VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Enumeration of Vibrio parahaemolyticus in the viable but nonculturable state using direct plate counts and recognition of individual gene fluorescence in situ hybridization

Vibrio parahaemolyticus is a gram-negative, halophilic bacterium indigenous to marine and estuarine environments and it is capable of causing food and water-borne illness in humans. It can also cause disease in marine animals, including cultured species. Currently, culture-based techniques are used for quantification of V. parahaemolyticus in environmental samples; however, these can be misleading as they fail to detect V. parahaemolyticus in a viable but nonculturable (VBNC) state which leads to an underestimation of the population density. In this study, we used a novel fluorescence visualization technique, called recognition of individual gene fluorescence in situ hybridization (RING-FISH), which targets chromosomal DNA for enumeration. A polynucleotide probe labeled with Cyanine 3 (Cy3) was created corresponding to the ubiquitous V. parahaemolyticus gene that codes for thermolabile hemolysin (tlh). When coupled with the Kogure method to distinguish viable from dead cells, RING-FISH probes reliably enumerated total, viable V. parahaemolyticus. The probe was tested for sensitivity and specificity against a pure culture of tlh⁺, tdh⁻, trh⁻V. parahaemolyticus, pure cultures of Vibrio vulnificus, Vibrio harveyi, Vibrio alginolyticus and Vibrio fischeri, and a mixed environmental sample. This research will provide additional tools for a better understanding of the risk these environmental organisms pose to human health.     

Detection of Vibrio parahaemolyticus in food samples using in situ loop-mediated isothermal amplification method

A novel in situ loop-mediated isothermal amplification (in situ LAMP) technique for rapid detection of the food-borne Vibrio parahaemolyticus strains had been developed and evaluated in this study. The sensitivity of the in situ LAMP assay was detected to be 10 CFU/reaction via test in serial 10-fold dilutions of V. parahaemolyticus cells, and high specificity had also been obtained through confirmation with 14 reference gram-positive and -negative strains. Application of the established in situ LAMP assay had been performed on 58 strains previously isolated from seafood samples, including 48 V. parahaemolyticus and 10 non-V. parahaemolyticus strains. Of 48 V. parahaemolyticus strains, 48, 45 and 34 strains were detected as positive by in situ LAMP, regular LAMP and PCR, respectively, with the detection rate and negative predictive value (NPV) found to be 100% vs 93.8% vs 70.8% and 100% vs 76.9% vs 41.7%. In addition, none of the tested non-V. parahaemolyticus strains showed positive result, indicating a 100% positive predictive value (PPV) for all of 3 assays. Compared with regular LAMP methods and PCR-based methods, the in situ LAMP assay is advantageous on rapidity, high specificity, less time consumption and ease in operation, and may provide a novel, useful and practical detection platform for pathogens in food safety laboratories.     

Ecological determinants of the occurrence and dynamics of Vibrio parahaemolyticus in offshore areas

The life cycle of Vibrio parahaemolyticus has been conventionally associated with estuarine areas characterized by moderate salinity and warm seawater temperatures. Recent evidence suggests that the distribution and population dynamics of V. parahaemolyticus may be shaped by the existence of an oceanic transport of communities of this organism mediated by zooplankton. To evaluate this possibility, the presence of V. parahaemolyticus in the water column of offshore areas of Galicia was investigated by PCR monthly over an 18-month period. Analysis of zooplankton and seawater showed that the occurrence of V. parahaemolyticus in offshore areas was almost exclusively associated with zooplankton and was present in 80% of the samples. The influence of environmental factors assessed by generalized additive models revealed that the abundance and seasonality of V. parahaemolyticus in zooplankton was favoured by the concurrence of downwelling periods that promoted the zooplankton patchiness. These results confirm that offshore waters may be common habitats for V. parahaemolyticus, including strains with virulent traits. Additionally, genetically related populations were found in offshore zooplankton and in estuaries dispersed along 1500 km. This finding suggests that zooplankton may operate as a vehicle for oceanic dispersal of V. parahaemolyticus populations, connecting distant regions and habitats, and thereby producing impacts on the local community demography and the spread of Vibrio-related diseases.     

Acanthamoeba polyphaga is a possible host for Vibrio cholerae in aquatic environments

Acanthamoeba is a genus of free-living amoebae found to be able to host many bacterial species living in the environment. Acanthamoebae and Vibrio cholerae are found in the aquatic environments of cholera endemic areas. Previously it has been shown that V. cholerae O1 and O139 can survive and grow in Acanthamoeba castellanii. The aim of this study was to examine the ability of Acanthamoeba polyphaga to host V. cholerae O1 and O139. The interaction between A. polyphaga and V. cholerae strains was studied by means of viable amoeba cell counts and viable count of the bacteria in the absence and presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Electron microscopy was used to determine the localization of V. cholerae inside A. polyphaga. The results showed that A. polyphaga enhanced growth and survival of V. cholerae, which grew and survived inside the amoeba cells for 2 weeks. The electron microscopy showed that A. polyphaga hosted intracellular V. cholerae localized in the vacuoles of amoeba cell. Neither the presence of V. cholerae together with A. polyphaga nor the intracellular localization of the bacteria inhibited growth and survival of A. polyphaga. The outcome of the interaction between these microorganisms may support strongly the role of A. polyphaga as host for V. cholerae O1 and O139.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F₀F₁ ATP synthase's delta subunit.Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40 min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.     

大连海域沉积物中可培养海洋链霉菌活性及其多样性

以大肠杆菌、金黄色葡萄球菌和尖孢镰刀枯萎病菌作为测试靶目标,采用9种分离培养基从大连海域13个不同采样点的海洋沉积物样品中分离到165株海洋链霉菌。从165株海洋放线菌中筛选到对金黄色葡萄球菌具有抑制活性的菌株85株,占总菌株数的51.5%;对大肠杆菌具有抑制活性的菌株27株,占总菌株数的16.4%;对尖孢镰刀枯萎病菌具有抑制活性的菌株仅有6株,占总菌株数的3.6%。因此,海洋链霉菌的活性更多地表现为对细菌的抗性,尤其对革兰氏阳性细菌具有更高的抑制活性。对其中具有抑制活性或形态独特的菌株进行了16S r DNA序列分析,并构建系统发育树,显示活性海洋链霉菌具有丰富的种类多样性和广谱抗菌活性。同种海洋链霉菌与土壤链霉菌活性比较结果也表明,海洋链霉菌多表现抗革兰氏阳性细菌活性。     

合浦珠母贝鳃的显微与超微结构

合浦珠母贝(Pinctada fucata)是典型的滤食性瓣鳃类动物,也是我国重要的海水珍珠养殖贝类。本研究用光学显微镜、扫描电镜和透射电镜观察了合浦珠母贝鳃的显微和超微结构。结果表明,合浦珠母贝鳃结构属于异丝鳃型,左右两侧各2个鳃瓣,每个鳃瓣由内鳃瓣和外鳃瓣组成。鳃瓣由主鳃丝和普通鳃丝构成,主鳃丝在鳃瓣中主要起支架作用,每2根主鳃丝之间的9～12根普通鳃丝由"簇内连接"(intrabunchial junction)相连成簇。普通鳃丝之间通过"丝间连接"(interfilament junction)相连,丝间连接的上皮细胞与普通鳃丝的扁平细胞结构一样,为鳃的呼吸上皮。丝间连接的存在扩大了鳃的表面积,这种结构有助于进行气体交换。主鳃丝和普通鳃丝表面有前纤毛和侧纤毛,与食物运送和气体交换有关。普通鳃丝表面的纤毛为典型的"9+2"型微管结构。     

Resistance of a recombinant Escherichia coli to dehydration

Dehydration of microorganisms, rendering them anhydrobiotic, is often an efficient method for the short and long term conservation of different strain-producers. However, some biotechnologically important recombinant bacterial strains are extremely sensitive to conventional treatment. We describe appropriate conditions during dehydration of the recombinant Escherichia coli strain HB 101 (GAPDH) that can result dry cells having a ∼88% viability on rehydration. The methods entails air-drying after addition of 100 mM trehalose to the cultivation medium or distilled water (for short term incubation).     

Isolation and distribution of iridescent Cellulophaga and other iridescent marine bacteria from the Charente-Maritime coast,French Atlantic

An intense colored marine bacterium, identified as Cellulophaga lytica, was isolated previously from a sea anemone surface on the Charente-Maritime rocky shore (Atlantic Coast, France), and iridescence of its colonies under direct light was recently described. In addition, iridescence intensities were found to differ strongly between C. lytica strains from different culture collections. However, importantly, the occurrence and distribution of iridescent bacteria in the marine environment were still unknown. Therefore, in this study, a search was undertaken for marine iridescent bacterial strains in different biotopes of the Charente-Maritime coast. Various marine samples (water, sediment, macroalgae, other macroorganisms and detritus) were collected from seven biotopes using a direct plate inoculation method. As a result, 34 iridescent strains related to the genus Cellulophaga, as well as 63 iridescent strains affiliated to the genera Tenacibaculum and Aquimarina, were isolated. Iridescent colors were different according to the genera but iridescent marine bacteria were widely distributed. However, a majority of strains were isolated from rocky shores and, in particular, red seaweed surfaces and mollusks. The data from the study suggested that isolates with iridescent properties were well conserved in stressful environments such as the coastal shoreline. This origin may provide an insight into the ecological and biological functions of iridescence.     

Long-Term Study of Vibrio parahaemolyticus Prevalence and Distribution in New Zealand Shellfish

The food-borne pathogen Vibrio parahaemolyticus has been reported as being present in New Zealand (NZ) seawaters, but there have been no reported outbreaks of food-borne infection from commercially grown NZ seafood. Our study determined the current incidence of V. parahaemolyticus in NZ oysters and Greenshell mussels and the prevalence of V. parahaemolyticus tdh and trh strains. Pacific (235) and dredge (21) oyster samples and mussel samples (55) were obtained from commercial shellfish-growing areas between December 2009 and June 2012. Total V. parahaemolyticus numbers and the presence of pathogenic genes tdh and trh were determined using the FDA most-probable-number (MPN) method and confirmed using PCR analysis. In samples from the North Island of NZ, V. parahaemolyticus was detected in 81% of Pacific oysters and 34% of mussel samples, while the numbers of V. parahaemolyticus tdh and trh strains were low, with just 3/215 Pacific oyster samples carrying the tdh gene. V. parahaemolyticus organisms carrying tdh and trh were not detected in South Island samples, and V. parahaemolyticus was detected in just 1/21 dredge oyster and 2/16 mussel samples. Numbers of V. parahaemolyticus organisms increased when seawater temperatures were high, the season when most commercial shellfish-growing areas are not harvested. The numbers of V. parahaemolyticus organisms in samples exceeded 1,000 MPN/g only when the seawater temperatures exceeded 19°C, so this environmental parameter could be used as a trigger warning of potential hazard. There is some evidence that the total V. parahaemolyticus numbers increased compared with those reported from a previous 1981 to 1984 study, but the analytical methods differed significantly.     

Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus

Background

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before.

Results

Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium.

Conclusions

We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1135) contains supplementary material, which is available to authorized users.     

In Vitro Protective Efficacy of Clostridium butyricum Against Fish Pathogen Infections

Most pathogens in intestine are opportunist, called “opportunistic pathogens” that usually do not cause disease in a healthy host. Only when the host’s resistance is lowered or the intestinal microecological balance is destroyed, the opportunistic pathogens are capable of causing disease. Here, two opportunistic pathogens, Salmonella enteritidis and Vibrio parahaemolyticus were chosen to test the possible antagonistic effect of the probiotic agent Clostridium butyricum on these pathogens infections in vitro using fish intestinal epithelial cells (FIECs). The C. butyricum and its spent culture supernatants exhibited significant inhibitory activity on S. enteritidis and V. parahaemolyticus growth and adherence to FIECs. The C. butyricum also showed significant inhibitory effects on S. enteritidis and V. parahaemolyticus induced apoptosis, which may due to its growth and adhesion inhibitory effects. These results indicated that the probiotic bacterium C. butyricum has preventive and therapeutic effects on S. enteritidis and V. parahaemolyticus infections in fish.     

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

Vigna unguiculata is nodulated in Spain by endosymbionts of Genisteae legumes and by a new symbiovar (vignae) of the genus Bradyrhizobium

Vigna unguiculata was introduced into Europe from its distribution centre in Africa, and it is currently being cultivated in Mediterranean regions with adequate edapho-climatic conditions where the slow growing rhizobia nodulating this legume have not yet been studied. Previous studies based on rrs gene and ITS region analyses have shown that Bradyrhizobium yuanmingense and B. elkanii nodulated V. unguiculata in Africa, but these two species were not found in this study. Using the same phylogenetic markers it was shown that V. unguiculata, a legume from the tribe Phaseolae, was nodulated in Spain by two species of group I, B. cytisi and B. canariense, which are common endosymbionts of Genisteae in both Europe and Africa. These species have not been found to date in V. unguiculata nodules in its African distribution centres. All strains from Bradyrhizobium group I isolated in Spain belonged to the symbiovar genistearum, which is found at present only in Genisteae legumes in both Africa and Europe. V. unguiculata was also nodulated in Spain by a strain from Bradyrhizobium group II that belonged to a novel symbiovar (vignae). Some African V. unguiculata-nodulating strains also belonged to this proposed new symbiovar.     

Vibrio bacteria in raw oysters: managing risks to human health

The human-pathogenic marine bacteria Vibrio vulnificus and V. parahaemolyticus are strongly correlated with water temperature, with concentrations increasing as waters warm seasonally. Both of these bacteria can be concentrated in filter-feeding shellfish, especially oysters. Because oysters are often consumed raw, this exposes people to large doses of potentially harmful bacteria. Various models are used to predict the abundance of these bacteria in oysters, which guide shellfish harvest policy meant to reduce human health risk. Vibrio abundance and behaviour varies from site to site, suggesting that location-specific studies are needed to establish targeted risk reduction strategies. Moreover, virulence potential, rather than simple abundance, should be also be included in future modeling efforts.     

副溶血弧菌的毒力基因表达调控的分子机制

副溶血弧菌是革兰氏阴性嗜盐细菌,是海洋脊椎动物和无脊椎动物中主要致病菌,也是引起人类急性肠胃炎、败血症和坏死性筋膜炎等疾病的主要病原体。在过去,由副溶血弧菌引起的致病感染在世界范围内有不断增加的趋势。副溶血弧菌的致病性与其自身产生的多种毒力因子有关,这些毒力因子包括粘附因子、脂多糖、溶血素、III型分泌系统、VI型分泌系统、铁摄取系统、蛋白酶、外膜蛋白等。然而,这些毒力因子的表达都受到环境因子以及宿主体内信号因子的调控。副溶血弧菌通过感知外界生存环境的各种信号因子,从而激活体内不同的信号通路,进而诱导不同的毒力因子的表达。本文主要对副溶血弧菌毒力因子表达调控的分子机制进行综述,为更好地理解宿主与病原体的相互作用对副溶血弧菌的致病机制的影响,以及为今后预防和治疗由副溶血弧菌所引起的疾病提供理论参考。     

Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni

Termites encounter a diverse array of potentially useful and harmful fungi in their subterranean habitats. These vary from symbiotic to harmful species with varying levels of virulence. How these hemiedaphic insects survive in habitats with infective fungi is not well understood. Possible mediation of olfactory signals in avoiding contact with entomopathogenic fungi has been explored by a number of workers. In the present study, we initially found that Macrotermes michaelseni detected a virulent isolate of Metarhizium anisopliae from some distance and avoided direct physical contact. We hypothesized that there may be a relationship between virulence and repellency of different isolates of M. anisopliae and Beauveria bassiana to the termite. We compared these for selected isolates of the two fungi. Positive correlations between the two parameters for both sets of isolates of the fungi were obtained. The results show an interesting co-evolutionary phenomenon in which the termite's response to either M. anisopliae or B. bassiana is directly related to potential harm these fungi can inflict on the insect and that the virulent strains are more likely to be recognized from some distance and avoided.     

Unusual iridoid glycosides in Veronica sects. Hebe and Labiatoides

In a chemosystematic investigation of three Southern hemisphere species of Veronica, namely the Australian Veronica derwentiana Andrews and Veronica perfoliata R.Br. (formerly Derwentia species), and the New Zealand Veronica catarractae G. Forster (formerly a species of Parahebe), the water-soluble constituents were isolated and identified by spectroscopic methods. Apart from other iridoid glucosides common to the genus, three unusual substituted benzoyl esters of aucubin (derwentiosides A–C) were obtained from V. derwentiana and a chlorinated iridoid glycoside (catarractoside) from V. catarractae in addition to other iridoids common to the genus. The chemical profile of V. perfoliata is similar to that of Northern hemisphere species of Veronica because of the presence of characteristic 6-O-catalpol esters. The profile of V. derwentiana is unique, since 6-O-esters of aucubin rather than of catalpol dominate, however, the acyl groups are the same as those present in catalpol esters found in some other Veronica sections. V. catarractae also contains one of the catalpol esters characteristic of Veronica, but in addition three 6-O-rhamnopyranosyl substituted iridoid glycosides, one of which is 6-O-rhamnopyranosylcatalpol. Esters of the latter compound are previously only known from the more derived species in recent phylogenetic trees of sect. Hebe to which V. catarractae now also belongs, but as a more basal member.