• 한국미생물·생명공학회지
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• 제47권2호
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• pp.310-316
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• 2019

Distribution of Salmonella enterica serovars and their associated virulence determinants is wide-spread among food animals, which are continuously implicated in periodic salmonellosis outbreaks globally. The aim of this study was to determine and evaluate the diversity of five Salmonella serovar virulence genes (invA, pefA, cdtB, spvC and iroN) isolated from food animals and humans. Using standard microbiological techniques, Salmonella spp. were isolated from the feces of humans and three major food animals. Virulence determinants of the isolates were assayed using PCR. Clonal relatedness of the dominant serovar was determined via pulsed-field gel electrophoresis (PFGE) using the restriction enzyme, Xbal. Seventy one Salmonella spp. were isolated and serotyped into 44 serovars. Non-typhoidal Salmonella (NTS; 68) accounted for majority (95.8%) of the Salmonella serovars. Isolates from chicken (34) accounted for 47.9% of all isolates, out of which S. Budapest (14) was predominant (34.8%). However, the dominant S. Budapest serovars showed no genetic relatedness. The invA gene located on SPI-1 was detected in all isolates. Furthermore, 94% of the isolates from sheep harbored the spvC genes. The iroN gene was present in 50%, 100%, 88%, and 91% of isolates from human, chicken, sheep, and cattle, respectively. The pefA gene was detected in 18 isolates from chicken and a single isolate from sheep. Notably, having diverse Salmonella serovars containing plasmid encoded virulence genes circulating the food chain is of public health significance; hence, surveillance is required.

• 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.

• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.25-33
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• 2012

The association of verotoxic E. coli and Acinetobacter spp. with various antibiotic-resistant, diarrhogenic, and nosocomial infections has been a cause for concern worldwide. E. coli and A. haemolyticus isolated on a number of selective media were screened for virulence factors, antibiotic resistance, and transformation of resistance genes. Out of 69 E. coli isolates obtained, 25 (35.23%), 14 (20.30%), and 28 (40.58%) were positive for Vtx1&2, Vtx1, and Vtx2, respectively, 49 (71.015%) for extendedspectrum beta-lactamases (ESBLs), 34 (49.28%) for serum resistance, 57 (82.61%) for cell surface hydrophobicity, 48 (69.57%) for gelatinase production, and 37 (53.62%) for hemolysin production. For the 14 A. haemolyticus isolates, only 2 (14.29%) in each case from all the samples investigated were positive for Vtx1, Vtx2 and Vtx1&2 respectively, 8 (57.14%) for ESBLs, 7 (50.00%) for serum resistance, 11 (78.57%) for cell surface hydrophobicity, 4 (28.57%) for gelatinase production, and 8 (57.14%) for hemolysin production. Although transformation occurred among the E. coli and Acinetobacter isolates (transformation frequency: $13.3{\times}10^{-7}-53.4^{-7}$), there was poor curing of the plasmid genes, a confirmation of the presence of stable antibiotic-resistant genes (DNA concentration between 42.7 and 123.8 ${\mu}g$) and intragenetic transfer of multidrug-resistant genes among the isolates. The isolates were potentially virulent and contained potentially transferable antibiotic resistance genes. Detection of virulence factors, antibiotic resistance genes, and transformation among these isolates is a very significant outcome that will influence approaches to proactive preventive and control measures and future investigations. However, continued surveillance for drug resistance among these bacteria and further investigation of the mechanism of action of their virulence factors are a necessity.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.898-903
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• 2009

Pullorum disease affecting poultry is caused by Salmonella enterica serovar Pullorum and results in severe economic loss every year, especially in countries with a developing poultry industry. The pathogenesis of S. Pullorum is not yet well defined, as the specific virulence factors still need to be identified. Thus, to isolate specific DNA fragments belonging to S. Pullorum, this study used suppression subtractive hybridization. As such, the genome of the S. Pullorum C79-13 strain was subtracted from the genome of Salmonella enterica serovar Gallinarum 9 and Salmonella enterica serovar Enteritidis CMCC(B) 50041, respectively, resulting in the identification of 20 subtracted fragments. A sequence homology analysis then revealed three types of fragment: phage sequences, plasmid sequences, and sequences with an unknown function. As a result, several important virulence-related genes encoding the IpaJ protein, colicin Y, tailspike protein, excisionase, and Rhs protein were identified that may play a role in the pathogenesis of S. Pullorum.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 춘계학술발표대회
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• pp.584-587
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• 2000

The invasion genes from Salmonella typhimurium were identified by the construction of a cosmid library and subcloning genes into a plasmid vector, pGEM-7Z. The 4.65 kb fragment of the invasion-conferring genomic region of the subclone, pSV6235 was sequenced in both direction. The three open reading frames, which were located at downstream of a promoter region, were designated as sir (Salmonella invasion region)A coding for the 36 amino acids, sirB coding for the 132 amino acids and sirC for the 82 amino acids, respectively. Interesingly, the genomic region of pSV6235 was highly homologous to Yersinia enterocolitica genomic DNA for a high pathogenicity island and Salmonella enteritidis insertion element IS1351 and IS200 DNA. These results show that there could be a significant relationship between S. typhimurium, Y. enterocolitica and S. enteritidis with respect to horizontal evolution process and acquisition of virulence determinants by means of transposon, plasmid or bacteriophage.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2000년도 International Meeting 2000
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• pp.58-65
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• 2000

Diarrheal diseases are a major cause of both illness and death in developing countries and are caused by rotavirus, Shigella spp., Salmonella spp., enterotoxigenic Escherichia coli (ETEC), and Vibrio spp. In this study, for the development of vaccine against diarrheal diseases caused by Shigella sonei, Salmonella typhimurium, E. coli O157, and Vibrio cholerae, cloning and nucleotide sequence analysis of genes and characteristics of their gene products in E. coli were performed. For construction of attenuated strain of S. sonnei KNIH104 and Salmonella typhimurium KNIH100, the aroA genes were cloned, respectively. The recombinant plasmid $_pJP{\Delta}A45$ containing aroA deleted region and suicide vector $(_pJP5603)$ was constructed. The aroA gene deleted mutants were constructed using this recombinant plasmid. For cloning gene encoding antigenic region of E. coli O157 KNIH317, the O-antigen synthesis gene cluster and sit gene was cloned. The E. coli XL1-Blue cells harboring this recombinant plasmid showed cytotoxicity in Vero cells. The ctx gene was cloned for tile purpose of antigenic region against V. cholerae KNIH002. Sequence analysis confirmed that the virulence gene cassette was consisted of ace, zot, ctxA and ctxB genes.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1460-1466
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• 2015

In this study, the prevalence of Shiga toxin-producing Escherichia coli (STEC) was investigated among raw meat or meat products from slaughterhouses and retail markets in South Korea, and their potential for antibiotic resistance and virulence was further analyzed. A total of 912 raw meats, including beef, pork, and chicken, were collected from 2008 to 2009. E. coli strains were frequently isolated in chicken meats (176/233, 75.9%), beef (102/217, 42.3%), and pork (109/235, 39.2%). Putative STEC isolates were further categorized, based on the presence or absence of the Shiga toxin (stx) genes, followed by standard O-serotyping. Polymerase chain reaction assays were used to detect the previously defined virulence genes in STEC, including Shiga toxins 1 and Shiga toxin 2 (stx1 and 2), enterohemolysin (ehxA), intimin (eaeA), STEC autoagglutination adhesion (saa), and subtilase cytotoxin (subAB). All carried both stx1 and eae genes, but none of them had the stx2, saa, or subAB genes. Six (50.0%) STEC isolates possessed the ehxA gene, which is known to be encoded by the 60-megadalton virulence plasmid. Our antibiogram profiling demonstrated that some STEC strains, particularly pork and chicken isolates, displayed a multiple drug-resistance phenotype. RPLA analysis revealed that all the stx1-positive STEC isolates produced Stx1 only at the undetectable level. Altogether, these results imply that the locus of enterocyte and effacement (LEE)-positive strains STEC are predominant among raw meats or meat products from slaughterhouses or retail markets in Korea.

• 한국식품위생안전성학회지
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• 제29권4호
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• pp.316-321
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• 2014

The aim of study was to investigate the virulence profile of Escherichia coli O157:H7 bacteriophages isolated from sewage and livestock stools. Among 23 E. coli O157:H7 bacteriophages, 14 strains were isolated from sewage and 9 were from animal stools collected from 10 livestock farms in Korea. For each bacteriophage DNA sample, the presence of stx1, stx2, eae, aafII, ial, elt, estI, estII, astA, afa, and cnf was examined by polymerase chain reaction. The detection rate of eae, stx2, estI, astA, and ial was 100%, 69.6%, 13.0%, 13.0%, 8.7%, respectively. While all E. coli O157:H7 bacteriophages isolated from stools carried eae+stx2, stx2+eae, eae+astA, eae, stx2+eae+estI, eae+estI, stx2+eae+ial, and eae+ial were observed in bacteriophages isolated from sewage. As several plasmid-carrying virulence factors (estI, astA, and ial) were found in E. coli O157:H7 bacteriophages obtained from sewage and stools, the microbial safety of bacteriophages should be investigated in further study.

• 한국식품위생안전성학회지
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• 제15권3호
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• pp.241-247
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• 2000

국내 유통식품에서 verotoxin(VT)을 생성하는 대장균의 오염도를 조사하고 이로 인한 식중독의 발생을 사전에 예방하기 위하여 1997년부터 1999년 사이 햄버거. 식육 및 채소류에서 VT생성 대장균의 분포조사를 실시하였으며, 이 결과 분리된 대장균에 대한 분자생물학적 특성을 조사하였다 식육, 햄버거 및 채소류 총 1,700건의 식품 중에서 VT를 생성하는 대장균이 검출된 것은 3건으로 매우 낮은 검출율을 보였는데, 분리주의 혈청형은 각각 026 : H4, 0157 : H7 및 055 : Hl2였다. 혈청형 026 : H4 분리주는 VT I과 VT II를 생성하였고,055 : Hl2주는 VT I을 각각 생성하였으며, 두 분리주에서 eae유전자와 60 MDa plasmid DNA는 검출되지 않았다. 혈청형 0157 : H7주는 VT II를 생성하였고 60 MDa plasmid DNA는 검출되었으나 eae유전자는 확인되지 않았다. 따라서 분리된 VT생성 대장균들은 eae 유전자가 검출되지 않아 질병유발 가능성은 낮은 것으로 추정되었다. 항생제 내성능에서 혈청형 0157 : H7 분리주는 ampicillin과 streptomycin에 내성을 나타낸 반면, 혈청형 026 : H4주와 혈청형 055 : Hl2주는 ampicillin, carbenicillin, cephalothin, trimethoprim/sulfamethoxazole과 tetracycline에 내성을 보여 분리주의 다재내성능을 확인하였다 세포배양액으로 실시한 vero cell과 HeLa cell에 대한 세포독성능은 3주의 분리주에서 확인되었다.

• 생명과학회지
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• 제29권1호
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• pp.97-104
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• 2019

Streptococcus iniae는 대표적인 어류병원균으로 인수공통의 질병을 일으킨다. S. iniae FP5228에 존재하는 병원성 인자를 찾고자 하는 연구과정에서 S. iniae를 24시간 이상 배양한 배양액에는 살아있는 균의 수가 급격하게 감소하는 현상을 발견하였다. 이 현상은 FP5228 균이 가지고 있는 14 kb plasmid 상에 있는 toxin/antitoxin (TA) system의 구성요소인 toxin ${\zeta}$와 antitoxin ${\varepsilon}$ 유전자가 관련이 있을 것이란 가설을 설정하였다. IPTG와 arabinose에 의해 toxin ${\zeta}$와 antitoxin ${\varepsilon}$의 발현이 조절되는 pBP1140 vector system을 구축하였다. E. coli/pBP1140 균주는 toxin이 발현되는 조건에서 초기 생육이 느려졌고, 현미경의 관찰에서 균체가 길어짐을 확인하였다. FP5228 균주가 가진 14 kb plasmid를 없앤 S. iniae CK287을 제조하였다. CK287 균은 배양 중 급격하게 사멸되는 현상을 보이지 않았고, biofilm 생성능력도 감소하였고 세포독성 시험과 물고기 시험에서 독성이 약화 된 것이 확인되었다. 이들 결과 들은 TA system이 생리적 조절 및 병원성 인자의 발현에 관련이 있음을 추정할 수 있다.