Occurrence and pathogenic potential of Bacillus cereus group bacteria in a sandy loam

The major part (94%) of the Bacillus cereus-like isolates from a Danish sandy loam are psychrotolerant Bacillus weihenstephanensis according to their ability to grow at temperatures below 7 °C and/or two PCR-based methods, while the remaining 6% are B. cereus. The Bacillus mycoides-like isolates could also be␣divided into psychrotolerant and mesophilic isolates. The psychrotolerant isolates of B. mycoides could␣be discriminated from the mesophilic by the two PCR-based methods used to characterize B.␣weihenstephanensis. It is likely that the mesophilic B. mycoides strains are synonymous with Bacillus pseudomycoides, while psychrotolerant B. weihenstephanensis, like B. mycoides, are B. mycoides senso stricto. B. cereus is known to produce a number of factors, which are involved in its ability to cause gastrointestinal and somatic diseases. All the B. cereus-like and B. mycoides like isolates from the sandy loam were investigated by PCR for the presence of 12 genes encoding toxins. Genes for the enterotoxins (hemolysin BL and nonhemolytic enterotoxin) and the two of the enzymes (cereolysin AB) were present in the major part of the isolates, while genes for phospolipase C and hemolysin III were present in fewer isolates, especially among B. mycoides like isolates. Genes for cytotoxin K and the hemolysin II were only present in isolates affiliated to B. cereus. Most of the mesophilic B. mycoides isolates did not possess the genes for the nonhemolytic enterotoxin and the cereolysin AB. The presence of multiple genes coding for virulence factors in all the isolates from the B. cereus group suggests that all the isolates from the sandy loam are potential pathogens.     

Evidence that <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> ahpC2 is essential for survival under high salinity by modulating intracellular level of ROS

Expression of ahpC2 encoding an alkyl hydroperoxide reductase of Vibrio vulnificus, a foodborne pathogen, was incrementally induced depending on NaCl concentrations in the culture. Growth of the ahpC2 mutant was significantly impaired with longer lag phase and lower growth rate when cultured under high salinity. ROS was accumulated in V. vulnificus cells when stressed by exposure to high salinity, and the ahpC2 mutant accumulated higher level of ROS as compared with the parental wild type. Consequently, the combined results suggest that AhpC2 contributes to the growth of V. vulnificus under high salinity by scavenging ROS in cells.     

Identification of the <Emphasis Type="Italic">Vibrio vulnificus ahpC</Emphasis>l gene and its influence on survival under oxidative stress and virulence

Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpCl, a homologue of Escherichia coli ahpC encoding a peroxiredoxin, was identified among the Vibrio vulnificus genes specifically induced by exposure to H₂O₂. In order to analyze the role of AhpCl in the pathogenesis of V. vulnificus, a mutant, in which the ahpCl gene was disrupted, was constructed by allelic exchanges. The ahpCl mutant was hypersusceptable to killing by reactive oxygen species (ROS) such as H₂O₂ and t-BOOH, which is one of the most commonly used hydroperoxides in vitro. The purified AhpCl reduced H₂O₂ in the presence of AhpF and NADH as a hydrogen donor, indicating that V. vulnificus AhpCl is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. Compared to wild type, the ahpCl mutant exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. In addition, the ahpCl mutant was significantly diminished in growth with INT-407 epithelial cells, reflecting that the ability of the mutant to grow, survive, and persist during infection is also impaired. Consequently, the combined results suggest that AhpCl and the capability of resistance to oxidative stresses contribute to the virulence of V. vulnificus by assuring growth and survival during infection.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

牛源致病性蜡样芽孢杆菌溶血素BL亚基的原核表达及其生物学活性分析

蜡样芽孢杆菌属于革兰氏阳性菌,分布广泛,具有一定的致病性。不同的蜡样芽孢杆菌携带有不同的毒力因子,这直接决定了蜡样芽孢杆菌株致病性的差异。研究和探讨毒力因子分布以及具体毒素的生物活性有助于对蜡样芽孢杆菌病采取更科学的防控。本研究通过原核表达系统将溶血素BL三亚基重组表达,并对表达蛋白进行了纯化和部分生物学活性的检测。研究结果表明,牛源致病性蜡样芽孢杆菌溶血素BL可以在原核表达系统中成功表达和纯化,牛源致病性蜡样芽孢杆菌溶血素BL拥有溶血性、细胞毒性、很好的免疫原性以及对小鼠具有一定的免疫保护性。本研究表达了溶血素BL三亚基,并探究了牛源致病性蜡样芽孢杆溶血素BL的生物学活性。本研究为进一步揭示蜡样芽孢杆菌溶血素BL的致病作用机制和建立针对牛源致病性蜡样芽孢杆菌病的检测方法奠定了理论基础。     

Protective effect of polygoni cuspidati radix and emodin on <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> cytotoxicity and infection

Vibrio vulnificus, a good model organism of bacterial septicemia, causes fatal septicemia manifesting a fulminating course and a high mortality rate within days. In order to identify new natural substances preventing V. vulnificus infection, a plant library was screened for inhibiting cytotoxicity to host cells by using Trypan blue staining and LDH assay. We found that Polygoni Cuspidati Radix potently suppressed the acute death of HeLa and RAW264.7 cells in a dose dependent manner. Further studies revealed that Polygoni Cuspidati Radix inhibited V. vulnificus growth and survival in HI broth and seawater, respectively. We confirmed that Polygoni Cuspidati Radix contained high level of emodin by thin layer chromatography (TLC). Emodin showed direct antibacterial activity against V. vulnificus. In addition, emodin prevented the morphologic damages and acute death of HeLa cells caused from V. vulnificus. The safety of Polygoni Cuspidati Radix and emodin to host cells was confirmed by MTT assay. Polygoni Cuspidati Radix and emodin protected mice from V. vulnificus infection.     

Cloning and Characterization of a Periplasmic Nuclease of Vibrio vulnificus and Its Role in Preventing Uptake of Foreign DNA

We have cloned a nuclease gene, vvn, from Vibrio vulnificus, an estuarine bacterium that causes wound infections and septicemia in humans and eels. The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either V. vulnificus or recombinant Escherichia coli strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities. Expression of Vvn in E. coli DH5α reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient V. vulnificus mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients had little effect on the conjugation frequency in either E. coli or V. vulnificus. Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that Vvn was not required for the virulence of V. vulnificus mice.     

The nucleotide sequence of the lipo-penicillinase gene of alkalophilic Bacillus sp. strain 170

The lipo-penicillinase (LIPEN) gene from an alkalophilic Bacillus sp. strain 170 was cloned in Escherichia coli using the vector pHSG399. A plasmid, pFAP121, was isolated from an ampicillin resistant transformant and the cloned LIPEN gene was found to be in a 2.2 kb DNA fragment. The nucleotide sequence of a 1.9 kb segment encoding the LIPEN was determined. This segment showed an open reading frame which would encode a polypeptide of 310 amino acids. The amino acid sequence of this LIPEN gene product has strong homology with those of the Bacillus cereus -lactamase III and Bacillus licheniformis penicillinase.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.     

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus. Part of the work was carried out at Biotechnology Programme, Jadavpur University, Calcutta 700 032, India.     

Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification

Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 °C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus.     

Factors affecting hemolysin production byVibrio vulnificus

Factors affecting the hemolytic activity ofVibrio vulnificus cultures supernatant fluids against sheep erythrocytes were studied in order to optimize conditions and develop an assay system to assess the pathogenic potential of marineV. vulnificus strains. The heat-labile hemolysin produced during logarithmic growth ofV. vulnificus was produced optimally in heart infusion broth (HIB). Lesser amounts of hemolysin were produced in brain-heart infusion and tryptic soy broths. Hemolytic activity of HIB cultures decreased with increasing NaCl concentrations. Higher NaCl concentrations were found to affect hemolysin production but not its activity. The assay system described herein is a simple and rapid method that is being applied to the study of the pathogenic potential ofV. vulnificus strains from marine environments.     

Prevalence and Heterogeneity of Hemolysin Gene <Emphasis Type="Italic">vhh</Emphasis> Among Hatchery Isolates of <Emphasis Type="Italic">Vibrio harveyi</Emphasis> in India

Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH. The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR.     

Identification and transcriptional analysis of genes involved in <Emphasis Type="Italic">Bacillus cereus</Emphasis>-induced systemic resistance in <Emphasis Type="Italic">Lilium</Emphasis>

Bacillus cereus C1L was demonstrated to induce systemic resistance in Lilium formosanum against leaf blight caused by Botrytis elliptica. Suppression subtractive hybridization library of L. formosanum triggered by B. cereus C1L were screened and 3 differentially expressed genes were identified. Based on sequence analysis, these genes encoding putative glycine-rich protein, metallothionein-like protein, and PsbR protein of photosystem 2, were designated LfGRP1, LfMT1, and LfPsbR, respectively. The results of Northern blot analysis showed that expressions of LfGRP1, LfMT1 and LfPsbR increased in response to B. elliptica infection. On the other hand, expression of LfMT1 increased but expressions of LfGRP1 and LfPsbR decreased when the rhizosphere of L. formosanum was drenched with suspension of B. cereus C1L with or without subsequent challenge with B. elliptica on lily leaves. Similar expression profiles of homologues of LfGRP1, LfMT1, and LfPsbR (named LsGRP1, LsMT1, and LsPsbR, respectively) were presented in Lilium oriental hybrid Star Gazer. In addition, application of the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, on the leaves reduced disease severity and expressions of LsGRP1 and LsPsbR just as that in response to B. cereus C1L treatment.     

Subcellular localisation of lipoproteins of <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> by the identification of outer membrane vesicles components

Vibrio vulnificus, a Gram-negative halophilic bacterium, is an opportunistic human pathogen that is responsible for the majority of seafood-associated deaths worldwide. Lipoproteins are important components of the bacterial cell envelope and have been shown to be involved in a wide variety of cellular processes. Little is known about the localisation or transport mechanism of lipoproteins in V. vulnificus. To assess the localisation of lipoproteins in V. vulnificus, we tested two established techniques for the rapid separation of membrane-associated proteins: detergent extraction with Sarkosyl and outer membrane vesicles (OMVs) preparation. The results showed that Sarkosyl extraction was not useful for the separation of lipoproteins from the different membranes of V. vulnificus. On the other hand, we confirmed that OMVs produced by V. vulnificus contained lipoproteins from the outer but not the inner membrane. Analysis of the OMVs components confirmed the localisation of several well-known lipoproteins to membranes that were different from expected, based on their predicted functions. Using this technique, we found that Asp at position +2 of mature lipoproteins can function as an inner membrane retention signal in V. vulnificus. Interestingly, the Escherichia coli “+2 rule” does not apply to the V. vulnificus lipoprotein IlpA (G2D) mutant, as a Ser to Asp mutation at position +2 of IlpA did not affect its outer membrane localisation. Furthermore, an IlpA tether-mRFP chimeric lipoprotein and its G2D mutant also behaved like IlpA. Together, these results suggest that the sorting rule of lipoproteins in V. vulnificus might be different from that in E. coli.     

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.     

Monoclonal Antibody Developed against a Hemolysin of Bacillus thuringiensis

A total of five hybridoma cell lines that produced monoclonal antibodies (MAb) against a hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis were established and characterized. All of these monoclonal antibodies reacted similarly not only to Bt-hemolysin but also to a hemolysin (Bc-hemolysin) produced by B. cereus, suggesting that the two hemolysins are immunologically indistinguishable. The MAb developed in this study was also successfully applied for rapid and simple purification of both Bt- and Bc-hemolysins by immunoaffinity column chromatography. The partial N-terminal amino acid sequence of the purified hemolysins was determined to be Ile-Glu-Gln-Thr.     

Effects of temperature,growth phase and luxO-disruption on regulation systems of toxin production in Vibrio vulnificus strain L-180, a human clinical isolate

Vibrio vulnificus is a halophilic estuarine bacterium while it causes fatal septicemia or necrotizing wound infections in humans. This pathogen secretes the metalloprotease (V. vulnificus protease: VVP) and the cytolysin (V. vulnificus hemolysin: VVH) as protein toxins; however, their production was coordinated in response to the bacterial cell density. This regulation is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). In the present study, we investigated effects of disruption of luxO encoding a central response regulator of the QS circuit, as well as effects of temperature and growth phase, on the toxin production by V. vulnificus. Disruption of luxO was found to increase VVP production and expression of its gene vvpE. The expression of smcR, crp and rpoS, of which products positively regulate vvpE expression, and luxS encoding the AI-2 synthetase were also significantly increased. On the other hand, the luxO disruption resulted in reduction of VVH production and expression of its gene vvhA. Expression of other two genes affecting the QS circuit, luxT and rpoN, were also significantly decreased. The regulation systems of VVP production were found to exert their action during the stationary phase of the bacterial growth and to be operated strongly at 26 °C. By contrast, those of VVH production apparently started at the log phase and were operated more effectively at 37 °C.