• Microbiology and Biotechnology Letters
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• v.51 no.4
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• pp.390-402
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• 2023

Salmonella are Gram-negative pathogenic bacteria commonly found in food animals such as poultry and swine and potentially constitute risks and threats to food safety and public health through transmissible virulence and antimicrobial resistance (AMR) genes. Although there are previous studies in the Philippines regarding genotypic and phenotypic AMR in Salmonella, there are very few on virulence and their associations. Hence, this study collected 700 Salmonella isolates from swine samples in abattoirs and wet markets among four districts in Metro Manila and characterized their genotypic virulence and β-lactam AMR profiles. Gene frequency patterns and statistical associations between virulence and bla genes and comparisons based on location types (abattoirs and wet markets) and districts were also determined. High prevalence (>50%) of virulence genes was detected encompassing Salmonella pathogenicity islands (SPIs) 1-5 suggesting their pathogenic potential, but none possessed plasmid-borne virulence genes spvR and spvC. For bla, bla_TEM was detected with high prevalence (>45%) and revealed significant associations to four SPI genes, namely, avrA, hilA, mgtC, and spi4R, which suggest high resistance potential particularly to β-lactam antibiotics and relationships with pathogenicity that remain mechanistically unestablished until now. Lastly, comparisons of location types and districts showed variations in gene prevalence suggesting effects from environmental factors throughout the swine production chain. This study provides vital data on the genotypic virulence and AMR of Salmonella from swine in abattoirs and wet markets that suggest their pathogenicity and resistance potential for policymakers to implement enforced surveillance and regulations for the improvement of the Philippine swine industry.

• Journal of Microbiology
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• v.45 no.4
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• pp.297-304
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• 2007

The detection of virulence factors of Aeromonas is a key component in determining potential pathogenicity because these factors act multifunctionally and multifactorially. In this study water samples were collected from a trout farm on a seasonal basis, and diseased fish and Aeromonas species were isolated and identified. For rapid detection of six virulence factors of isolated Aeromonas, a hexaplex-polymerase chain reaction (hexaplex-PCR) assay was used. The detected virulence factors include aerolysin (aer), GCAT (gcat), serine protease (ser), nuclease (nuc) lipase (lip) and lateral flagella (laf). The dominant strain found in our isolates was Aeromonas sobria, and the dominant virulence factors were aer and nuc for all seasons. We confirmed that A. sobria and two of the virulence genes (aer and nuc) are related. We proposed a method by which one can identify the major strains of Aeromonas: A. hydrophila, A. sobria, A. caviae, and A. veronii, using hexaplex-PCR.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.161-170
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• 2017

Meningitis caused by Streptococcus suis serotype 2 (S. suis 2) is a great threat to the pig industry and human health. Virulence factors associated with the pathogenesis of meningitis have yet to be clearly defined, even though many potential S. suis 2 virulence factors have been identified. This greatly hinders the progress of S. suis 2 meningitis pathogenesis research. In this study, a co-culture blood-brain barrier (BBB) model was established using primary porcine brain microvascular endothelial cells and astrocytes, and the whole genome library of S. suis 2 was constructed using phage display technology. Finally, a total of 14 potential virulence factors contributing to S. suis 2 adherence to and invasion of the BBB were selected by analyzing the interactions between the phage library and the co-culture model. Twelve of these factors have not been previously reported in meningitis-related research. The data provide valuable insight into the pathogenesis of S. suis 2 meningitis and potential targets for the development of drug therapies.

• The Plant Pathology Journal
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• v.22 no.1
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• pp.28-35
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• 2006

Virulence and genetic diversity were studied using 21 isolates of Sclerospora graminicola, the pearl millet downy mildew pathogen collected from major pearl millet growing areas of India. Variability for virulence was determined by inoculating a set of 10 differential hosts with the S. graminicola isolates in a greenhouse. The isolates varied for latent period (6.4 to 11 days), disease incidence (0 to $98\%$), virulence index (0 to 18.7) and oospore-production potential (1 to 4). Among the 21 isolates, Sg 139 (Rajasthan) was the most virulent and Sg 110 (Tamil Nadu) the least virulent. Based on virulence index (disease incidence$\time$slatent $period^{-1}$), the 21 isolates were classified into eight virulence groups. Genetic diversity among isolates was studied using AFLP markers. Based on similarity index of banding pattern, the 21 isolates were clustered into eight genotypic groups. The AFLP groupings, however, did not match with that of the virulence groupings, and these two were found independent. The isolate Sg 139 that remained distinct in both pathogenic and genetic groupings indicated its highly virulent nature. Implications of these results in downy mildew resistance breeding are discussed.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1475-1481
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• 2009

Anthrax is a zoonotic disease caused by Bacillus anthracis, and well recognized as a potential agent for bioterrorism. B. anthracis can be identified by detecting the virulence factors genes located on two plasmids, pXO1 and pXO2. The aim of the present study was to determine the presence of virulence genes in 27 isolates of B. anthracis isolated from clinical and environmental samples. For this purpose, multiplex PCR and DNA probes were designed to detect protective antigen (pag), edema factor (cya), lethal factor (lef), and capsule (cap) genes. Our results indicated that all the isolates contained all the above virulence genes, suggesting that the isolates were virulent. To the best our knowledge, this is the first study about the determination of virulence marker genes in clinical and environmental isolates of B. anthracis using multiplex PCR and DNA probes in India. We suggest that the above methods can be useful in specific identification of virulent B. anthracis in clinical and environmental samples.

• Genomics & Informatics
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• v.20 no.4
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• pp.41.1-41.9
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• 2022

The pathogen Gallibacterium anatis has caused heavy economic losses for commercial poultry farms around the world. However, despite its importance, the functions of its hypothetical proteins (HPs) have been poorly characterized. The present study analyzed the functions and structures of HPs obtained from Gallibacterium anatis (NCTC11413) using various bioinformatics tools. Initially, all the functions of HPs were predicted using the VICMpred tool, and the physicochemical properties of the identified virulence proteins were then analyzed using Expasy's ProtParam server. A virulence protein (WP_013745346.1) that can act as a potential drug target was further analyzed for its secondary structure, followed by homology modeling and three-dimensional (3D) structure determination using the Swiss-Model and Phyre2 servers. The quality assessment and validation of the 3D model were conducted using ERRAT, Verify3D, and PROCHECK programs. The functional and phylogenetic analysis was conducted using ProFunc, STRING, KEGG servers, and MEGA software. The bioinformatics analysis revealed 201 HPs related to cellular processes (n = 119), metabolism (n = 61), virulence (n = 11), and information/storage molecules (n = 10). Among the virulence proteins, three were detected as drug targets and six as vaccine targets. The characterized virulence protein WP_013745346.1 is proven to be stable, a drug target, and an enzyme related to the citrate cycle in the present pathogen. This enzyme was also found to facilitate other metabolic pathways, the biosynthesis of secondary metabolites, and the biosynthesis of amino acids.

• The Korean Journal of Mycology
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• v.26 no.3 s.86
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• pp.387-395
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• 1998

Mycelial growth, production of microsclerotia and perithecia, and presence of double-stranded RNA were examined in Calonectria ilicicola isolates from several hosts to detect morphological and/or genetic markers for comparison with levels of virulence. Variability in disease severity, production of microsclerotia and perithecia, and mycelial growth was observed across all isolates. None of 35 isolates of C. ilicicola examined contained detectable levels of double-stranded RNA. Disease severity on soybean cultivars correlated positively with production of both microsclerotia and perithecia. Mycelial growth correlated negatively with production of perithecia. Virulence on the cultivars and production of microsclerotia and perithecia were greater in isolates of C. ilicicola from soybean than those from peanut. These results suggest that the ability of isolates to produce microsclerotia and perithecia is a component of inoculum potential. Perithecia production may serve as a useful marker for characterizing virulence or host specialization in this homothallic fungus.

• Korean Journal of Veterinary Research
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• v.32 no.2
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• pp.181-194
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• 1992

Two hundred and eighty nine strains of Yersinia enterocolitica isolated from healthy pigs were tested for the presence of 40~50 Megadalton virulence-associated plasmids and plasmidmediated in vitro virulence-associated properties, i.e., congo red uptake, calcium dependency, autoagglutination, CRMOX reaction, crystal violet binding and pyrazinamidase reaction. The correlationships between in vitro virulence-associated properties and the presence of 220 Kdalton outer membrane protein(OMP) were examined in strains with or without virulence-associated plasmids. The correlationships between the presence of plasmids on the production of the OMP and the expression of in vitro virulence-associated properties were studied with $CRMOX^+$ strains and acridine orangecured $CRMOX^-$ mutants. The results were as follows : 1. Of the in vitro virulence-associated tests with 289 strains of Y enterocolitica, 275 strains (95.2%) were positive for pyrazinamidase test, and followed by in order of crystal violet binding test, 226 (79.2% ) ; CRMOX test, 190 (65.7%) ; autoagglutination test, 1.85(64.0%) : calcium dependency test, 86 (29.8%) and congo red uptake test, 47(16.3%). 2. The correlationship between autoagglutination and CRMOX test(r=0.90) was highly significant (p<0.01). 3. In 190 strains(65.7%) bearing the virulence-associated plasmids(MW 40~50 Mdalton), the correlation between the presence of plasmids and their in vitro virulence-associated properties were highest with CRMOX test(r=0.93) and followed by in orders of AAG test(0.81), CV test(0.46), PYZ test(0.37) and CD test(0.18), but no correlationship between the presence of plasmids and CR test(-0.11). 4. The $CRMOX^+$ strains produced the 220 Kdalton OMP when they were cultured at $37^{\circ}C$, but not at $26^{\circ}C$. The presence of 220 Kdalton OMP was correlated significantly with in vitro virulence properties and the presence of virulence-associated plasmid, respectively. 5. In the isogenic $CRMOX^-$ mutant strains, of which plasmid were cured by treatment with acridine orange not only in vitro virulence-associated properties(CR 100%, CD 100%, AAG 82.6%, CV 58.3%) disappeared but also 220 Kdalton OMP(100%) was not produced. These results indicate that the positive CRMOX reaction is plasmid-mediated and the CRMOX test is potential as an in vitro virulence tests with Y enterocolitica.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.651-661
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• 2019

Targeting the pathogen viability using drugs is associated with development of drug resistance due to selective pressure. Hence, there is an increased interest in developing agents that target bacterial virulence. In this study, the inhibitory effect of ciclopirox, an antifungal agent with iron chelation potential, on the microbial virulence factors was evaluated in 26 clinical MDR Pseudomonas aeruginosa isolates collected from Alexandria Main University Hospital, a tertiary hospital in Egypt. Treatment with 9 ㎍/ml ciclopirox inhibited the hemolytic activity in 70% isolates, reduced pyocyanin production, decreased protease secretion in 46% isolates, lowered twitching and swarming motility, and decreased biofilm formation by 1.5- to 4.5-fold. The quantitative real-time PCR analysis revealed that treatment with ciclopirox downregulated the expression levels of alkaline protease (aprA) and pyocyanin (phzA1). Ciclopirox is used to treat hematological malignancies and the systemic administration of ciclopirox is reported to have adequate oral absorption with a satisfactory drug safety profile. It is important to calculate the appropriate clinical dose and therapeutic index to reposition ciclopirox from a topical antifungal agent to a promising virulence-modifying agent agent against P. aeruginosa, a problematic Gram-negative pathogen.

• Korean Journal of Veterinary Service
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• v.41 no.3
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• pp.157-163
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• 2018

In order for monitoring of pathogenic bacterial contamination in the freshly slaughtered poultry meats produced in Gyeong-Nam province, we first isolated 4 strains of Salmonella spp. and 32 strains of Enterococcus faecalis from the total 164 samples, then we analyzed potential virulence gene profiles of the bacterial isolates by PCR using species-specific primer. The potential virulence genes we selected in this study were stn, invA, fimA, spvR, and spvC for the isolates of Salmonella spp. and those of esp, cylM, cylA, cylB, gelE, fsrA, fsrB, and fsrC were for the isolates of E. faecalis. The PCR results showed that all 5 virulence genes were detected simultaneously in the all isolates of Salmonella spp. However, there was a diverse occurrence pattern of the virulence genes in the case of E. faecalis. The gene for enterococcal surface protein (esp) was not detected among the isolates (0/32), and the haemolysin gene prevalence rate of cylA, cylB, and cylM were 3.1% (1/32), 9.3% (3/32), and 9.3% (3/32), respectively. Moreover, the genes of gelE, fsrA, fsrB, and fsrC that associated with gelatinase activity were detected in the rate of 53.1% (17/32), 53.1% (17/32), 53.1% (17/32), and 53.1% (17/32), respectively. In conclusion, in the isolates of Salmonella spp., all possessed 5 virulence genes tested, suggesting that they are all related with each other clonally. However, in the case of E. faecalis isolates, the occurrence of the haemolysin genes (cylM, cylA, cylB) and the gelatinase genes (gelE, fsrABC) was highly variable among the isolates.