• 한국동물위생학회지
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• 제33권4호
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• pp.341-344
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• 2010

Poultry and poultry products have been implicated as a major source of Salmonella infection in human, and infection due to Salmonella serotypes continue to be a major health problem. The presence of two virulence genes, invA and spvC, in 34 Salmonella isolates obtained from duck farms was investigated. All isolates contained the invA gene, and spvC gene was found in 20 (58.8%) of 34 Salmonella isolates : S. Typhimurium (n=8), S. Fyris (n=5), S. Enteritidis (n=3), S. Typhimurium var. copenhagen (n=1), S. Haardt (n=1) and S. Mbandaka (n=1). This study showed the presence of the spvC gene was widely distributed in between different Salmonella enterica isolates.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1353-1358
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• 2005

A rapid and quantitative real-time PCR was developed to target the invasion A (invA) gene of Salmonella spp. We developed quantitative standard curves based on plasmids containing the invA gene. Based on these curves, we detected Salmonella spp. in artificially contaminated buffered peptone water (BPW) and milk samples. We were able to determine the invA gene copy number per ml of food samples, with the minimum detection limit of $4.1{\times}10^{3}$ copies/ml of BPW and $3.3{\times}10^{3}$ copies/ml of milk. When applied directly to detect and quantify Salmonella spp. in BPW and milk, the present real-time PCR assay was as sensitive as the plate count method; however, copy numbers were one to two logs higher than the colony-forming units obtained by the plate count methods. In the present work, the real-time PCR assay was shown to significantly reduce the total time necessary for the detection of Salmonella spp. in foods and to provide an important model for other foodborne pathogens.

• 한국미생물·생명공학회지
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• 제40권1호
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• pp.70-75
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• 2012

살모넬라(Salmonella)의 염색체에 존재하는 병원성 유전자의 집합체인 Salmonella pathogenicity island(SPI)1 과 2는 살모넬라가 유발하는 다양한 질병에 중요한 역할을 한다. SPI1의 발현을 유도하는 HilD는 Luria-Bertani(LB) 배지 조건에서 SPI2의 발현 활성인자로 작용하는 것으로 알려져 있으나 LB 배지 내에서 hilD 유전자의 발현 양상은 아직까지 연구되지 않았다. 본 연구에서는 LB 배지에 살모넬라를 배양하면서 hilD 유전자의 발현과 단백질 양을 조사하였으며 SPI2 유전자인 sseA의 발현과 비교하였다. hilD의 발현은 대수 증식기 경과 후 정지기(stationary phase)로 전환되는 시기에 비약적으로 증가하였으나 sseA의 발현은 정지기 후반부에 최대로 증가하였다. 즉, 후반 정지기에서 HilD 단백질은 낮은 수준으로 존재함에도 불구하고 SPI2의 발현을 유도한다는 것을 알 수 있었다. SPI1의 다른 발현 조절인자인 hilA와 invF의 변이체에서 sseA의 발현을 살펴본 결과 invF의 변이는 hilD와는 다르게 배지 조건에 상관없이 오히려 sseA의 발현을 증가시켰다. 또한, InvF의 과발현은 sseA 발현을 정상 수준으로 복원시켰지만 추가적인 감소는 일으키지 않는다는 것을 알 수 있었다. SPI1은 HilD를 이용하여 SPI2의 발현을 유도하지만 반대로 InvF를 이용하여 발현을 억제하기도 하는 이중적인 조절 기전을 가지고 있는 것으로 판단된다.

• 한국식품위생안전성학회지
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• 제22권4호
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• pp.248-256
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• 2007

돼지고기에 오염된 살모넬라의 수를 직접 측정하는 방법으로 경쟁적 PCR(competitive PCR)을 사용하였다. 세가지 DNA추출방법을 비교한 후 guanidine thiocyanate-phenol-chloroform 방법을 일부 수정하여 인위적으로 살모넬라를 오염시킨 돼지고기로부터 DNA를 직접 추출하는 방법으로 선택하였다. Rahn 등(Mol. Cell. Probes 1992년)이 이미 보고한 284-bp iuvA gene의 특이성을 평가하기 위해 살모넬라 57균주와 살모넬라가 아닌 균주 24개를 사용하였다. 시험해 본 모든 살모넬라 균주는 invA 양성으로 살모넬라가 아닌 모든 균주는 invA 음성으로 판명되었으며 살모넬라가 아닌 균주 중 양성으로 잘 못 판명된 경우는 없었다. 돼지고기 0.1 g을 사용할 경우 검출한계는 1,460 cfu였다. 경쟁적 PCR을 위해 invA gene중 82-bp를 결실시킨 DNA조각을 pGEM-4Z백터에 클로닝을 하였다. 인위적으로 오염된 돼지고기로부터 추출한 살모넬라 염색체 DNA와 이와 같은 primer결합자리를 가진 경쟁 DNA를 동시에 경쟁적 PCR로 증폭하였다. 경쟁적 PCR을 사용하여 결정한 균수와 MPN방법으로 결정한 균수는 거의 유사하였으며 전체 과정을 수행하는 데 약 5시간이 소요되었다.

• Food Science and Biotechnology
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• 제16권5호
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• pp.778-782
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• 2007

This study assessed the effects of the antimicrobial substances sulforaphane, grapefruit seed extracts (GSE), and reuterin on the expression of Salmonella HilA and InvF virulence gene using a LacZY assay (${\beta}$-galactosidase assay) with hilA:lacZY and invF:lacZY fusion strains of Salmonella typhimurium SL1344. Salmonella was grown for 8 hr at $37^{\circ}C$ in the presence of diluted antimicrobial substances ($2\;{\mu}g/mL$ sulforaphane, $20\{\mu}g/mL$ GSE, and 0.26 mM reuterin) at concentrations that did not inhibit the cellular growth of Salmonella. Sulforaphane inhibited the expression of HilA and InvF by 50-90 and 20-80%, respectively. GSE also inhibited the expression of both genes, but to a lesser degree. Among the 3 antimicrobial substances, reuterin showed the least inhibition, which was abolished after 3-4 hr. None of the antimicrobial substances inhibited the ${\beta}$-galactosidase enzyme activity of S. typhimurium. The assay used in this study represents a very sensitive method for screening bioactive substances that inhibit the expression of virulence genes in Salmonella.

• Journal of Veterinary Science
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• 제24권6호
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• pp.82.1-82.12
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• 2023

Background: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. Objective: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. Methods: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). Results: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [bla_TEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3")-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. Conclusion: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

• 한국동물위생학회지
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• 제26권1호
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• pp.39-50
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• 2003

Non-typhoidal Salmonella serovars remain a potential threat to human health and many animals including beef cattle, broiler chickens, and pigs which possible sources of non-typhoidal salmonellosis in human. In this study, the cecal contents of slaughtered pigs were examined for Salmonella serovar prevalence. The characteristics of the isolates, including antimicrobial resistance patterns and virulence genes, were studied along with the reference strain S typhimurium ATCC 13311. Out of 640 sample, 137 Salmonella(21.4%) were isolated and their serovar were identified S typhimurium 83 strains(60.6%), S agona 10 strains(7.3%), S schwarzengrund 4 strains(2.9%), S derby 4 strains(2.9%), S ayinde 1 strains(0.7%), and untypable 35 strains(25.5%). All 83 S typhimurium strains(100%) were multi-drug resistance to at least 7 antibiotics, and 20 strains(24.1%) of 83 isolates were R-type ACSSuT. Examination of virulent gene by PCR revealed that 73 S typimurium field isolates(88%) have a invA gene and 24 strains(28.9%) have a spvC gene. Consequently, S typhimurium infection in slaughtered pigs was relatively to appear high prevalence in their herds which suggested that it should be necessary for herd health monitoring and surveillance.

• Animal Bioscience
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• 제36권4호
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• pp.654-670
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• 2023

Objective: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples. Methods: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method. Results: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63℃ for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×10² CFU/mL (6 CFU/reaction) and 9.01 pg/μL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×10⁴ CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method. Conclusion: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1664-1672
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• 2013

In Salmonella enterica serovar Typhimurium, many genes encoded within Salmonella pathogenicity island 1 (SPI1) are required to induce intestinal/diarrheal disease. In this study, we compared the expression of four SPI1 genes (hilA, invF, prgH, and sipC) under shaking and standing culture conditions and found that the expression of these genes was highest during the transition from the exponential to stationary phase under shaking conditions. To identify regulators associated with the stationary phase-dependent activation of SPI1, the effects of selected regulatory genes, including relA/spoT (ppGpp), luxS, ihfB, hfq, and arcA, on the expression of hilA and invF were compared under shaking conditions. Mutations in the hfq and arcA genes caused a reduction in hilA and invF expression (more than 2-fold) in the early stationary phase only, whereas the lack of ppGpp and IHF decreased hilA and invF gene expression during the entire stationary phase. We also found that hfq and arcA mutations caused a reduction of hilD expression upon entry into the stationary phase under shaking culture conditions. Taken together, these results suggest that Hfq and ArcA regulate the hilD promoter, causing an accumulation of HilD, which can trigger a stationary phase-dependent activation of SPI1 genes under shaking culture conditions.

• Journal of Microbiology and Biotechnology
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• 제17권10호
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• pp.1616-1621
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• 2007

We have established a SYBR Green-based realtime PCR method using AnyDirect solution, which enhances PCR from whole blood, for direct amplification of the virA gene of Shigella flexneri and the invA gene of Salmonella typhimurium from human feces without prior DNA purification. When we compared the efficiency of conventional or realtime PCR amplification of the virA and invA genes from the supernatant of boiled feces supplemented with S. flexneri and S. typhimurium in the presence or absence of AnyDirect solution, amplification products were detected only in reactions to which AnyDirect solution had been added. The detection limit of real-time PCR was $1{\times}10^4\;CFU/g$ feces for S. flexneri and $2{\times}10^4\;CFU/g$ feces for S. typhimurium; this sensitivity level was comparable to other studies. Our real-time PCR assay with AnyDirect solution is simple, rapid, sensitive, and specific, and allows simultaneous detection of S. flexneri and S. typhimurium directly from fecal samples without prior DNA purification.