-
Journal of Applied Microbiology Sep 2022We aimed at developing a fast and accurate method to detect Vibrio mimicus using real-time recombinase polymerase amplification assay.
AIM
We aimed at developing a fast and accurate method to detect Vibrio mimicus using real-time recombinase polymerase amplification assay.
METHODS AND RESULTS
Specific primers and probe were designed to target V. mimicus haemolysin (vmh) gene. Target DNA was successfully amplified at 41°C within 20 min. The method exhibited a high level of specificity and the sensitivity was 2.1 × 10 copies/25 μl or 8.4 copies/μl, which is in line with real-time polymerase chain reaction (PCR). The calibration curve plotted by the second-order polynomial regression showed better than the linear curve, as the correlation coefficient was raised to 0.9907, which suggested that the second-order polynomial regressions might be considered to apply to the quantification of real-time recombinase polymerase amplification (RPA). The limit of detection (LOD) was predicted to be 77 copies/25 μl or 3 copies/μl by a probit model. The limit of quantification (LOQ) was calculated to be 28 copies /25 μl or 1 copies/μl by a receiver operating characteristic (ROC) curve, which firstly make LOQ could be available to real-time RPA. For the performance of the real-time RPA in plasma samples, the detection sensitivity of real-time RPA was as good as the real-time PCR. For pretreatment of plasma samples, the boiling method was better than using kits, as it further shortened the time of the real-time RPA in detecting V. mimicus.
CONCLUSIONS
The real-time RPA assay developed in our study shows multiple advantages over currently available DNA diagnostic method, including a quicker time-to-result for a single sample, requiring minimal infrastructure and technical support and being tolerant to inhibitors in plasma samples.
SIGNIFICANCE AND IMPACT OF THE STUDY
The real-time RPA assay developed here is a potentially valuable tool for point-of-care (POC) diagnosis of V. mimicus infection in endemic field, especially in the resources-limited settings, as combined with portable devices.
Topics: DNA Primers; Humans; Limit of Detection; Nucleic Acid Amplification Techniques; Real-Time Polymerase Chain Reaction; Recombinases; Sensitivity and Specificity
PubMed: 35702884
DOI: 10.1111/jam.15666 -
Frontiers in Microbiology 2022The potentially pathogenic species of the genus pose a threat to both humans and animals, creating medical burdens and economic losses to the mariculture industry....
The potentially pathogenic species of the genus pose a threat to both humans and animals, creating medical burdens and economic losses to the mariculture industry. Improvements in surveillance and diagnosis are needed to successfully manage vibriosis outbreaks. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can provide rapid diagnosis and has been widely used in the identification of spp. The main weakness of this technology is the limited number of strains and species of in the existing commercial database. Here, we develop a new in-house database named PVBase containing 790 main spectra projections (MSP) of ten species that come from various regions of China and include abundant clinical and environmental strains. PVBase was validated through a blind test of 65 strains. The identification accuracy and scoring of strains was greatly improved through the addition of PVBase. Identification accuracy increased from 73.4 to 100%. The number of strains with identification scores above 2.2 increased from 53.1% to 96.9% and 53.1% of strains had an identification score above 2.59. Moreover, perfect discrimination was obtained when using all of the MSPs created for the species, even for very closely related species such as , , and or , , and . In addition, we used phyloproteomic analysis to study whether there are differences in protein fingerprints of different regions or pathogenic strains. We found that MSP characteristics of species were not related to their region or source. With the construction of PVBase, the identification efficiency of potentially pathogenic species has been greatly improved, which is an important advance for epidemic prevention and control, and aquaculture disease detection.
PubMed: 35656002
DOI: 10.3389/fmicb.2022.872825 -
Frontiers in Microbiology 2022The study aimed to determine the prevalence of different species of spp. in fish and shellfish sold in subtropical-arid countries (United Arab Emirates). It also...
The study aimed to determine the prevalence of different species of spp. in fish and shellfish sold in subtropical-arid countries (United Arab Emirates). It also examined the antimicrobial resistance of the isolated species and their growth behavior upon environmental changes concerning temperature, pH, and salinity. The prevalence of spp. in fish and shellfish samples, was 64.5 and 92%, respectively. However, were detected in a mere 7.5 and 13.0% of the samples, respectively. On the other hand, was detected in 1.5 and 8.5% of the samples, respectively. None of the six antibiotics studied except for Sulfamethoxazole-trimethoprim were effective against fish spp. isolates. On a similar note, three antibiotics, namely Penicillin, Daptomycin, and Vancomycin, were ineffective against the shellfish isolates. The growth of the microorganisms did not show any significant trend with changes in pH and salinity. The optimum temperature for spp. growth was observed to be 37°C.
PubMed: 35464960
DOI: 10.3389/fmicb.2022.861547 -
Frontiers in Microbiology 2022species are important pathogens of marine animals and aquaculture populations and some of them can cause serious infections in humans through consumption of...
species are important pathogens of marine animals and aquaculture populations and some of them can cause serious infections in humans through consumption of contaminated seafood and aquaculture products. Lytic bacteriophages can potentially alleviate contamination in the aquaculture organisms and in the processing of aquatic products and have gained significant scientific attention in recent years. In the present study, bacteriophages were isolated from sewage of local aquatic products markets and grown using CICC 21613 as host cells. The lytic vibriophage OY1 belonging to the newly proposed family and the genus was identified by observation under electron microscope and comparative genomic analysis. The phage OY1 showed lytic activity against 24 among 32 tested strains belonging to eight species. The complete phage OY1 genome consists of a single circular double-stranded DNA of 43,479 bp with a total GC content of 49.27% and was predicted to encode 40 open reading frames (ORFs). To evaluate its potential against vibrios, the one-step growth curve, thermal and pH stability, host range, and lytic activity of the OY1 phage against species were evaluated. The results showed that phage OY1 had a range of thermal and pH tolerance, and exhibited a significant inhibitory effect on the growth of tested species. Bacterial growth in the fish muscle extract juice (FMEJ) inoculated with CICC 21613, CICC 21617, VJ14, and the mixed bacterial culture was reduced by 2.65 log CFU/ml, 2.42 log CFU/ml, 1.93 log CFU/ml, and 2.01 log CFU/ml, respectively, by incubation with phage OY1 at 25°C for 36 h. Phage OY1 also showed a strong ability to prevent biofilm formation and destroy formed species biofilms. These results indicate that phage OY1 is a potential biocontrol agent against species in the aquaculture industry and in food safety control.
PubMed: 35464931
DOI: 10.3389/fmicb.2022.830692 -
Frontiers in Microbiology 2022Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in...
Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in almost every aspect of metabolism, of both eukaryotes and prokaryotes. Several global lysine acetylome studies have been carried out in various bacteria, but thus far, there have been no reports of lysine acetylation for the commercially important aquatic animal pathogen In the present study, we used anti-Ac-K antibody beads to highly sensitive immune-affinity purification and combined high-resolution LC-MS/MS to perform the first global lysine acetylome analysis in , leading to the identification of 1,097 lysine-acetylated sites on 582 proteins, and more than half (58.4%) of the acetylated proteins had only one site. The analysis of acetylated modified peptide motifs revealed six significantly enriched motifs, namely, KacL, KacR, L(-2) KacL, LKacK, L(-7) EKac, and IEKac. In addition, bioinformatic assessments state clearly that acetylated proteins have a hand in many important biological processes in , such as purine metabolism, ribosome, pyruvate metabolism, glycolysis/gluconeogenesis, the TCA cycle, and so on. Moreover, 13 acetylated proteins were related to the virulence of . To sum up, this is a comprehensive analysis whole situation protein lysine acetylome in and provides an important foundation for in-depth study of the biological function of lysine acetylation in .
PubMed: 35250932
DOI: 10.3389/fmicb.2022.816968 -
International Maritime Health 2021Vibrio infections are becoming more frequent in the Baltic Sea region, which is caused by an increase in the sea surface temperature. Climate change creates the...
BACKGROUND
Vibrio infections are becoming more frequent in the Baltic Sea region, which is caused by an increase in the sea surface temperature. Climate change creates the conditions for the emergence of new environmental niches that are beneficial for Vibrio spp., especially in the summer months. Vibrio vulnificus, which causes wound infections and septicaemia, represents a particularly dangerous species of Vibrio spp. There are numerous publications on the prevalence of V. vulnificus in various regions of the Baltic Sea, but there is a lack of such data for the Polish coast. This prompted us to conduct a pilot study into the prevalence of the bacteria in the Gulf of Gdansk. The study aimed to detect Vibrio spp. in the coastal waters and the wet sand at the beaches and bathing areas in the Gulf of Gdansk.
MATERIALS AND METHODS
During the period from June 16th to September 23rd 2020, 112 samples of seawater and 105 samples of wet sand were collected at 16 locations along the coast of the Gulf of Gdansk and Hel peninsula. Isolation of Vibrio spp. was conducted by filtering method and the isolated bacteria was cultured on CHROM agar Vibrio and TCBS agar. Final genus identification was performed by the MALDI TOF technique.
RESULTS
In the present study, 10 isolates of Vibrio spp. were obtained from seawater and wet sand samples collected in the Gulf of Gdansk and Hel peninsula coast. Three of the isolates were identified as V. vulnificus; the presence of the species was confirmed in the seawater samples which had been collected in Hel (1 isolate), Jastarnia (1 isolate), and Chalupy (1 isolate). One strain of Vibrio alginolyticus was isolated from the seawater sample collected in Hel. Moreover, identification was incomplete for 6 of the isolated strains, these were identified as Vibrio cholerae/mimicus These strains were collected in Jastarnia (1 isolate), Kuznica (1 isolate), Gdansk-Brzezno (1 isolate), Puck (2 isolates), Chalupy (1 isolate).
CONCLUSIONS
Our preliminary research study confirmed the presence of potentially pathogenic V. vulnificus in the Gulf of Gdansk in the summer months. Therefore, further monitoring of the presence of Vibrio spp. in the Baltic coast area is necessary.
Topics: Agar; Humans; Pilot Projects; Sand; Seawater; Vibrio; Vibrio vulnificus
PubMed: 35147210
DOI: 10.5603/IMH.2021.0048 -
Fish & Shellfish Immunology Mar 2022Vibrio mimicus (V. mimicus) is a pathogen causing serious vibriosis in aquatic animals. Hepcidin and β-Defensin1 are two important antibacterial peptides (AMPs) with...
Vibrio mimicus (V. mimicus) is a pathogen causing serious vibriosis in aquatic animals. Hepcidin and β-Defensin1 are two important antibacterial peptides (AMPs) with broad-spectrum antibacterial activity in fish. In mammals, some evidences demonstrated that interleukin-1β (IL-1β) primarily promote AMPs expression via activating classical NF-κB pathway, but it still remains unclear in fish. Here, the temporal and spatial expression patterns of grass carp IL-1β (gcIL-1β) gene and two AMPs genes (gchepcidin and gcβ-defensin1) in tissues post-V. mimicus infection and anti-V. mimicus activity of these two AMPs in vitro were detected, showing that V. mimicus infection significantly elevated the mRNA levels of these three genes in the immune-related tissues although their expression patterns were not entirely consistent, and both gcHepcidin and gcβ-Defensin1 possessed anti-V. mimicus activity in vitro. Subsequently, the recombinant gcIL-1β (rgcIL-1β) was expressed prokaryotically in an inclusion body, which could promote proliferation of grass carp head kidney leukocytes (gcHKLs) and enhance respiratory burst activity and phagocytic activity of head kidney macrophages. Stimulation with rgcIL-1β was able to significantly regulate the mRNA expression of key regulatory genes (il-1RI, traf6, tak1, ikkβ, iκBα and p65) involved in the activation of classical NF-κB pathway, and then induce gcTAK1 phosphorylation, promote gcp65 nuclear translocation and enhance endogenous gcIL-1β expression at both mRNA and protein levels, implying NF-κB pathway was activated. More importantly, exogenous rgcIL-1β stimulation also significantly up-regulated both gcHepcidin and gcβ-Defensin1 mRNA levels against V. mimicus, and the regulatory effect was blocked or inhibited by NF-κB inhibitor PDTC. Taken together, our results demonstrated for the first time that grass carp IL-1β stimulation could significantly enhance the expression of these two anti-V.mimicus AMPs via activating classical NF-κB pathway.
Topics: Animals; Antimicrobial Peptides; Carps; Fish Diseases; Fish Proteins; Interleukin-1beta; Mammals; NF-kappa B; Signal Transduction; Vibrio mimicus
PubMed: 34922017
DOI: 10.1016/j.fsi.2021.12.020 -
Scientific Reports Nov 2021Adequate water supply is one of the public health issues among the population living in low-income settings. Vibriosis remain a significant health challenge drawing the...
Adequate water supply is one of the public health issues among the population living in low-income settings. Vibriosis remain a significant health challenge drawing the attention of both healthcare planners and researchers in South West districts of Uganda. Intending to clamp down the disease cases in the safest water deprive locality, we investigated the virulent toxins as contaminants and epidemiologic potentials of Vibrio species recovered from surface waters in greater Bushenyi districts, Uganda. Surface water sources within 46 villages located in the study districts were obtained between June and October 2018. Standard microbiological and molecular methods were used to analyse samples. Our results showed that 981 presumptive isolates retrieved cell counts of 10-100 CFU/g, with, with (640) 65% confirmed as Vibrio genus using polymerase chain reaction, which is distributed as follows; V. vulnificus 46/640 (7.2%), V. fluvialis 30/594 (5.1), V. parahaemolyticus 21/564 (3.7), V. cholera 5/543 (0.9), V. alginolyticus 62/538 (11.5) and V. mimicus 20/476 (4.2). The virulence toxins observed were heat-stable enterotoxin (stn) 46 (82.10%), V. vulnificus virulence gene (vcgCPI) 40 (87.00%), extracellular haemolysin gene {vfh 21 (70.00)} and Heme utilization protein gene {hupO 5 (16.70)}. The cluster analysis depicts hupO (4.46% n = 112); vfh (18.75%, n = 112); vcgCPI and stn (35.71%, & 41.07%, n = 112). The principal component analysis revealed the toxins (hupO, vfh) were correlated with the isolate recovered from Bohole water (BW) source, while (vcgCPI, stn) toxins are correlated with natural raw water (NRW) and open springs (OS) water sources isolates. Such observation indicates that surface waters sources are highly contaminated with an odds ratio of 1.00, 95% CI (70.48-90.5), attributed risk of (aR = 64.29) and relative risk of (RR = 73.91). In addition, it also implies that the surface waters sources have > 1 risk of contamination with vfh and > six times of contamination with hupO (aR = 40, - 66). This is a call of utmost importance to the population, which depends on these water sources to undertake appropriate sanitation, personal hygienic practices and potential measures that ensure water quality.
Topics: Bacterial Toxins; DNA, Bacterial; Genes, Bacterial; Hemolysin Proteins; Natural Springs; Polymerase Chain Reaction; Uganda; Vibrio; Vibrio Infections; Virulence; Virulence Factors
PubMed: 34789791
DOI: 10.1038/s41598-021-01375-3 -
Frontiers in Cellular and Infection... 2021The study investigated the occurrence of antimicrobial resistance genes and virulence determinants in species recovered from different freshwater sheds in rustic...
The study investigated the occurrence of antimicrobial resistance genes and virulence determinants in species recovered from different freshwater sheds in rustic milieu. A total of 118 isolates comprising (n=41), (n=40) and (n=37) was identified by amplification of , and genes. The amplification of virulence genes indicated that . (, , , , and ) genes were detected in 12.5%, 32.5%, 45%, 37.5% and 10% respectively. . genes (, and ) were harboured in 48.8%, 14.6% and 19.5% isolates congruently. The other virulence genes that include and were observed in 63.1% and 29% of isolates belonging to . . With the exceptions of imipenem, meropenem and ciprofloxacin, most isolates exhibited more than 50% resistance to antibiotics. The antimicrobial resistance was more prevalent for polymyxin B (100%), azithromycin (100%) and least in ciprofloxacin (16.1%). Multiple antibiotic resistance index range was 0.3 and 0.8 with most isolates showing MARI of 0.8. The TEM, AmpC, GES, IMP, OXA-48 and KPC genes were detected in 53.3%, 42%, 29.6%, 16.6%, 15%, 11.3% and 5.6% of the isolates. Non-beta lactamases such as streptomycin resistance ( and ), gentamicin resistance () and quinolone resistance gene () were found in 5.2%, 44.3%, 26% and 2.8%. Chloramphenicol resistance genes ( and ) were found in 5.2% and 44.3% among the isolates. Our findings reveal the presence of antimicrobial resistance genes and virulent species in aquatic environment which can have potential risk to human and animal's health.
Topics: Animals; Anti-Bacterial Agents; Drug Resistance, Microbial; Fresh Water; Humans; Microbial Sensitivity Tests; Vibrio; Virulence
PubMed: 34490150
DOI: 10.3389/fcimb.2021.732001 -
MSphere Aug 2021Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a...
Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a specialized protein secretion machinery known as the type III secretion system 2 (T3SS2) to cause disease. The T3SS2 mediates the delivery of effector proteins into the cytosol of infected cells, where they subvert multiple cellular pathways. Here, we identify a new T3SS2 effector protein encoded by VPA1328 (VP_RS21530) in V. parahaemolyticus RIMD2210633. Bioinformatic analysis revealed that VPA1328 is part of a larger family of uncharacterized T3SS effector proteins with homology to the VopG effector protein in Vibrio cholerae AM-19226. These VopG-like proteins are found in many but not all T3SS2 gene clusters and are distributed among diverse species, including V. parahaemolyticus, V. cholerae, V. mimicus, and V. diabolicus and also in Shewanella baltica. Structure-based prediction analyses uncovered the presence of a conserved C-terminal kinase domain in VopG orthologs, similar to the serine/threonine kinase domain found in the NleH family of T3SS effector proteins. However, in contrast to NleH effector proteins, in tissue culture-based infections, VopG did not impede host cell death or suppress interleukin 8 (IL-8) secretion, suggesting a yet undefined role for VopG during V. parahaemolyticus infection. Collectively, our work reveals that VopG effector proteins, a new family of likely serine/threonine kinases, is widely distributed in the T3SS2 effector armamentarium among marine bacteria. Vibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function. In this study, we identified a novel Vibrio parahaemolyticus effector protein that is similar to the VopG effector of Vibrio cholerae. VopG-like effectors were found in diverse species and contain a conserved serine/threonine kinase domain that bears similarity to the kinase domain in the enterohemorrhagic Escherichia coli (EHEC) and NleH effectors that manipulate host cell survival pathways and host immune responses. Together our findings identify a new family of effector proteins and highlight the role of horizontal gene transfer events among marine bacteria in shaping T3SS gene clusters.
Topics: Bacterial Proteins; Caco-2 Cells; Computational Biology; Gene Expression Regulation, Bacterial; Humans; Interleukin-8; Multigene Family; Protein Serine-Threonine Kinases; Protein Transport; Serine; Type III Secretion Systems; Vibrio parahaemolyticus
PubMed: 34346702
DOI: 10.1128/mSphere.00599-21