A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression.

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods.

Complete genome sequence of serotype 3 Streptococcus suis INT-01, isolated from a domestic pig in Korea.

Streptococcus suis is a major pig pathogen causing severe economic losses to the swine industry. This study aimed to analyze the genome of S. suis strain INT-01 isolated from a domestic pig in Korea. We found that the genome of strain INT-01 contains 2,092,054 bp, with a guanine (G) + cytosine (C) content of 41.3%, and the capsular polysaccharide synthesis locus of this strain is almost identical to that of serotype 3 S. suis strain 4961 isolated from China, suggesting that these isolates can be classified as serotype 3. Genomic analyses revealed that strain INT-01 is an extracellular protein factor (epf)-/ muraminidase-released protein (mrp)+/ suilysin (sly)- S. suis, which is the most prevalent genotype in Korea, and several virulence-related genes associated with the pathogenicity of S. suis were also detected. The genomic information of strain INT-01 may provide important insights into the development of control strategies against S. suis infections in Korea.

Specific bacteriophage of Bordetella bronchiseptica regulates B. bronchiseptica-induced microRNA expression profiles to decrease inflammation in swine nasal turbinate cells.

BACKGROUND: Respiratory diseases in pigs are the main health concerns for swine producers. Similar to the diseases in human and other animals, respiratory diseases are primary related to morbidity and are the result of infection with bacteria, viruses, or both. B. bronchiseptica causes serious respiratory diseases in the swine airway track. However, the B. bronchiseptica-specific bacteriophage has diverse advantages such as decreasing antibiotic overuse and possible therapeutic potential against bacteria. OBJECTIVE: The objects of this study were to investigate the therapeutic effect of specific B. bronchiseptica bacteriophages and to identify genes related to bacteriophage signaling utilizing RNA microarrays in swine nasal turbinate cells. METHODS: Bor-BRP-1 phages were applied 24 h prior to B.bronchiseptica infection (1 × 107 cfu/ml) at several concentrations of bacterial infection. Cells were incubated to detect cytokines and 24 h to detect mucin production. And real-time quantitative PCR was performed to examine related genes expression. To determine the change of total gene expression based on B.bronchiseptica and Bor-BRP-1 treatment, we performed RNA sequencing experiments. RESULTS: The results showed that B. bronchiseptica induced increased expression of several inflammatory genes such as IL-1ß, IL-6, and Muc1 in a dose-dependent manner. However, Bor-BRP-1 induced reduction of gene expression compared to the B. bronchiseptica induction group. In addition, microarrays detected Bor-BRP-1-altered inflammatory gene expression against B. bronchiseptica, reducing B. bronchiseptica-induced airway inflammation in swine epithelial cells. CONCLUSION: These results suggest that the specific bacteriophage has a therapeutic potential to defend against B. bronchiseptica infection by altering inflammatory gene expression profiles.

Pasteurella multocida specific bacteriophage suppresses P. multocida-induced inflammation: identification of genes related to bacteriophage signaling by Pasteurella multocida-infected swine nasal turbinate cells.

BACKGROUND: Although Pasteurella multocida is highly prevalent pathogen in animals and plays an important role in swine respiratory diseases, only a few studies on the use of bacteriophages specific to Pasteurella multocida disease have been reported. OBJECTIVE: The object of this study was to investigate the therapeutic effect of specific P. multocida bacteriophages and to identify genes related to bacteriophage signaling utilizing RNA microarrays in swine nasal turbinate cells. METHODS: Pas-MUP-1 phages were applied 24 h prior to P. multocida infection (1 × 107 cfu/ml) at several concentrations of bacterial infection. Cells were incubated to detect cytokines and 24 h to detect mucin production. And real-time quantitative PCR was performed to examine related genes expression. To determine the change of total gene expression based on P. multocida and Pas-MUP-1 treatment, we performed RNA sequencing experiments. RESULTS: We found that P. multocida-infected PT-K75 cells show increased gene expression of IL-1ß, IL-6, and Muc1 in a dose-dependent manner. Interestingly, these genes resulted in decreased expression in P. multocida pretreated with the P. multocida-specific Pas-MUP-1 bacteriophage. RNA sequencing analysis revealed that bacteriophage administration regulated genes associated with immune and inflammatory responses, and the regulated genes were dramatically concentrated in the cytokine/chemokine-based signaling pathways. Pas-MUP-1 treatment was shown to regulate P. multocida induced gene expression in the bacteria. CONCLUSION: These results suggest the specific bacteriophage has therapeutic potential as an alternative to antibiotic treatment to defend against P. multocida infection by altering inflammatory gene expression profiles.

Complete Genome Sequence of Aeromonas rivipollensis KN-Mc-11N1, Isolated from a Wild Nutria (Myocastor coypus) in South Korea.

We report here the complete genome sequence of Aeromonas rivipollensis KN-Mc-11N1, which was isolated from a wild nutria (Myocastor coypus) in South Korea. Genomic analysis indicated that A. rivipollensis may have zoonotic potential similar to that of other aeromonads, and nutria could be one of the sources of transmission of zoonotic pathogens to humans.

Bordetella bronchiseptica bateriophage suppresses B. bronchiseptica-induced inflammation in swine nasal turbinate cells.

The development of therapeutic bacteriophages will provide several benefits based on an understanding the basic physiological dynamics of phage and bacteria interactions for therapeutic use in light of the results of antibiotic abuse. However, studies on bacteriophage therapeutics against microbes are very limited, because of lack of phage stability and an incomplete understanding of the physiological intracellular mechanisms of phage. The major objective of this investigation was to provide opportunity for development of a novel therapeutic treatment to control respiratory diseases in swine. The cytokine array system was used to identify the secreted cytokines/chemokines after Bordetella bronchiseptica infection into swine nasal turbinate cells (PT-K75). We also performed the real-time quantitative PCR method to investigate the gene expression regulated by B. bronchiseptica infection or bacteriophage treatment. We found that B. bronchiseptica infection of PT-K75 induces secretion of many cytokines/chemokines to regulate airway inflammation. Of them, secretion and expression of IL-1ß and IL-6 are increased in a dose-dependent manner. Interestingly, membrane-bound mucin production via expression of the Muc1 gene is increased in B. bronchiseptica-infected PT-K75 cells. However, cytokine production and Muc1 gene expression are dramatically inhibited by treatment with a specific B. bronchiseptica bacteriophage (Bor-BRP-1). The regulation of cytokine profiles in B. bronchiseptica-induced inflammation by B. bronchiseptica bacteriophage is essential for avoiding inappropriate inflammatory responses. The ability of bacteriophages to downregulate the immune response by inhibiting bacterial infection emphasizes the possibility of bacteriophage-based therapies as a novel anti-inflammatory therapeutic strategy in swine respiratory tracts.

Evaluation of the broad-spectrum lytic capability of bacteriophage cocktails against various Salmonella serovars and their effects on weaned pigs infected with Salmonella Typhimurium.

The broad-spectrum lytic capability of Salmonella bacteriophages against various Salmonella species was evaluated to determine their potential as an alternative for antibiotics, and the safety and preventive effects of the bacteriophages were assessed on mice and pigs. Four bacteriophage cocktails were prepared using 13 bacteriophages, and the lytic capability of the four bacteriophage cocktails was tested using Salmonella reference strains and field isolates. Bacteriophage cocktail C (SEP-1, SGP-1, STP-1, SS3eP-1, STP-2, SChP-1, SAP-1, SAP-2; ≥109 pfu/ml) showed the best lytic activity against the Salmonella reference strains (100% of 34) and field isolates (92.5% of 107). Fifty mice were then orally inoculated with bacteriophage cocktail C to determine the distribution of bacteriophages in various organs, blood and feces. The effects of bacteriophages on Salmonella infection in weaned pigs (n=15) were also evaluated through an experimental challenge with Salmonella Typhimurium after treatment with bacteriophage cocktail C. All mice exhibited distribution of the bacteriophages in all organs, blood and feces until 15 days post infection (dpi). After 35 dpi, bacteriophages were not detected in any of these specimens. As demonstrated in a pig challenge study, treatment with bacteriophage cocktail C reduced the level of Salmonella shedding in feces. The metagenomic analyses of these pig feces also revealed that bacteriophage treatment decreased the number of species of the Enterobacteriaceae family without significant disturbance to the normal fecal flora. This study showed that bacteriophages effectively controlled Salmonella in a pig challenge model and could be a good alternative for antibiotics to control Salmonella infection.

Characterisation of the antibacterial properties of the recombinant phage endolysins AP50-31 and LysB4 as potent bactericidal agents against Bacillus anthracis.

The recombinant phage endolysins AP50-31 and LysB4 were developed using genetic information from bacteriophages AP50 and B4 and were produced by microbial cultivation followed by chromatographic purification. Subsequently, appropriate formulations were developed that provided an acceptable stability of the recombinant endolysins. The bacteriolytic properties of the formulated endolysins AP50-31 and LysB4 against several bacterial strains belonging to the Bacillus genus including Bacillus anthracis (anthrax) strains were examined. AP50-31 and LysB4 displayed rapid bacteriolytic activity and broad bacteriolytic spectra within the Bacillus genus, including bacteriolytic activity against all the B. anthracis strains tested. When administered intranasally, LysB4 completely protected A/J mice from lethality after infection with the spores of B. anthracis Sterne. When examined at 3 days post-infection, bacterial counts in the major organs (lung, liver, kidney, and spleen) were significantly lower compared with those of the control group that was not treated with endolysin. In addition, histopathological examinations revealed a marked improvement of pathological features in the LysB4-treated group. The results of this study support the idea that phage endolysins are promising candidates for developing therapeutics against anthrax infection.

Simultaneous RNA-seq based transcriptional profiling of intracellular Brucella abortus and B. abortus-infected murine macrophages.

Brucella is a zoonotic pathogen that survives within macrophages; however the replicative mechanisms involved are not fully understood. We describe the isolation of sufficient Brucella abortus RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular B. abortus and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection. Our results revealed that 25.12% (801/3190) and 16.16% (515/3190) of the total B. abortus genes were up-regulated and down-regulated at >2-fold, respectively as compared to the free-living B. abortus. Among >5-fold differentially expressed genes, the up-regulated genes are mostly involved in DNA, RNA manipulations as well as protein biosynthesis and secretion while the down-regulated genes are mainly involved in energy production and metabolism. On the other hand, the host responses during B. abortus infection revealed that 14.01% (6071/43,346) of BMM genes were reproducibly transcribed at >5-fold during infection. Transcription of cytokines, chemokines and transcriptional factors, such as tumor necrosis factor (Tnf), interleukin-1α (Il1α), interleukin-1ß (Il1ß), interleukin-6 (Il6), interleukin-12 (Il12), chemokine C-X-C motif (CXCL) family, nuclear factor kappa B (Nf-κb), signal transducer and activator of transcription 1 (Stat1), that may contribute to host defense were markedly induced while transcription of various genes involved in cell proliferation and metabolism were suppressed upon B. abortus infection. In conclusion, these data suggest that Brucella modulates gene expression in hostile intracellular environment while simultaneously alters the host pathways that may lead to the pathogen's intracellular survival and infection.

Transcriptomic analysis of genes modulated by cyclo(L-phenylalanine-L-proline) in Vibrio vulnificus.

Diketopiperazine is produced by various organisms, including bacteria, fungi, and animals, and has been suggested as a novel signal molecule involved in the modulation of genes with various biological functions. Vibrio vulnificus, which causes septicemia in humans, produces cyclo(L-phenylalanine-L-proline) (cFP). To understand the biological roles of cFP, the effect of the compound on the expression of the total mRNA in V. vulnificus was assessed by nextgeneration sequencing. Based on the transcriptomic analysis, we classified the cFP-regulated genes into functional categories and clustered them according to the expression patterns resulted from treatment with cFP. From a total of 4,673 genes, excepting the genes encoding tRNA in V. vulnificus, 356 genes were up-regulated and 602 genes were down-regulated with an RPKM (reads per kilobase per million) value above 3. The genes most highly induced by cFP comprised those associated with the transport and metabolism of inorganic molecules, particularly iron. The genes negatively regulated by cFP included those associated with energy production and conversion, as well as carbohydrate metabolism. Noticeably, numerous genes related with biofilm formation were modulated by cFP. We demonstrated that cFP interferes significantly with the biofilm formation of V. vulnificus.

The fur-iron complex modulates expression of the quorum-sensing master regulator, SmcR, to control expression of virulence factors in Vibrio vulnificus.

The gene vvpE, encoding the virulence factor elastase, is a member of the quorum-sensing regulon in Vibrio vulnificus and displays enhanced expression at high cell density. We observed that this gene was repressed under iron-rich conditions and that the repression was due to a Fur (ferric uptake regulator)-dependent repression of smcR, a gene encoding a quorum-sensing master regulator with similarity to luxR in Vibrio harveyi. A gel mobility shift assay and a footprinting experiment demonstrated that the Fur-iron complex binds directly to two regions upstream of smcR (-82 to -36 and -2 to +27, with respect to the transcription start site) with differing affinities. However, binding of the Fur-iron complex is reversible enough to allow expression of smcR to be induced by quorum sensing at high cell density under iron-rich conditions. Under iron-limiting conditions, Fur fails to bind either region and the expression of smcR is regulated solely by quorum sensing. These results suggest that two biologically important environmental signals, iron and quorum sensing, converge to direct the expression of smcR, which then coordinates the expression of virulence factors.

Iron and quorum sensing coordinately regulate the expression of vulnibactin biosynthesis in Vibrio vulnificus.

Vibrio vulnificus is a halophilic marine pathogen associated with human diseases such as septicemia and serious wound infections. Genes vvsA and vvsB, which are co-transcribed and encode a member of the nonribosomal peptide synthase family, are required for vulnibactin biosynthesis in V. vulnificus. In this study, we found that quorum sensing represses the transcription of a vvsAB-lux reporter fusion. Gel shift assay and DNaseI footprinting experiments show that the main regulator of quorum sensing, SmcR, binds to a 22-bp region located between -40 and -19 with respect to the vvsA transcription start site. Mutation of the SmcR binding site abolishes the repression of vvsA::luxAB by SmcR. Fur represses vvsAB transcription in the presence of iron by binding to a 47-bp region located between -45 and +2 with respect to the vvsA transcription start site. A competition gel shift assay and footprinting experiment using Fur and SmcR showed that Fur binds to the vvsA promoter region with higher affinity than SmcR. Studies with the vvsAB::luxAB transcriptional fusion demonstrate that in the presence of iron, Fur is the key repressor of vvsAB transcription, whereas in iron-limited conditions, SmcR is the key regulator repressing vvsAB transcription. This study demonstrates that the Fe-Fur complex and quorum sensing cooperate to repress the transcription of vvsAB in response to iron conditions, suggesting that fine tuning of the intracellular iron level is important for the survival and pathogenicity of V. vulnificus.

Complete genome sequence of bacteriophage phiAS7, a T7-like virus that infects Aeromonas salmonicida subsp. salmonicida.

To date, a number of Myoviridae bacteriophages that infect Aeromonadaceae have been identified and characterized. However, the genome sequences of Aeromonas phages that not belong to the Myoviridae have not been investigated yet. Herein, we report the complete genome sequence of Aeromonas phage phiAS7, which belongs to the Podoviridae and infects Aeromonas salmonicida subsp. salmonicida.

Complete genome sequence and characterization of a broad-host range T4-like bacteriophage phiAS5 infecting Aeromonas salmonicida subsp. salmonicida.

In this study, we report one lytic Myoviridae bacteriophage (phage) infecting Aeromonas salmonicida subsp. salmonicida. The phage (named as phiAS5) was isolated from environmental river waters in Korea, and showed broad infectivity to other bacterial species in the family Aeromonadaceae as well as antibiotic-resistant A. salmonicida subsp. salmonicida strains. The biological properties and complete genome of phiAS5 were simultaneously investigated. The complete genome of phiAS5 composed of linear double-stranded DNA of 225,268 bp with G+C content of 43.0%, and encoded 343 putative ORFs, 69 putative promoters, 33 transcriptional terminator regions and 24 tRNA-encoding genes. A high degree of similarity to other T4-like Aeromonas phage was found in most ORFs of phiAS5. Therefore, the genome of phiAS5 was further compared with T4 phage and the closest relative, Aeromonas phage Aeh1, and the result demonstrated that it could be classified as a new member of the T4-like group. The bacteriolytic activity of phiAS5 against A. salmonicida subsp. salmonicida was evaluated at different doses of multiplicity of infection using one each of virulent strain that possesses the ascV gene and multi-drug resistant strain, and the results proved to be efficient for the reduction of bacterial growth. Based on these results, phiAS5 may have the potential for reducing the impacts of virulent or antibiotic-resistant A. salmonicida subsp. salmonicida in aquaculture and may also advance our understanding of the biodiversity of T4-like Aeromonas phages.

Identification of a peptide that interacts with Nestin protein expressed in brain cancer stem cells.

Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.

Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea.

The antibiotic resistance of 16 Aeromonas (A.) salmonicida strains isolated from diseased fish and environmental samples in Korea from 2006 to 2009 were investigated in this study. Tetracycline or quinolone resistance was observed in eight and 16 of the isolates, respectively, based on the measured minimal inhibitory concentrations. Among the tetracycline-resistant strains, seven of the isolates harbored tetA gene and one isolate harbored tetE gene. Additionally, quinolone-resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A. salmonicida strains, 15 harbored point mutations in the gyrA codon 83 which were responsible for the corresponding amino acid substitutions of Ser(83)→Arg(83) or Ser(83)→Asn(83). We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84. Genetic similarity was assessed via pulsed-field gel electrophoresis, and the results indicated high clonality among the Korean antibiotic-resistant strains of A. salmonicida.

Comparison of the antibacterial properties of phage endolysins SAL-1 and LysK.

In spite of the high degree of amino acid sequence similarity between the newly discovered phage endolysin SAL-1 and the phage endolysin LysK, SAL-1 has an approximately 2-fold-lower MIC against several Staphylococcus aureus strains and higher bacterial cell-wall-hydrolyzing activity than LysK. The amino acid residue change contributing the most to this enhanced enzymatic activity is a change from glutamic acid to glutamine at the 114th residue.

Identification of virulence factors in vibrio vulnificus by comparative transcriptomic analyses between clinical and environmental isolates using cDNA microarray.

We compared the gene expression among four clinical and five environmental V. vulnificus isolates, using a cDNA microarray containing 131 genes possibly associated with pathogenicity, transport, signal transduction, and gene regulations in the pathogen. cDNAs from total RNAs of these isolates were hybridized into the cDNA microarray using the cDNA of the wild-type strain MO6/24-O as a reference. We focused on selecting differentially expressed (DE) genes between clinical and environmental isolates using a modified t-statistic. We could detect two statistically significant DE genes between virulent isolates and less-virulent isolates with a marginal statistical significance (pvalue of 0.008). These were genes putatively encoding pilin and adenlyate cylase. Real time-PCR confirmed that these two selected genes transcribed in significantly higher levels in virulent isolates than in less-virulent isolates. Mutants with lesions in the gene encoding pilin showed significantly higher LD50 values than that of wild type.

Antibacterial and biofilm removal activity of a podoviridae Staphylococcus aureus bacteriophage SAP-2 and a derived recombinant cell-wall-degrading enzyme.

Antibacterial and biofilm removal activity of a new podoviridae Staphylococcus aureus bacteriophage (SAP-2), which belongs to the phi29-like phage genus of the Podoviridae family, and a cell-wall-degrading enzyme (SAL-2), which is derived from bacteriophage SAP-2, have been characterized. The cell-wall-degrading enzyme SAL-2 was expressed in Escherichia coli in a soluble form using a low-temperature culture. The cell-wall-degrading enzyme SAL-2 had specific lytic activity against S. aureus, including methicillin-resistant strains, and showed a minimum inhibitory concentration of about 1 microg/ml. In addition, this enzyme showed a broader spectrum of activity within the Staphylococcus genus compared with bacteriophage SAP-2 in its ability to remove the S. aureus biofilms. Thus, the cell-wall-degrading enzyme SAL-2 can be used to prevent and treat biofilm-associated S. aureus infections either on its own or in combination with other cell-wall-degrading enzymes with anti-S. aureus activity.