vector pRE112 was used to overcome this limitation. The efficiency of generating double recombinants was average 74.3%, and the conjugation protocol may be applied to other P. shigelloides strains. We also identified that the SipD protein of P. shigelloides G5884 (serotype O45) is 65% similar to the SipD in Salmonella pathogenicity island 1 (SPI-1), which is a key element of the type III secretion system related to Salmonella invasion. A P. shigelloides sipD null mutant was generated via the conjugation system, using the suicide vector pRE112. The isogenic mutant strain lacking sipD showed a 50% reduction in its capacity to invade Caco-2 cells.
Assuntos
Proteínas de Bactérias/genética , Técnicas de Transferência de Genes , Plesiomonas/genética , Conjugação Genética , MutaçãoRESUMO
Iron availability plays an important role in the virulence of micro-organisms, which develops different systems for iron acquisition. The expression of genes involved in iron uptake systems is usually regulated by Fur, a transcriptional regulator. Plesiomonas shigelloides is a Gram-negative food- and water-borne enteropathogen. Even though the mechanisms involved in the pathogenicity of P. shigelloides are not properly elucidated, iron seems to be implicated in the development of human infections and in the production of potential virulence factors; however, detection and characterization of fur gene has not been performed in this bacterium. In this work the presence of a conserved fur gene was determined in six strains of P. shigelloides. The expression of fur was studied under different culture conditions and it was demonstrated to be higher when the micro-organism was cultured under iron-restricted than under iron-abundance conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: This study provides evidence of the presence of a conserved fur gene in strains of Plesiomonas shigelloides. Expression of this gene is higher when the micro-organism is cultured under iron-restricted conditions. The study provides clues to understand the role of iron in the regulation of important activities of P. shigelloides.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Ferro/metabolismo , Plesiomonas/genética , Plesiomonas/patogenicidade , Proteínas Repressoras/genética , Proteínas de Bactérias/biossíntese , Transporte Biológico/genética , Humanos , Plesiomonas/metabolismo , Proteínas Repressoras/biossíntese , Virulência , Fatores de Virulência/genéticaRESUMO
Plesiomonas shigelloides (P. shigelloides) is implicated as an aetiological agent of human gastroenteritis in humans, for which reliable laboratory detection of P. shigelloides is clinically and epidemiologically desirable. A simple molecular method for rapid detection of P. shigelloides using crosspriming amplification (CPA) has been developed, with hugA as the target. The hugA gene is required for haem iron utilisation and is critical for the survival and growth of P. shigelloides. The assay output was visualised as a colour change with no need to open the reaction tubes, and no falsepositive results were detected for the 33 non P. shigelloides strains examined to assess assay specificity. The limit of detection was 200 fg P. shigelloides DNA per reaction and 3x103 CFU per g in human stools, which was 100 and 10fold more sensitive than polymerase chain reaction, respectively. The CPA method was used to detect the presence of P. shigelloides in stool specimens from 70 patients with diarrhoea and 30 environmental water samples, with no difference in accuracy between the CPA assay and the biological culture. The present study, therefore, suggests that the P. shigelloides hugA CPA assay may represent a valuable tool for rapid and sensitive detection of P. shigelloides in primary care facilities and clinical laboratories.