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Microorganisms Mar 2024With rising infection rates in recent years, poses an increasing threat to public safety in the coastal brackish Baltic Sea. It is therefore important to monitor this...
With rising infection rates in recent years, poses an increasing threat to public safety in the coastal brackish Baltic Sea. It is therefore important to monitor this organism and assess the infection risk on a more regular basis. However, as the coastline of the Baltic Sea is 8000 km long and shared by nine nations, a convenient, fast, inexpensive, yet efficient identification method is essential. We evaluated the effectiveness of a two-step agar-based approach consisting of successive isolation and cultivation on thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagarâ„¢ for in comparison with , , and . Our study contains isolates from water and sediment across a broad expanse of the Baltic Sea including 13 locations and two different summers, the time of year during which infections are usually much more frequent. Confirmation of isolate species identity was carried out using molecular analyses. The two-step agar plating method performed well across different locations and timeframes in correctly identifying by more than 80%, but the sensitivity in other species varied. Thus, our approach yielded promising results as a potential tool for early detection across a broad timeframe and transect of the Baltic Sea and potentially other brackish environments.
PubMed: 38543665
DOI: 10.3390/microorganisms12030614 -
Frontiers in Cellular and Infection... 2023Our patient, a 48-year-old man from Guangdong's coastal region, worked selling and processing oysters and other seafood. He started experiencing swelling and pain in his...
Our patient, a 48-year-old man from Guangdong's coastal region, worked selling and processing oysters and other seafood. He started experiencing swelling and pain in his left knee on October 4, 2022, and they got worse over time. The findings of mNGS test showed Vibrio vulnificus infection. The patient had AIDS, hepatitis A and hepatitis B concurrently. He was admitted to the hospital's intensive care unit (ICU) for treatment as his symptoms worsened. We refrained from performing an amputation because the family members expressed a desire to keep the limb. The limb was managed with regular dressing changes, thorough debridement, wound closure, ongoing VSD drainage, and local antibiotic irrigation. The patient's organ function eventually returned to normal, and the systemic infection got better. On November 1, the wound's new granulation tissue had grown well and had gradually crept to cover 80% of the wound. The tissue's blood flow had also improved, indicating a trend of growth and healing.
Topics: Male; Humans; Middle Aged; Vibrio vulnificus; Coinfection; Vibrio Infections; Hepatitis B; Hepatitis C; HIV Infections
PubMed: 38146312
DOI: 10.3389/fcimb.2023.1290636 -
The Veterinary Quarterly Dec 2023an opportunistic human pathogen native to marine/estuarine environment, is one of the leading causes of death due to seafood consumption and exposure of wounds to...
an opportunistic human pathogen native to marine/estuarine environment, is one of the leading causes of death due to seafood consumption and exposure of wounds to seawater worldwide. The present study involves the whole genome sequence analysis of an environmental strain of (clinical genotype) isolated from seafood along the Mangaluru coast of India. The sequenced genome data was subjected to analysis of phylogeny, virulence genes, antimicrobial resistance determinants, and secretary proteins using suitable bioinformatics tools. The sequenced isolate had an overall genome length of 4.8 Mb and GC content of 46% with 4400 coding DNA sequences. The sequenced strain belongs to a new sequence type (Multilocus sequence typing) and was also found to branch with a phylogenetic lineage that groups the most infectious strains of . The seafood isolate had complete genes involved in conferring serum resistance yet showed limited serum resistance. The study identified several genes against the antibiotics that are commonly used in their treatment, highlighting the need for alternative treatments. Also, the secretory protein analysis revealed genes associated with major pathways like ABC transporters, two-component systems, quorum sensing, biofilm formation, cationic antimicrobial peptide (CAMP) resistance, and others that play a critical role in the pathogenesis of the . To the best of our knowledge, this is the first report of a detailed analysis of the genomic information of a isolated from the Indian subcontinent and provides evidence that raises public health concerns about the safety of seafood.
Topics: Humans; Animals; Vibrio vulnificus; Virulence; Phylogeny; Genotype; Seafood
PubMed: 37478018
DOI: 10.1080/01652176.2023.2240389 -
Italian Journal of Food Safety Feb 2024The majority of human diseases attributed to seafood are caused by spp., and the most commonly reported species are , , and . The conventional methods for the detection...
Detection of pathogenic spp. in foods: polymerase chain reaction-based screening strategy to rapidly detect pathogenic , , and in bivalve mollusks and preliminary results.
The majority of human diseases attributed to seafood are caused by spp., and the most commonly reported species are , , and . The conventional methods for the detection of species involve the use of selective media, which are inexpensive and simple but time-consuming. The present work aimed to develop a rapid method based on the use of multiplex real-time polymerase chain reaction (PCR) to detect , , and in bivalve mollusks. 30 aliquots of bivalve mollusks () were experimentally inoculated with two levels of , , and . ISO 21872-1:2017 was used in parallel for qualitative analysis. The limit of detection of 50% was 7.67 CFU/g for , 0.024 CFU/g for , and 1.36 CFU/g for . For and , the real-time PCR protocol was demonstrated to amplify the pathogens in samples seeded with the lowest and highest levels. The molecular method evaluated showed a concordance rate of 100% with the reference microbiological method. was never detected in samples contaminated with the lowest level, and it was detected in 14 samples (93.33%) seeded with the highest concentration. In conclusion, the developed multiplex real-time PCR proved to be reliable for and Results for are promising, but further analysis is needed. The proposed method could represent a quick monitoring tool and, if used, would allow the implementation of food safety.
PubMed: 38623280
DOI: 10.4081/ijfs.2024.11635 -
Microbiology Spectrum Aug 2023Enteropathogenic bacteria express two-component systems (TCSs) to sense and respond to host environments, developing resistance to host innate immune systems like...
Enteropathogenic bacteria express two-component systems (TCSs) to sense and respond to host environments, developing resistance to host innate immune systems like cationic antimicrobial peptides (CAMPs). Although an opportunistic human pathogen Vibrio vulnificus shows intrinsic resistance to the CAMP-like polymyxin B (PMB), its TCSs responsible for resistance have barely been investigated. Here, a mutant exhibiting a reduced growth rate in the presence of PMB was screened from a random transposon mutant library of V. vulnificus, and response regulator CarR of the CarRS TCS was identified as essential for its PMB resistance. Transcriptome analysis revealed that CarR strongly activates the expression of the , , and operons. In particular, the operon plays a major role in developing the CarR-mediated PMB resistance. Phosphorylation of CarR by the sensor kinase CarS is required for the regulation of its downstream genes, leading to the PMB resistance. Nevertheless, CarR directly binds to specific sequences in the upstream regions of the and operons, regardless of its phosphorylation. Notably, the CarRS TCS alters its own activation state by responding to several environmental stresses, including PMB, divalent cations, bile salts, and pH change. Furthermore, CarR modulates the resistance of V. vulnificus to bile salts and acidic pH among the stresses, as well as PMB. Altogether, this study suggests that the CarRS TCS, in responding to multiple host environmental signals, could provide V. vulnificus with the benefit of surviving within the host by enhancing its optimal fitness during infection. Enteropathogenic bacteria have evolved multiple TCSs to recognize and appropriately respond to host environments. CAMP is one of the inherent host barriers that the pathogens encounter during the course of infection. In this study, the CarRS TCS of V. vulnificus was found to develop resistance to PMB, a CAMP-like antimicrobial peptide, by directly activating the expression of the operon. Although CarR binds to the upstream regions of the and operons regardless of phosphorylation, phosphorylation of CarR is required for the regulation of the operons, resulting in the PMB resistance. Furthermore, the CarRS TCS determines the resistance of V. vulnificus to bile salts and acidic pH by differentially regulating its own activation state in response to these environmental stresses. Altogether, the CarRS TCS responds to multiple host-related signals, and thus could enhance the survival of V. vulnificus within the host, leading to successful infection.
Topics: Humans; Polymyxin B; Vibrio vulnificus; Gene Expression Profiling; Bile Acids and Salts
PubMed: 37289068
DOI: 10.1128/spectrum.00305-23 -
BioRxiv : the Preprint Server For... Jan 2024CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific...
CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible and genes into the genome using Tn transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple species: , and . We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in species to systematically probe gene function and essentiality in various behaviors and native environments.
PubMed: 38293084
DOI: 10.1101/2024.01.17.575898 -
BioRxiv : the Preprint Server For... Feb 2024In species, quorum sensing signaling culminates in the production of a TetR-type master transcription factor collectively called the LuxR/HapR family, which regulates...
UNLABELLED
In species, quorum sensing signaling culminates in the production of a TetR-type master transcription factor collectively called the LuxR/HapR family, which regulates genes required for colonization and infection of host organisms. These proteins possess a solvent accessible putative ligand binding pocket. However, a native ligand has not been identified, and the role of ligand binding in LuxR/HapR function in is unknown. To probe the role of the ligand binding pocket, we utilize the small molecule thiophenesulfonamide inhibitor PTSP (3- henyl-1-( hiophen-2-yl ulfonyl)-1 - yrazole) that we previously showed targets LuxR/HapR proteins. Amino acid conservation in the ligand binding pocket determines the specificity and efficacy of PTSP inhibition across species. Here, we used structure-function analyses to identify PTSP-interacting residues in the ligand binding pocket of SmcR - the LuxR/HapR homolog - that are required for PTSP inhibition of SmcR activity . Forward genetic screening combined with X-ray crystallography structural determination of SmcR bound to PTSP identified substitutions at eight residues that were sufficient to reduce or eliminate PTSP-mediated SmcR inhibition. Small-angle X-ray scattering and computational modeling determined that PTSP drives allosteric unfolding at the N-terminal DNA binding domain. We discovered that SmcR is degraded by the ClpAP protease in the presence of PTSP ; substitution of key PTSP-interacting residues stabilized or increased SmcR levels in the cell. This mechanism of inhibition is observed for all thiophenesulfonamide compounds tested and against other species. We conclude that thiophenesulfonamides specifically bind in the ligand binding pocket of LuxR/HapR proteins, promoting protein degradation and thereby suppressing downstream gene expression, implicating ligand binding as a mediator of LuxR/HapR protein stability and function to govern virulence gene expression in pathogens.
SIGNIFICANCE
LuxR/HapR proteins were discovered in the 1990s as central regulators of quorum sensing gene expression and later discovered to be conserved in all studied species. LuxR/HapR homologs regulate a wide range of genes involved in pathogenesis, including but not limited to genes involved in biofilm production and toxin secretion. As archetypal members of the broad class of TetR-type transcription factors, each LuxR/HapR protein has a predicted ligand binding pocket. However, no ligand has been identified for LuxR/HapR proteins that control their function as regulators. Here, we used LuxR/HapR-specific chemical inhibitors to determine that ligand binding drives proteolytic degradation , the first demonstration of LuxR/HapR function connected to ligand binding for this historical protein family.
PubMed: 38405947
DOI: 10.1101/2024.02.15.580527 -
Foods (Basel, Switzerland) Sep 2023can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic is important for early disease prevention. In this study, a...
can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic is important for early disease prevention. In this study, a novel recombinase-aided amplification (RAA) combined test strip with double T-lines (RAA-TS-DTL) was developed for the rapid detection of in aquatic products. Pathogenic was detected using the virulence gene and the housekeeping gene gene as the dual target of the test strip. The RAA-TS-DTL method showed 100% specificity for , and no cross-reaction was observed with spp. or other bacteria ( = 14). Furthermore, sensitive detection of in oysters was achieved. The LODs of the and genes were 6 CFU/mL and 23 CFU/mL, respectively, which was about five times higher than that of the commercial test strip. The method was validated with spiked samples ( = 60) of fish, shrimp and oyster. The consistency between RAA-TS-DTL and the traditional culture method was 97.9%. In addition, the entire process of detection, including preparation of the sample, could be completed within 50 min. Our results indicated that the developed RAA-TS-DTL was a reliable and useful tool for rapid screening or on-site detection of pathogenic in aquatic products and aquaculture water.
PubMed: 37835259
DOI: 10.3390/foods12193605 -
PloS One 2023Heterogeneity of ribosomal RNA (rRNA) sequences has recently emerged as a mechanism that can lead to subpopulations of specialized ribosomes. Our previous study showed...
Heterogeneity of ribosomal RNA (rRNA) sequences has recently emerged as a mechanism that can lead to subpopulations of specialized ribosomes. Our previous study showed that ribosomes containing highly divergent rRNAs expressed from the rrnI operon (I-ribosomes) can preferentially translate a subset of mRNAs such as hspA and tpiA in the Vibrio vulnificus CMCP6 strain. Here, we explored the functional conservation of I-ribosomes across Vibrio species. Exogenous expression of the rrnI operon in another V. vulnificus strain, MO6-24/O, and in another Vibrio species, V. fischeri (strain MJ11), decreased heat shock susceptibility by upregulating HspA expression. In addition, we provide direct evidence for the preferential synthesis of HspA by I-ribosomes in the V. vulnificus MO6-24/O strain. Furthermore, exogenous expression of rrnI in V. vulnificus MO6-24/O cells led to higher mortality of infected mice when compared to the wild-type (WT) strain and a strain expressing exogenous rrnG, a redundant rRNA gene in the V. vulnificus CMCP6 strain. Our findings suggest that specialized ribosomes bearing heterogeneous rRNAs play a conserved role in translational regulation among Vibrio species. This study shows the functional importance of rRNA heterogeneity in gene expression control by preferential translation of specific mRNAs, providing another layer of specialized ribosome system.
Topics: Mice; Animals; Vibrio; RNA, Ribosomal; Ribosomes; Vibrio vulnificus; Operon
PubMed: 38051731
DOI: 10.1371/journal.pone.0289072 -
Italian Journal of Food Safety May 2024In this study, two Mediterranean coastal lagoons (Lesina and Varano) of southern Italy, located in the north of the Apulia region, were investigated for the presence of...
In this study, two Mediterranean coastal lagoons (Lesina and Varano) of southern Italy, located in the north of the Apulia region, were investigated for the presence of Shiga toxin (STEC) and potentially enteropathogenic species in parallel with norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the presence of potentially pathogenic bacteria and viruses in the water and sediments of these ecosystems. From March 2022 to February 2023, a total of 98 samples were collected: 49 water samples and 49 sediment samples. STEC strains were isolated in three samples (3.1%), of which one (2%) was water ( and positive) and two (4.1%) were sediment (both positive) samples. spp. were detected in twenty samples (20.4%), of which nine were water (18.4%) and eleven were sediment (22.4%) samples. The species detected included , , and . NoV was detected in 25 (25.5%) samples, while none of the water or sediment samples were positive for HAV, HEV, and SARS-CoV-2. The results of this study provide an overview of the presence of potentially pathogenic microorganisms in areas influenced by anthropogenic pressure. Monitoring the circulation of these pathogens could be useful to evaluate the water flowing into the lagoons, in particular discharge waters (, urban, agricultural, and livestock runoff), considering the presence of fish and shellfish farms in these sites.
PubMed: 38887590
DOI: 10.4081/ijfs.2024.12218