Phenotypic changes and gene expression profiles of Vibrio parahaemolyticus in response to low concentrations of ampicillin.

Vibrio parahaemolyticus is a leading cause of seafood-associated gastroenteritis and possesses intrinsic resistance to ampicillin. While ampicillin can trigger transcriptional responses of global genes, the behavioral and molecular changes that occur in V. parahaemolyticus when exposed to ampicillin are not fully understood. In this work, we investigated the effects of low concentrations of ampicillin on the physiology and gene expression of V. parahaemolyticus by combining phenotypic assays and RNA sequencing (RNA-seq) analysis. Our results showed that the growth of V. parahaemolyticus were notably delayed, and both motility and c-di-GMP production were significantly inhibited in the response to low concentrations of ampicillin stress. In contrast, biofilm formation by V. parahaemolyticus was enhanced by exposure to low concentrations of ampicillin. However, low concentrations of ampicillin had no effect on the cytotoxicity or adherence activity of V. parahaemolyticus. The RNA-seq data revealed that a low concentration of ampicillin significantly affected the expression levels of 676 genes, including those involved in antibiotic resistance, virulence, biofilm formation, and regulation. This work contributes to our understanding of how V. parahaemolyticus alters its behavior and gene expression in response to ampicillin exposure.

GefB, a GGDEF domain-containing protein, affects motility and biofilm formation of Vibrio parahaemolyticus and is regulated by quorum sensing regulators.

Vibrio parahaemolyticus (V. parahaemolyticus) stands as the predominant etiological agent responsible for gastroenteritis associated with the consumption of seafood. Cyclic di-guanosine monophosphate (c-di-GMP), a secondary messenger in bacteria, controls multiple bacterial behaviors including pathogenesis, the development of biofilms, and motility. The protein GefB (VPA1478), characterized by the presence of a GGDEF domain, inhibits the swarming motility of V. parahaemolyticus. In this study, we showed that deletion of gefB remarkably reduced cellular c-di-GMP level and biofilm formation by V. parahaemolyticus, but significantly enhanced the swimming and swarming motility. In addition, GefB inhibited the polar and lateral flagellar genes but activated genes associated with exopolysaccharide production of V. parahaemolyticus. The data also demonstrated that vpa1477 and gefB are co-transcribed as a single transcriptional unit, designated as vpa1477-gefB. Transcription of vpa1477-gefB was under the collective regulation of the master quorum sensing (QS) regulators AphA and OpaR, which function at low (LCD) and high cell density (HCD), respectively. AphA positively regulated vpa1477-gefB transcription at LCD, whereas OpaR negatively regulated its transcription at HCD. The findings significantly enhance our comprehension of the metabolism and regulatory mechanisms of c-di-GMP in V. parahaemolyticus.

VPA0198, a GGDEF domain-containing protein, affects the motility and biofilm formation of Vibrio parahaemolyticus and is regulated by quorum sensing associated regulators.

Cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger in bacteria, affects multiple bacterial behaviors including motility and biofilm formation. c-di-GMP is synthesized by diguanylate cyclase harboring a GGDEF domain and degraded by phosphodiesterase harboring an either EAL or HD-GYP domain. Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, harbors more than 60 genes involved in c-di-GMP metabolism. However, roles of most of these genes including vpa0198, which encodes a GGDEF-domain containing protein, are still completely unknown. AphA and OpaR are the master quorum sensing (QS) regulators operating at low (LCD) and high cell density (HCD), respectively. QsvR integrates into QS to control gene expression via direct regulation of AphA and OpaR. In this study, we showed that deletion of vpa0198 remarkably reduced c-di-GMP production and biofilm formation, whereas promoted the swimming motility of V. parahaemolyticus. Overexpression of VPA0198 in the vpa0198 mutant strain significantly reduced the swimming and swarming motility and enhanced the biofilm formation ability of V. parahaemolyticus. In addition, transcription of vpa0198 was under the collective regulation of AphA, OpaR and QsvR. AphA activated the transcription of vpa0198 at LCD, whereas QsvR and OpaR coordinately and directly repressed vpa0198 transcription at HCD, thereby leading to a cell density-dependent expression of vpa0198. Therefore, this work expanded the knowledge of synthetic regulatory mechanism of c-di-GMP in V. parahaemolyticus.

AcsS Negatively Regulates the Transcription of type VI Secretion System 2 Genes in Vibrio parahaemolyticus.

The type VI secretion system 2 (T6SS2) gene cluster of Vibrio parahaemolyticus comprises three operons: VPA1027-1024, VPA1043-1028, and VPA1044-1046. AcsS is a LysR-like transcriptional regulator that play a role in activating flagella-driven motility in V. parahaemolyticus. However, its potential roles in other cellular pathways remain poorly understood. In this study, we conducted a series of experiments to investigate the regulatory effects of AcsS on the transcription of VPA1027 (hcp2), VPA1043, and VPA1044. The findings revealed that AcsS indirectly inhibits the transcription of these genes. Additionally, deletion of acsS resulted in enhanced adhesion of V. parahaemolyticus to HeLa cells. However, disruption of T6SS2 alone or in conjunction with AcsS significantly diminished the adhesion capacity of V. parahaemolyticus to HeLa cells. Therefore, it is suggested that AcsS suppresses cell adhesion in V. parahaemolyticus by downregulating the transcription of T6SS2 genes.

Assessment of Vibrionaceae prevalence in seafood from Qidong market and analysis of Vibrio parahaemolyticus strains.

The aim of this study was to investigate the prevalence of Vibrionaceae family in retail seafood products available in the Qidong market during the summer of 2023 and to characterize Vibrio parahaemolyticus isolates, given that this bacterium is the leading cause of seafood-associated food poisoning. We successfully isolated a total of 240 Vibrionaceae strains from a pool of 718 seafood samples. The breakdown of the isolates included 146 Photobacterium damselae, 59 V. parahaemolyticus, 18 V. campbellii, and 11 V. alginolyticus. Among these, P. damselae and V. parahaemolyticus were the predominant species, with respective prevalence rates of 20.3% and 8.2%. Interestingly, all 59 isolates of V. parahaemolyticus were identified as non-pathogenic. They demonstrated proficiency in swimming and swarming motility and were capable of forming biofilms across a range of temperatures. In terms of antibiotic resistance, the V. parahaemolyticus isolates showed high resistance to ampicillin, intermediate resistance to cefuroxime and cefazolin, and were sensitive to the other antibiotics evaluated. The findings of this study may offer valuable insights and theoretical support for enhancing seafood safety measures in Qidong City.

Effect of capsular polysaccharide phase variation on biofilm formation, motility and gene expression in Vibrio vulnificus.

Vibrio vulnificus, a significant marine pathogen, undergoes opaque (Op)-translucent (Tr) colony switching based on whether capsular polysaccharide (CPS) is produced. CPS phase variation is sometime accompanied by genetic variation or down-regulation of particular genes, such as wzb. In addition, CPS prevents biofilm formation and is important to the virulence of V. vulnificus. However, the extent to which there is a difference in gene expression between Tr and Op colonies and the impact of CPS phase variation on other behaviors of V. vulnificus remain unknown. In this work, the data have shown that CPS phase variation of V. vulnificus is affected by incubation time. Tr and Op strains exhibited similar growth rates. However, Tr strains had enhanced biofilm formation capacities but reduced swimming motility compared to Op strains. The RNA-seq assay revealed 488 differentially expressed genes, with 214 downregulated and 274 upregulated genes, between Tr and Op colonies. Genes associated with Tad pili and CPS were downregulated, whereas those involved in flagellum were upregulated, in Tr colonies compared with Op colonies. In addition, 9 putative c-di-GMP metabolism-associated genes and 28 genes encoding putative regulators were significantly differentially expressed, suggesting that CPS phase variation is probably strictly regulated in V. vulnificus. Moreover, 8 genes encoding putative porins were also differentially expressed between the two phenotypic colonies, indicating that bacterial outer membrane was remodeled during CPS phase variation. In brief, this work highlighted the gene expression profiles associated with CPS phase variation, but more studies should be performed to disclose the intrinsic mechanisms in the future.

The effect of environmental calcium on gene expression, biofilm formation and virulence of Vibrio parahaemolyticus.

Calcium (Ca2+) can regulate the swarming motility and virulence of Vibrio parahaemolyticus BB22. However, the effects of Ca2+ on the physiology of V. parahaemolyticus RIMD2210633, whose genomic composition is quite different with that of BB22, have not been investigated. In this study, the results of phenotypic assays showed that the biofilm formation, c-di-GMP production, swimming motility, zebrafish survival rate, cytoxicity against HeLa cells, and adherence activity to HeLa cells of V. parahaemolyticus RIMD2210633 were significantly enhanced by Ca2+. However, Ca2+ had no effect on the growth, swarming motility, capsular polysaccharide (CPS) phase variation and hemolytic activity. The RNA sequencing (RNA-seq) assay disclosed 459 significantly differentially expressed genes (DEGs) in response to Ca2+, including biofilm formation-associated genes and those encode virulence factors and putative regulators. DEGs involved in polar flagellum and T3SS1 were upregulated, whereas majority of those involved in regulatory functions and c-di-GMP metabolism were downregulated. The work helps us understand how Ca2+ affects the behavior and gene expression of V. parahaemolyticus RIMD2210633.

Identification of an LysR family transcriptional regulator that activates motility and flagellar gene expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus utilizes a polar flagellum for swimming in liquids and employs multiple lateral flagella to swarm on surfaces and in viscous environments. The VPA0961 protein is an LysR family transcriptional regulator that can regulate the swimming and swarming motility of V. parahaemolyticus, but the detailed regulatory mechanisms are not yet fully understood. Herein, we designated the protein as AcsS, which stands for activator of swimming and swarming motility. Our data provided evidence that deleting the acsS gene significantly reduced both swimming and swarming motility of V. parahaemolyticus. Furthermore, AcsS was found to activate the expression of both polar (flgA, flgM, flgB, and flgK) and lateral (motY, fliM, lafA, and fliD) flagellar genes. Overexpression of AcsS in Escherichia coli induced the expression of flgA, motY, and lafA, but did not affect the expression of flgB, flgK, flgM, fliM, and fliD. Interestingly, His-tagged AcsS did not bind to the upstream DNA regions of all the tested genes, suggesting indirect regulation. In conclusion, AcsS positively regulated the swimming and swarming motility of V. parahaemolyticus by activating the transcription of polar and lateral flagellar genes. This work enriched our understanding of the gene expression regulation within the dual flagellar systems of V. parahaemolyticus.

IcmF2 of the type VI secretion system 2 plays a role in biofilm formation of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and HeLa cells and for the antibacterial activity of V. parahaemolyticus, while T6SS2 mainly contributes to HeLa cell adhesion. However, it remains unclear whether the T6SS systems have other physiological roles in V. parahaemolyticus. In this study, we demonstrated that the deletion of icmF2, a structural gene of T6SS2, reduced the biofilm formation capacity of V. parahaemolyticus under low salt conditions, which was also influenced by the incubation time. Nonetheless, the deletion of icmF2 did not affect the biofilm formation capacity in marine-like growth conditions, nor did it impact the flagella-driven swimming and swarming motility of V. parahaemolyticus. IcmF2 was found to promote the production of the main components of the biofilm matrix, including extracellular DNA (eDNA) and extracellular proteins, and cyclic di-GMP (c-di-GMP) in V. parahaemolyticus. Additionally, IcmF2 positively influenced the transcription of cpsA, mfpA, and several genes involved in c-di-GMP metabolism, including scrJ, scrL, vopY, tpdA, gefA, and scrG. Conversely, the transcription of scrA was negatively impacted by IcmF2. Therefore, IcmF2-dependent biofilm formation was mediated through its effects on the production of eDNA, extracellular proteins, and c-di-GMP, as well as its impact on the transcription of cpsA, mfpA, and genes associated with c-di-GMP metabolism. This study confirmed new physiological roles for IcmF2 in promoting biofilm formation and c-di-GMP production in V. parahaemolyticus.

Environmental magnesium ion affects global gene expression, motility, biofilm formation and virulence of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is widely distributed in marine ecosystems. Magnesium ion (Mg2+) is the second most abundant metal cation in seawater, and plays important roles in the growth and gene expression of V. parahaemolyticus, but lacks the detailed mechanisms. In this study, the RNA sequencing data demonstrated that a total of 1494 genes was significantly regulated by Mg2+. The majority of the genes associated with lateral flagella, exopolysaccharide, type III secretion system 2, type VI secretion system (T6SS) 1, T6SS2, and thermostable direct hemolysin were downregulated. A total of 18 genes that may be involved in c-di-GMP metabolism and more than 80 genes encoding putative regulators were also significantly and differentially expressed in response to Mg2+, indicating that the adaptation process to Mg2+ stress may be strictly regulated by complex regulatory networks. In addition, Mg2+ promoted the proliferative speed, swimming motility and cell adhesion of V. parahaemolyticus, but inhibited the swarming motility, biofilm formation, and c-di-GMP production. However, Mg2+ had no effect on the production of capsular polysaccharide and cytoxicity against HeLa cells. Therefore, Mg2+ had a comprehensive impact on the physiology and gene expression of V. parahaemolyticus.

QsvR and OpaR coordinately regulate the transcription of cpsS and cpsR in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, has a strong capacity to form biofilms on surfaces, which is strictly regulated by the CpsS-CpsR-CpsQ regulatory cascade. OpaR, a master regulator of quorum sensing, is a global regulator that controls multiple cellular pathways including biofilm formation and virulence. QsvR is an AraC-type regulator that works coordinately with OpaR to control biofilm formation and virulence gene expression of V. parahaemolyticus. QsvR and OpaR activate cpsQ transcription. OpaR also activates cpsR transcription, but lacks the detailed regulatory mechanisms. Furthermore, it is still unknown whether QsvR regulates cpsR transcription, as well as whether QsvR and OpaR regulate cpsS transcription. In this study, the results of quantitative real-time PCR and LacZ fusion assays demonstrated that deletion of qsvR and/or opaR significantly decreased the expression levels of cpsS and cpsR compared to the wild-type strain. However, the results of two-plasmid lacZ reporter and electrophoretic mobility-shift assays showed that both QsvR and OpaR were unable to bind the regulatory DNA regions of cpsS and cpsR. Therefore, transcription of cpsS and cpsR was coordinately and indirectly activated by QsvR and OpaR. This work enriched our knowledge on the regulatory network of biofilm formation in V. parahaemolyticus.

Complete genome sequence and comparative analysis of a Vibrio vulnificus strain isolated from a clinical patient.

Vibrio vulnificus is an opportunistic, global pathogen that naturally inhabits sea water and is responsible for most vibriosis-related deaths. We investigated the genetic characteristics of V. vulnificus isolated from the clinical blood culture specimen of a patient with hepatitis B virus cirrhosis in 2018 (named as V. vulnificus VV2018) by whole genome sequencing (WGS). VV2018 belonged to a novel sequencing type 620 (ST620) and comprised two circular chromosomes, containing 4,389 potential coding sequences (CDSs) and 152 RNA genes. The phylogenetic tree of single nucleotide polymorphisms (SNPs) using 26 representative genomes revealed that VV2108 grouped with two other V. vulnificus strains isolated from humans. The pan-genome of V. vulnificus was constructed using 26 representative genomes to elucidate their genetic diversity, evolutionary characteristics, and virulence and antibiotic resistance profiles. The pan-genome analysis revealed that VV2018 shared a total of 3,016 core genes (≥99% presence), including 115 core virulence factors (VFs) and 5 core antibiotic resistance-related genes, and 309 soft core genes (≥95 and <99% presence) with 25 other V. vulnificus strains. The varG gene might account for the cefazolin resistance, and comparative analysis of the genetic context of varG revealed that two genes upstream and downstream of varG were conserved. The glycosylation (pgl) like genes were found in VV2018 compared with Pgl-related proteins in Neisseria that might affect the adherence of the strain in hosts. The comparative analysis of VV2018 would contribute to a better understanding of the virulence and antibiotic resistance profiles of V. vulnificus. Meanwhile much work remains to be done to better understand the function of pgl-like genes in V. vulnificus.

The phase variation between wrinkly and smooth colony phenotype affects the virulence of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the causative agent of seafood-associated gastroenteritis, undergoes wrinkly and smooth colony switching on the plate. The wrinkly spreader grew faster, had stronger motility and biofilm capacity when compared with the smooth one. However, whether the two phenotypes differ in their virulence still needs to be further investigated. In this study, the data showed that the smooth spreader had stronger virulence phenotypes, including the cytotoxicity against HeLa cells, antibacterial activity against E. coli, adhesive capacity toward HeLa cells, and lethality in zebrafish, relative to the wrinkly one. However, the colony morphology variation had no influence on the haemolytic activity. The mRNA levels of major virulence genes including T3SS1, T6SS1, and T6SS2 were significantly enhanced in the smooth colonies relative to those in the wrinkly colonies. Taken together, the presented work highlighted the different virulence profiles of the wrinkly and smooth colony phenotypes.

Effect of sublethal dose of chloramphenicol on biofilm formation and virulence in Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates are generally very sensitive to chloramphenicol. However, it is usually necessary to transfer a plasmid carrying a chloramphenicol resistance gene into V. parahaemolyticus to investigate the function of a specific gene, and the effects of chloramphenicol on bacterial physiology have not been investigated. In this work, the effects of sublethal dose of chloramphenicol on V. parahaemolyticus were investigated by combined utilization of various phenotypic assays and RNA sequencing (RNA-seq). The results showed that the growth rate, biofilm formation capcity, c-di-GMP synthesis, motility, cytoxicity and adherence activity of V. parahaemolyticus were remarkably downregulated by the sublethal dose of chloramphenicol. The RNA-seq data revealed that the expression levels of 650 genes were significantly differentially expressed in the response to chloramphenicol stress, including antibiotic resistance genes, major virulence genes, biofilm-associated genes and putative regulatory genes. Majority of genes involved in the synthesis of polar flagellum, exopolysaccharide (EPS), mannose-sensitive haemagglutinin type IV pilus (MSHA), type III secretion systems (T3SS1 and T3SS2) and type VI secretion system 2 (T6SS2) were downregulated by the sublethal dose of chloramphenicol. Five putative c-di-GMP metabolism genes were significantly differentially expressed, which may be the reason for the decrease in intracellular c-di-GMP levels in the response of chloramphenicol stress. In addition, 23 genes encoding putative regulators were also significantly differentially expressed, suggesting that these regulators may be involved in the resistance of V. parahaemolyticus to chloramphenicol stress. This work helps us to understand how chloramphenicol effect on the physiology of V. parahaemolyticus.

Transcriptomic Profiles of Vibrio parahaemolyticus During Biofilm Formation.

Vibrio parahaemolyticus, the leading cause of bacterial seafood-associated gastroenteritis, can form biofilms. In this work, the gene expression profiles of V. parahaemolyticus during biofilm formation were investigated by transcriptome sequencing. A total of 183, 503, and 729 genes were significantly differentially expressed in the bacterial cells at 12, 24 and 48 h, respectively, compared with that at 6 h. Of these, 92 genes were consistently activated or repressed from 6 to 48 h. The genes involved in polar flagellum, chemotaxis, mannose-sensitive haemagglutinin type IV pili, capsular polysaccharide, type III secretion system 1 (T3SS1), T3SS2, thermostable direct hemolysin (TDH), type VI secretion system 1 (T6SS1) and T6SS2 were downregulated, whereas those involved in V. parahaemolyticus pathogenicity island (Vp-PAI) (except for T3SS2 and TDH) and membrane fusion proteins were upregulated. Three extracellular protease genes (vppC, prtA and VPA1071) and a dozen of outer membrane protein encoding genes were also significantly differentially expressed during biofilm formation. In addition, five putative c-di-GMP metabolism-associated genes were significantly differentially expressed, which may account for the drop in c-di-GMP levels after the beginning of biofilm formation. Moreover, many putative regulatory genes were significantly differentially expressed, and more than 1000 putative small non-coding RNAs were detected, suggesting that biofilm formation was tightly regulated by complex regulatory networks. The data provided a global view of gene expression profiles during biofilm formation, showing that the significantly differentially expressed genes were involved in multiple cellular pathways, including virulence, biofilm formation, metabolism, and regulation.

L-arabinose affects the growth, biofilm formation, motility, c-di-GMP metabolism, and global gene expression of Vibrio parahaemolyticus.

The L-arabinose inducible pBAD vectors are commonly used to turn on and off the expression of specific genes in bacteria. The utilization of certain carbohydrates can influence bacterial growth, virulence factor production, and biofilm formation. Vibrio parahaemolyticus, the causative agent of seafood-associated gastroenteritis, can grow in media with L-arabinose as the sole carbon source. However, the effects of L-arabinose on V. parahaemolyticus physiology have not been investigated. In this study, we show that the growth rate, biofilm formation capacity, capsular polysaccharide production, motility, and c-di-GMP production of V. parahaemolyticus are negatively affected by L-arabinose. RNA-seq data revealed significant changes in the expression levels of 752 genes, accounting for approximately 15.6% of V. parahaemolyticus genes in the presence of L-arabinose. The affected genes included those associated with L-arabinose utilization, major virulence genes, known key biofilm-related genes, and numerous regulatory genes. In the majority of type III secretion system, two genes were upregulated in the presence of L-arabinose, whereas in those of type VI secretion system, two genes were downregulated. Ten putative c-di-GMP metabolism-associated genes were also significantly differentially expressed, which may account for the reduced c-di-GMP levels in the presence of L-arabinose. Most importantly, almost 40 putative regulators were significantly differentially expressed due to the induction by L-arabinose, indicating that the utilization of L-arabinose is strictly regulated by regulatory networks in V. parahaemolyticus. The findings increase the understanding of how L-arabinose affects the physiology of V. parahaemolyticus. Researchers should use caution when considering the use of L-arabinose inducible pBAD vectors in V. parahaemolyticus. IMPORTANCE The data in this study show that L-arabinose negatively affects the growth rate, biofilm formation, capsular polysaccharide production, motility, and c-di-GMP production of V. parahaemolyticus. The data also clarify the gene expression profiles of the bacterium in the presence of L-arabinose. Significantly differentially expressed genes in response to L-arabinose were involved in multiple cellular pathways, including L-arabinose utilization, virulence factor production, biofilm formation, motility, adaptation, and regulation. The collective findings indicate the significant impact of L-arabinose on the physiology of V. parahaemolyticus. There may be similar effects on other species of bacteria. Necessary controls should be established when pBAD vectors must be used for ectopic gene expression.

Vibrio vulnificus septicemia in a hospitalized patient with hepatitis B virus-associated cirrhosis: A case report.

Vibrio vulnificus is usually transmitted by consumption of raw or undercooked seafood or exposure to seawater and can causes gastroenteritis, wound infection, and even sepsis. However, atypical or unclear sources of V. vulnificus infection have been reported. Here, we report a case of V. vulnificus infection presenting as septicemia in a 53-year-old man with hepatitis B virus-associated cirrhosis. The source of infection remained unclear as the patient reported no consumption of seafood or contact with seawater. Treatment with antibiotics was initiated prior to confirmation of V. vulnificus infection. This report provides an important reference for the diagnosis and treatment of V. vulnificus infection.

Rapid point-of-care detection of BK virus in urine by an HFman probe-based loop-mediated isothermal amplification assay and a finger-driven microfluidic chip.

Background: BK virus (BKV)-associated nephropathy (BKVN) is one of the leading causes of renal dysfunction and graft loss in renal transplant recipients. Early monitoring of BKV in urine is crucial to minimize the deleterious effects caused by this virus on preservation of graft function. Methods: We report a simple, rapid, sensitive loop-mediated isothermal amplification (LAMP) assay using an HFman probe for detecting BKV in urine. To evaluate the performance of the assay, a comparison of the HFman probe-based LAMP (HF-LAMP) assay with two qPCR assays was performed using urine samples from 132 HIV-1 infected individuals. We further evaluated the performance of HF-LAMP directly using the urine samples from these HIV-1 infected individuals and 30 kidney transplant recipients without DNA extraction. Furthermore, we combined the HF-LAMP assay with a portable finger-driven microfluidic chip for point-of-care testing (POCT). Results: The assay has high specificity and sensitivity with a limit of detection (LOD) of 12 copies/reaction and can be completed within 30 min. When the DNA was extracted, the HF-LAMP assay showed an equivalent and potentially even higher sensitivity (93.5%) than the qPCR assays (74.2-87.1%) for 132 urine samples from HIV-1 infected individuals. The HF-LAMP assay can be applied in an extraction-free format and can be completed within 45 min using a simple heat block. Although some decreased performance was seen on urine samples from HIV-1 infected individuals, the sensitivity, specificity, and accuracy of the extraction-free BKV HF-LAMP assay were 95%, 100%, and 96.7% for 30 clinical urine samples from kidney transplant recipients, respectively. Conclusion: The assay has high specificity and sensitivity. Combined with a portable finger-driven microfluidic chip for easy detection, this method shows great potential for POCT detection of BKV.

Quorum sensing and QsvR tightly control the transcription of vpa0607 encoding an active RNase II-type protein in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a Gram-negative, halophilic bacterium, is a leading cause of acute gastroenteritis in humans. AphA and OpaR are the master quorum sensing (QS) regulators operating at low cell density (LCD) and high cell density (HCD), respectively. QsvR is an AraC-type protein that integrates into the QS system to control gene expression by directly controlling the transcription of aphA and opaR. However, the regulation of QsvR itself remains unclear to date. In this study, we show that vpa0607 and qsvR are transcribed as an operon, vpa0607-qsvR. AphA indirectly activates the transcription of vpa0607 at LCD, whereas OpaR and QsvR directly repress vpa0607 transcription at HCD, leading to the highest expression levels of vpa0607 occurs at LCD. Moreover, VPA0607 acts as an active RNase II-type protein in V. parahaemolyticus and feedback inhibits the expression of QsvR at the post-transcriptional level. Taken together, this work deepens our understanding of the regulation of QsvR and enriches the integration mechanisms of QsvR with the QS system in V. parahaemolyticus.

QsvR and OpaR coordinately repress biofilm formation by Vibrio parahaemolyticus.

Mature biofilm formation by Vibrio parahaemolyticus requires exopolysaccharide (EPS), type IV pili, and capsular polysaccharide (CPS). Production of each is strictly regulated by various control pathways including quorum sensing (QS) and bis-(3'-5')-cyclic di-GMP (c-di-GMP). QsvR, an AraC-type regulator, integrates into the QS regulatory cascade via direct control of the transcription of the master QS regulators, AphA and OpaR. Deletion of qsvR in wild-type or opaR mutant backgrounds altered the biofilm formation by V. parahaemolyticus, suggesting that QsvR may coordinate with OpaR to control biofilm formation. Herein, we demonstrated both QsvR and OpaR repressed biofilm-associated phenotypes, c-di-GMP metabolism, and the formation of V. parahaemolyticus translucent (TR) colonies. QsvR restored the biofilm-associated phenotypic changes caused by opaR mutation, and vice versa. In addition, QsvR and OpaR worked coordinately to regulate the transcription of EPS-associated genes, type IV pili genes, CPS genes and c-di-GMP metabolism-related genes. These results demonstrated how QsvR works with the QS system to regulate biofilm formation by precisely controlling the transcription of multiple biofilm formation-associated genes in V. parahaemolyticus.