Expression of virulence genes in luminescent and nonluminescent isogenic vibrios and virulence towards gnotobiotic brine shrimp (<i>Artemia franciscana</i>)

Aims: This study aimed to evaluate the expression levels of virulence gene regulators (luxR and toxR) and virulence factors (serine protease, metalloprotease and haemolysin) in luminescent and nonluminescent isogenic Vibrio harveyi and Vibrio campbellii.Method and Results: Nonluminescent variants have been reported before to become dominant in cultures of luminescent vibrios when grown under static conditions in the dark. Wild-type V. harveyi BB120, V. campbellii LMG 21363, quorum sensing mutants of V. harveyi BB120 and their previously reported nonluminescent isogenic counterparts were used in this study. The expression level of the virulence genes srp serine protease, vhp metalloprotease and vhh haemolysin, the quorum sensing master regulator gene luxR and the virulence regulator gene toxR in isogenic luminescent and nonluminescent strains were quantified using reverse transcriptase real-time PCR. These experiments revealed that the nonluminescent strains produced lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene (which is known to be regulated by quorum sensing). Finally, challenge tests with gnotobiotic brine shrimp (Artemia franciscana) larvae revealed that the nonluminescent strains are less virulent than their luminescent isogenic counterparts.Conclusion: Nonluminescent variants of V. harveyi and V. campbellii strains produce lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene and are less virulent to brine shrimp than their isogenic luminescent counterparts.Significance and Impact of the study: These results indicate that adaptation of luminescent vibrios to specific growth conditions that result in a dominant nonluminescent phenotype is accompanied by a decreased adaptation to a host environment because of altered virulence gene regulation

Microbial community management in aquaculture

Microbial community management in aquaculture creates benefits at the nutritional as well as at health level for cultured species. In addition, in case of biofloc application, it allows to link species at different trophic levels, making bioflocs the potential link in integrated multispecies aquaculture

Evaluation of microbiological quality of commercially available bottled drinking water in Colombo district, Sri Lanka

In recent times, the consumption of bottled water has dramatically increased in Sri Lanka. However, compliance by the producers with the bottled water regulations is debatable, which poses questions about bottled water quality. This study aimed at evaluating the microbiological quality of bottled water in the Colombo district, Sri Lanka. Twenty-six brands of drinking water were collected from the Colombo district (19 locations) microbial quality was detected by checking the total coliforms (TC), fecal coliforms (FC), heterotrophic bacteria, fungi and algae. The results revealed that 50 % of drinking water brands violated the Sri Lanka Standards Institution (SLSI) and WHO guidelines, and the Sri Lanka Health Ministry regulation (0 cfu/100 ml). Twenty-three percent of brands exceeded the limits for presumptive FC (0 cfu/100 ml in accordance with WHO guidelines, SLSI, and the Sri Lanka Health Ministry requirement). Moreover, 35% showed higher heterotrophic bacteria which exceeded the WHO guidelines (50 cfu/ml). The dominant fungi found in the bottled water were Aspergillus spp., Rhizopus sp., Trichoderma sp. and Mucor sp. Chlorella vulgaris was identified as the algal species that was present in the drinking water samples and it was 8 % of the samples checked. Additionally, the statistical analysis of water sources revealed no significant differences in the levels of fecal and total coliforms in samples across springs, tube wells, and dug wells. However, the tube wells have a significant difference in HPC and algae than dug wells and springs. The findings of this study concluded that the bottled water industry needs to be closely supervised by competent authorities to provide customers with more healthy bottled water in Sri Lanka

Expression and Quorum Sensing Regulation of Type III Secretion System Genes of Vibrio harveyi during Infection of Gnotobiotic Brine Shrimp.

Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells

Quorum sensing regulation of virulence gene expression in Vibrio harveyi during its interaction with marine diatom Skeletonema marinoi

Communication between species from different kingdoms may be as important as intra-kingdom communication. It has recently been confirmed that co-existing bacteria and phytoplankton in aquatic ecosystems do cross-talk. This study examined the signs of possible cross signalling between V. harveyi, one of the predominant bacterial species of the marine ecosystem and a dominant diatom species, S.marinoi, to understand communication over species borders. It is known that V.harveyi employ quorum sensing for cell-to-cell communication, bioluminescence (luxR), and the regulation of the virulence gene (vhp, chiA). Former studies have also shown, this kind of interactions being disrupted by compounds secreted by a few algal species existing in the aquatic ecosystem. We investigated the QS communication by quantifying the expression levels of virulence regulator luxR and virulence factors metalloprotease (vhp) and chitinase (chiA) in four different V. harveyi strains grown in the presence of S. marinoi strain. Results obtained in this study indicate that quorum sensing was activated in strains of V. harveyi analysed but did not regulate the expressions of vhp and chiA virulence factors. This observation suggests that the existence of S. marinoi did not interfere with the QS behavior of V. harveyi and its interaction with marine diatom; it may be due to the commensalism relationship

<i>In vivo</i> expression of the type III secretion system genes <i>vopD</i>, <i>vcrD</i> and <i>vscP</i>.

<p><i>In vivo</i> expression of the type III secretion system genes <i>vopD</i> <b>(A)</b>, <i>vcrD</i> <b>(B)</b> and <i>vscP</i> <b>(C)</b> in wild type <i>V</i>. <i>harveyi</i> (WT) and mutants with the quorum sensing system locked in high cell density configuration (QS^c) and the quorum sensing system locked in low cell density configuration (QS^-), respectively, during infection of brine shrimp larvae. The expression in wild type <i>V</i>. <i>harveyi</i> at the 0.5h time point was set at 1 and the expression in all strains at all time points was normalised accordingly using the 2^-ΔΔCT method. The error bars represent the standard deviation of three independent shrimp cultures (each time based on bacterial mRNAs extracted from 500 larvae). The RNA polymerase A subunit (<i>rpoA</i>) mRNA was used to normalise between strains.</p

Expression and Quorum Sensing Regulation of Type III Secretion System Genes of <i>Vibrio harveyi</i> during Infection of Gnotobiotic Brine Shrimp

<div><p>Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of <i>Vibrio harveyi</i>, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in <i>V</i>. <i>harveyi</i> increased within the first 12h after challenge and decreased again thereafter. The <i>in vivo</i> expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, <i>in vivo</i> expression levels of the type III secretion system genes were much (> 1000 fold) higher than the <i>in vitro</i> expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells.</p></div

Quorum sensing in <i>Vibrio harveyi</i>.

<p>The LuxM, LuxS and CqsA enzymes synthesise the signal molecules HAI-1, AI-2 and CAI-1, respectively. These signal molecules are detected at the cell surface by the LuxN, LuxQ and CqsS two-component receptor proteins, respectively. Detection of AI-2 by LuxQ requires the periplasmic protein LuxP. <b>(A)</b> In the absence of signal molecules, the receptors autophosphorylate and transfer phosphate to LuxO via LuxU. Phosphorylation activates LuxO, which together with σ⁵⁴ activates the production of five small regulatory RNAs (sRNAs). These sRNAs, together with the chaperone Hfq, destabilise the mRNA encoding the transcriptional regulator LuxR. Therefore, in the absence of autoinducers, the LuxR protein is not produced. LuxR is a repressor of ExsA. Hence, in the absence of signal molecules, ExsA is produced and in turn activates expression of the TTSS operons. <b>(B)</b> In the presence of high concentrations of the signal molecules, the receptor proteins switch from kinases to phosphatases, which results in dephosphorylation of LuxO. Dephosphorylated LuxO is inactive and therefore, the sRNAs are not formed and the transcriptional regulator LuxR is produced. LuxR represses ExsA, and the TTSS operons are not expressed. “P” denotes phosphotransfer.</p

Decontaminating Effect of Organic Acids and Natural Compounds on Broiler Chicken Meat Contaminated with Salmonella typhimurium

Aims: The study was carried out to investigate the effect of natural compounds and organic acids on Salmonella typhimurium in broiler chicken meat. Study Design: This is a laboratory-controlled experimental design. Place and Duration of Study: Department of Livestock Production, Sabaragamuwa University of Sri Lanka, between February 2014 and April 2014. Methodology: Approximately equal sized (4x4x1.5 cm), shaped (cuboidal) and same weight 10g±0.2) chicken meat samples were taken, sterilized using UV and contaminated with Salmonella typhimurium (ATCC 14028) at two cell densities (102 and 108 Colony Forming Units (CFU)/g of meat). Subsequently, meat samples were treated with 1% solutions of both Chemical (Citric, Acetic and Lactic) and Natural compounds (Cardamom, Nutmeg and Mace) for 30 seconds. Treated samples were ground and serially diluted for the enumeration of recovered bacteria. Data were analyzed using SAS followed by a Tukey’s post-hoc test at 95% significance level. Results: Lactic, Acetic and Citric acids have shown 66% (25 CFU/g), 55% (33.33 CFU/g) and 51% (36.6 CFU/g) reduction of colony count at 102 CFU/g inoculation and 88% (8.13x106 CFU/g), 87% (9.3x106 CFU/g) and 72% (2.03x107CFU/g) reduction at 108CFU/g inoculation level. Treatment with Nutmeg, Mace and Cardamom showed 85% (10 CFU/g), 83% (11.6 CFU/g) and 66% (23.3 CFU/g) reduction at 102 CFU/g and 89% (7.3 x 106 CFU/g), 89% (7.5 x 106 CFU/g) and 72% (1.96 x 107 CFU/g) reduction at 108CFU/g inoculation level. Natural compounds showed a 20% greater reduction of colony count in broiler chicken compared to chemical compounds. Conclusion: It is concluded that the natural compounds and weak organic acids have an anti-S. typhimurium effect, hence it can be used for decontamination process of poultry meat

Assessment of the Inhibitory Effect of Nisin (E234) on Salmonella typhimurium and Bacillus subtilis in Chicken Sausage

Aims: Nitrite is used as the main curing agent in the process of sausage production, but Nitrite has limited usage in food processing due to its, carcinogenic effect. Thus, much attention has been given to find alternative compounds to replace the Nitrite over the past decades. Nisin, a natural polypeptide compound extracted from Lactococcus lactis with no toxin production has been identified as a potential compound in this regard. This study aimed at assessing the inhibitory effect of nisin (E234) against Salmonella typhimurium and Bacillus subtilis and to investigate the potential of nisin to replace the antimicrobial property of Nitrite in broiler chicken sausage production. Study Design:&nbsp; This is a laboratory-controlled experimental design. Place and Duration of Study: The study was conducted at the Laboratory of Livestock Production, Faculty of Agricultural Sciences Sabaragamuwa University of Sri Lanka during the period of March 2014 to May 2014. Methodology: Four types of sausages were prepared i.e with nitrite, nisin, nisin+nitrite combination, and one without any preservatives (control). Antibacterial effect of Nisin was investigated by artificial contamination of chicken meat with Salmonella typhimurium (ATCC 14028) and Bacillus subtilis (ATCC 6633) at two different cell densities (102 and 108 Colony Forming Units (CFU)/g of meat) followed by treatment with nisin and nitrite. The recovery of bacteria was determined by spread plate method using ground sausages samples at precooked, cooked and frozen status. Data analysis was done using statistical analysis system for windows 9.0 software at 95% significant level. Results: The precooked sausage batter with added nisin, nitrite and combination (nisin and nitrite)&nbsp; which were contaminated with S. typhimurium at the level of 102&nbsp; and 108 CFU/g meat cell density showed statistically significant reduction in S. typhimurium cell number (88%, 82.8% and 90% for102 and&nbsp; 89%, 84%, 91%&nbsp; for the latter) reduction respectively and&nbsp; there is reduction of B. subtilitis cells also at both concentrations, having 84%, 81% and 86% reduction at low and 89%, 83% and 90% at high cell density. Cooked as well as frozen treatments exhibited&nbsp; a significance reduction (100%) of average colony counts in preservative added samples (nisin, nitrite and combine – nisin with nitrite) compared to control in sausages contaminated with S. typhimurium at both cell densities (102 CFU/g meat, 108 CFU/g meat). In the samples which had low contamination with B. subtilis (102 CFU/g meat)&nbsp; showed 89.5%, 84.2%, 90.5% reduction compared to control (without nisin and nitrite) in nisin, Nitrite and combine (nisin + nitrite) respectively and in the samples contaminated with high number of cells showed 89.2%, 88.1%, and 91.9% respectively subsequently to cooking. In frozen samples at both contamination levels, both nisin and nitrite revealed a reduction in bacterial count compared to the untreated control. Conclusion: It is concluded that nisin has the inhibitory effect against Salmonella typhimurium and Bacillus subtilis and further it can be concluded that, there is a potential of nisin to replace the antimicrobial property of Nitrite in broiler chicken sausage production