Comparative Genomics of Clinical and Environmental Isolates of Vibrio spp. of Colombia: Implications of Traits Associated with Virulence and Resistance

There is widespread concern about the increase in cases of human and animal infections caused by pathogenic Vibrio species due to the emergence of epidemic lineages. In Colombia, active surveillance by the National Institute of Health (INS) has confirmed the presence of Vibrio; however, in routine surveillance, these isolates are not genomically characterized. This study focused on the pangenome analysis of six Vibrio species: V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. fluvialis, V. diabolicus and V. furnissii to determine the genetic architectures of potentially virulent and antimicrobial resistance traits. Isolates from environmental and clinical samples were genome sequenced, assembled and annotated. The most important species in public health were further characterized by multilocus sequence typing and phylogenomics. For V. parahaemolyticus, we found the virulent ST3 and ST120 genotypes. For V. vulnificus, we identified isolates belonging to lineages 1 and 2. Virulence gene homologues between species were found even in non-pathogenic species such as V. diabolicus. Annotations related to the mobilome, integrative mobile and conjugative elements and resistance genes were obtained from environmental and clinical isolates. This study contributes genomic information to the intensified surveillance program implemented by the INS to establish potential sources of vibriosis in Colombia.

Evaluation of probiotic bacteria Leucosnostoc mensenteroides against White Gut Disease in shrimp aquaculture

With increasing demand for environment friendly aquaculture, use of beneficial bacteria to displace pathogens by competitive processes is being used in the animal industry as a better remedy and is now gaining acceptance for pathogen control in aquaculture. With this concern, the present study was designed for isolation of suitable probiotic bacteria from natural sources, application in an effective dose in the rearing environment is expected to control the blowout of White Gut Disease in aquaculture systems. The interaction of pathogenic Vibrio anguillarum and the inner surface of the digestive tract of P. monodon, with a specific focus on their in-situ morphology, aggregation and attachment characteristics presented with pathogenic bacterial species and under the control of probiotic bacteria were analyzed through. The percentage of granular cells of treated group was higher than the control. Histological studies revealed that the treated group has optimistic effect which helps in the reduction of tissue damage and decrease the mortality rate than the infected shrimps. Leuconostoc sp., can survive in the saline condition rather than other Lactobacillus sp., and its tolerance of acidic environment of the shrimp intestine and their adherence level at the intestine will progressively replace the V. anguillarum from the infected shrimps and commendably control the White Gut Disease.

Alginate oligosaccharide/polysaccharide and lactic acid bacteria (Lactobacillus bulgaricus FNCC–0041 & Streptococcus thermophilus FNCC–0040) as immunostimulants against pathogenic Vibrio spp. using Artemia bio model

Alginate polysaccharide/oligosaccharide (APS/AOS) has been proven as a good immunostimulant. FNCC–0041 & FNCC–0040 are lactic acid bacteria (LAB) producing exopolysaccharides. Vibrio bacteria are pathogenic for aquatic cultivans. This study determines the performance of APS/AOS in combination with LAB as immunostimulants. These were conducted in Artemia challenge test assay against three species of Vibrio spp., namely Vibrio parahaemolyticus (Vp), V. harveyi (Vh), and V. vulnificus (Vv). The treatments were prepared by Factorial Design with two factors (APS/AOS doses and Vibrio spp.) and replicated three times. The APS/AOS concentration was 0, 300, 600, 900 ppm. There were 8 levels of vibrio challenges, namely non-Vibrio, Vp, Vh, Vv, Vp-Vh, Vh-Vp, Vv-Vp, and Vp-Vh-Vv. LAB and APS/AOS were fermented with seawater encapsulated by newly hatched Artemia’s nauplii for one hour. Ten nauplii were taken out and challenged with 108 cells/mL Vibrio. Its survival rate (SR) was counted every six hours until reached 100% mortality. Results show that SR of all nauplii Artemia bio encapsulated treatments was higher than control (p<0.05). The best survival rate was reached from 400 ppm AOS. It has appeared that there is a synergically positive effect among the bio encapsulated AOS and LAB to accelerate the Artemia’s immune system.

Hazard Identification Related to the Presence of Vibrio spp., Biogenic Amines, and Indole-Producing Bacteria in a Non-Filter Feeding Marine Gastropod (Tritia mutabilis) Commercialized on the Italian Market

Tritia mutabilis is a carrion-feeder edible marine gastropod with an open circulatory system. Therefore, biological, and chemical contaminants associated with the feed can reach all body tissues. The aim of the present study was to investigate the possible association of these characteristics with some food safety hazards. Vibrio spp. load, and the prevalence of pathogenic V. parahaemolyticus, V. vulnificus, and V. cholerae, were investigated. Moreover, biogenic amines (BAs) and indole-producing bacteria (IPB), markers of seafood decomposition, were quantified for the first time in an edible carrion-feeder. Overall, 49 batches were analyzed (38 from retail, and 11 from primary production). The Vibrio spp. load resulted of 5.64 ± 0.69 log10 CFU g−1 at retail, and 5.27 ± 0.74 at harvest but all batches resulted negative for pathogenic Vibrio. Histamine, putrescine, cadaverine, and tyramine were detected both at harvest and at the retail level. Their sum (BAs Index) showed a mean value of 50.45 and 65.83 mg Kg−1 in batches at harvest and at retail, respectively. IPB were detected at harvest and upon refrigeration for three days (T1–T3). The mean load resulted in 2.52 ± 0.85 log10 MPN g−1 at T0, 3.31 ± 1.23 at T3 in batches immediately refrigerated, and 3.22 ± 1.18 at T3 in batches previously immersed in clean seawater. Our results contribute to identifying food-borne hazards for T. mutabilis that may be related to the retention of biogenic amines and indole-producing bacteria due to carrion feeding.

Characteristics of anti-Vibrio harveyi compounds produced by Bacillus spp. isolated from shrimp ponds

Rusmana I, Isramilda, Akhdiya A. 2021. Characteristics of anti-Vibrio harveyi compounds produced by Bacillus spp. isolated from shrimp ponds. Biodiversitas 22: 4872-4879. Pathogenic Vibrio spp. such as Vibrio vulnificus, V. alginolyticus, V. fluvialis, V. anguillarum, and V. harveyi caused shrimp diseases. The application of bacterial probiotics can control the growth of pathogenic Vibrio in shrimp. Bacillus spp. can produce antimicrobial compounds that inhibit the growth of pathogenic Vibrio spp. Isolation of Bacillus from several pond water samples, pond sediment, and shrimp intestines was successfully collected 175 isolates of Bacillus spp. Three isolates, i.e., Ltw54, Lts36, dan Lts40 had a high inhibitory index and stability in inhibiting the growth of Vibrio harveyi. Verification assay showed that Bacillus sp. Lts40 had the highest antimicrobial activity. The competition assay showed that Bacillus sp Lts40 isolates inhibited the growth of V. harveyi up to 81,8%. The antimicrobial compound produced by Bacillus sp. Lts40 was stable at the pH range of 3-11 and remained stable after heating at 100ºC for 20 minutes. The purification results using the chromatographic filtration technique showed that the protein fraction with a molecular weight of 47,38 kDa effectively inhibited V. harveyi growth. Bacillus sp. Lts40 isolate has potential application as a probiotic agent in shrimp ponds to control the growth of V. harveyi that causes luminous vibriosis diseases and the antimicrobial substance is potentially to be developed and produced as an anti-V. harveyi product that can be applied in a shrimp hatchery.

Producing Active Secondary Metabolite Against Pathogenic Vibrio spp. by Actinobacteria-Sodium Alginate Co-Culture

Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio harveyi have been found in aquatic environments and suspected as the primary trigger of WFD (White Feces Disease) outbreaks in aquaculture. This Vibrio spp. has an antibiotic resistance to Ampicillin, Co-Amoxiclav, Amoxicillin, Azithromycin, Actinobacteria and Ciprofloxacin HCL. Actinobacteria and alginate have been reported to increase the marine biota resistance against diseases through prebiotic and probiotic mechanisms. This study aims to discover and increase the secondary metabolite production of Actinobacteria-Alginate and its ability as anti-vibrio. Alginate extraction in the samples dated September 2020 originally from Teluk Awur Bays, Jepara, Central Java, Indonesia (33.73±1.84%) was considerably higher than in May 2021 (22.67±0.3%). Samples were taken from sediment and mangrove root. Actinobacteria strains are macroscopically and microscopically similar to the genus Streptomyces. The most well-known antibiotics were produced by Streptomyces spp. The anti-vibrio test was carried out by Kirby-Bauer disc diffusion. The results were observed by measuring the inhibition zone surrounding the paper disc using a digital calliper. Co-culture strain 90 together with alginate have an approved antibacterial activity against all Vibrio spp. in the concentration of 10.disc-1 mg and 5 mg.disc-1. Co-culture Actinobacteria with alginate has remarkably changed the green-yellow color to olive green/dark red-orange (strains 3, 62, 63, 72, and 90), indicating the transformation of the formation alginate with pigments into other compounds through the biosynthetic pathway. Therefore, alginate enables to support of Actinobacteria by induction the active secondary metabolite as an anti-vibrio to counteract the bacterial pathogen diseases.

Spirulina water extract and Lactobacillus bulgaricus FNCC– 0041, Streptococcus thermophilus FNCC–0040 secretion as immunostimulants in gnotobiotic Artemia challenge tests against pathogenic Vibrio parahaemolyticus, V. vulnificus, and V. harveyi

Spirulina water extract (SWE) has a good potency as an immunostimulant. Lactobacillus bulgaricus & Streptococcus thermophilus are lactic acid bacteria (LAB) that produce exopolysaccharide exudate. Vibriosis is a common infectious disease for aquatic cultivans caused by Vibrio spp. This study determines the ability of SWE in combination with L. bulgaricus & S. thermophilus as immunostimulant (Artemia challenge test) assay against Vibrio parahaemolyticus, V. vulnificus, and V. harveyi. Factorial Design with two factors namely SWE doses (0, 300, 600, and 900 ppb) and Vibrio spp. treatment (non-Vibrio spp., Vibrio harveyi (Vh), Vibrio parahaemolyticus (Vp), Vibrio vulnificus (Vv), Vh-Vp, Vv-Vp, Vp-Vh, and Vp-Vv-Vh) were applicated. SWE was diluted and LAB at a concentration of 108 cell/mL were fermented in three days at 30°C. Ten newly hatched Artemia nauplii were enriched with fermented and non-fermented SWE for one hour, then challenged with 108 cell/mL Vibrio spp. The survival of Artemia was recorded every 6 hours. Results showed that the survival rate of Artemia enriched with 300 ppb concentration of SWE and LAB was significant than control (p<0.05). It is concluded that there is a positive effect on the bioencapsulation of the minimum concentration of SWE and LAB secretion to accelerate Artemia’s immune response.

In Silico Prediction of Novel Probiotic Species Limiting Pathogenic Vibrio Growth Using Constraint-Based Genome Scale Metabolic Modeling

The prevalence of bacterial diseases and the application of probiotics to prevent them is a common practice in shrimp aquaculture. A wide range of bacterial species/strains is utilized in probiotic formulations, with proven beneficial effects. However, knowledge of their role in inhibiting the growth of a specific pathogen is restricted. In this study, we employed constraint-based genome-scale metabolic modeling approach to screen and identify the beneficial bacteria capable of limiting the growth of V. harveyi, a common pathogen in shrimp culture. Genome-scale models were built for 194 species (including strains from the genera Bacillus, Lactobacillus, and Lactococcus and the pathogenic strain V. harveyi) to explore the metabolic potential of these strains under different nutrient conditions in a consortium. In silico-based phenotypic analysis on 193 paired models predicted six candidate strains with growth enhancement and pathogen suppression. Growth simulations reveal that mannitol and glucoronate environments mediate parasitic interactions in a pairwise community. Furthermore, in a mannitol environment, the shortlisted six strains were purely metabolite consumers without donating metabolites to V. harveyi. The production of acetate by the screened species in a paired community suggests the natural metabolic end product’s role in limiting pathogen survival. Our study employing in silico approach successfully predicted three novel candidate strains for probiotic applications, namely, Bacillus sp 1 (identified as B. licheniformis in this study), Bacillus weihaiensis Alg07, and Lactobacillus lindneri TMW 1.1993. The study is the first to apply genomic-scale metabolic models for aquaculture applications to detect bacterial species limiting Vibrio harveyi growth.