ABSTRACT
Introduction@#PCR to detect and amplificate the virulence genes of STEC is specific and more sensitive, however, it takes five to six hours for whole test procedure and requires special lab instruments such as thermocycler. Loop-mediated isothermal amplification is a simple, rapid, specific and cost-effective nucleic acid amplification method by using four to six primers when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification.@*Goal@#Detection and comparison of STEC by PCR and LAMP@*Materials and Methods@#In our study, we analyzed comparison of PCR and LAMP results on standard strain used quality control strain solution which diluted 1pg/µL DNA, 10 pg/µL DNA, 100 pg/µL DNA, 1 ng/μL DNA, 10 ng/μL DNA, and 100 ng/μL DNA concentration from LB agar cultures. @*Research ethics@#Permission to submit the survey was granted by the Ethics Review Committee of the MNUMS and the survey was conducted in accordance with the rules and regulations.@*Result@#Sensitivity of Stx1 and stx2 genes in PCR results are positive in 10 pg/µL DNA solution and negative in 1pg/µL DNA. In LAMP test results showed that positive for all concentration. It shows that LAMP method sensitivity is 10 times more than PCR. @*Conclusion@#It shows that LAMP method sensitivity is 10 times more than PCR. All in allLAMP test is cost effective test with sensitive for detection STEC.
ABSTRACT
Introduction@#PCR to detect and amplificate the virulence genes of STEC is specific and more sensitive, however, it takes five to six hours for whole test procedure and requires special lab instruments such as thermocycler. Loop-mediated isothermal amplification is a simple, rapid, specific and cost-effective nucleic acid amplification method by using four to six primers when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification.@*Materials and Methods@#In our study, we analyzed comparison of PCR and LAMP results on standard strain used quality control strain solution which diluted 1pg/µL DNA, 10 pg/µL DNA, 100 pg/µL DNA, 1 ng/μL DNA, 10 ng/μL DNA, and 100 ng/μL DNA concentration from LB agar cultures. @*Research ethics@#Permission to submit the survey was granted by the Ethics Review Committee of the MNUMS and the survey was conducted in accordance with the rules and regulations. @*Goal@#Detection and comparison of STEC by PCR and LAMP@*Result@#Sensitivity of Stx1 and stx2 genes in PCR results are positive in 10 pg/µL DNA solution and negative in 1pg/µL DNA. In LAMP test results showed that positive for all concentration. It shows that LAMP method sensitivity is 10 times more than PCR. @*Conclusion@#It shows that LAMP method sensitivity is 10 times more than PCR. All in allLAMP test is cost effective test with sensitive for detection STEC.
ABSTRACT
ABSTRACT: The present study was aimed at subtyping of Stx1 and Stx2 genes and characterization of antimicrobial resistance in 106 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle and sheep feces. PCR was used to determine the subtypes, and the disk-diffusion method was used to evaluate the antimicrobial resistance. Ten antibiotics from five different classes were tested. Among the isolates of bovine origin, two subtypes of Stx1 (Stx1a and Stx1c), and four subtypes of Stx2 (Stx2a, Stx2b, Stx2c, and Stx2d) were identified. In isolates of sheep origin, two subtypes of Stx1 (Stx1a and Stx1c), and four subtypes of Stx2 (Stx2a, Stx2b, Stx2c, and Stx2 g) were identified. The results obtained suggest the presence of high diversity in Stx1 and Stx2 genes. Further, 96.6% (57/59) of bovine fecal strains and 89.4% (42/47) of sheep fecal strains showed resistance to at least one tested antibiotic. In both animal species, most strains were multidrug-resistant (MDR) (67.8% in cattle and 59.6% in sheep), with no significant difference between host animals. Adult animals were eight times more likely to have STEC with greater pathogenic potential. STEC with the highest pathogenic potential were three times more likely to be multidrug-resistant than STEC with the lowest pathogenic potential. The data reported in this study suggests the occurrence of strains with high potential pathogenicity in the region studied. Therefore, the ruminants of this region are carriers of strains that can cause infections in humans.
RESUMO: O presente estudo teve como objetivo subtipar os genes Stx1 e Stx2 e caracterizar a resistência antimicrobiana em 106 isolados de Escherichia coli produtoras de toxinas Shiga (STEC) isoladas de fezes de bovinos e ovinos. A PCR foi utilizada para determinar os subtipos e o método de difusão em disco foi utilizado para avaliar a resistência antimicrobiana. Dez antibióticos de cinco classes diferentes foram testados. Entre os isolados de origem bovina, foram identificados dois subtipos de Stx1 (Stx1a e Stx1c) e quatro subtipos de Stx2 (Stx2a, Stx2b, Stx2c e Stx2d). Nos isolados de origem ovina, foram identificados dois subtipos de Stx1 (Stx1a e Stx1c) e quatro subtipos de Stx2 (Stx2a, Stx2b, Stx2c e Stx2g). Os resultados obtidos sugerem a presença de alta variabilidade nos genes Stx1 e Stx2. Além disso, 96,6% (57/59) dos isolados fecais de bovinos e 89,4% (42/47) dos isolados de ovinos mostraram resistência a pelo menos um antibiótico testado. Em ambas as espécies animais, a maioria das cepas foi multirresistente (MDR) (67,8% em bovinos e 59,6% em ovinos), sem diferença significativa entre as espécies animais do reservatório. Os animais adultos tiveram oito vezes mais chances de apresentar STEC com maior potencial patogênico. STEC com o maior potencial patogênico teve três vezes mais chances de ser multirresistente do que o STEC com o menor potencial patogênico. Os dados relatados neste estudo sugerem a ocorrência de cepas com alto potencial de patogenicidade na região estudada. Portanto, os ruminantes dessa região são hospedeiros de isolados que podem causar infecções em humanos.
ABSTRACT
The present study was aimed at subtyping of Stx1 and Stx2 genes and characterization of antimicrobial resistance in 106 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle and sheep feces. PCR was used to determine the subtypes, and the disk-diffusion method was used to evaluate the antimicrobial resistance. Ten antibiotics from five different classes were tested. Among the isolates of bovine origin, two subtypes of Stx1 (Stx1a and Stx1c), and four subtypes of Stx2 (Stx2a, Stx2b, Stx2c, and Stx2d) were identified. In isolates of sheep origin, two subtypes of Stx1 (Stx1a and Stx1c), and four subtypes of Stx2 (Stx2a, Stx2b, Stx2c, and Stx2 g) were identified. The results obtained suggest the presence of high diversity in Stx1 and Stx2 genes. Further, 96.6% (57/59) of bovine fecal strains and 89.4% (42/47) of sheep fecal strains showed resistance to at least one tested antibiotic. In both animal species, most strains were multidrug-resistant (MDR) (67.8% in cattle and 59.6% in sheep), with no significant difference between host animals. Adult animals were eight times more likely to have STEC with greater pathogenic potential. STEC with the highest pathogenic potential were three times more likely to be multidrug-resistant than STEC with the lowest pathogenic potential. The data reported in this study suggests the occurrence of strains with high potential pathogenicity in the region studied. Therefore, the ruminants of this region are carriers of strains that can cause infections in humans.(AU)
O presente estudo teve como objetivo subtipar os genes Stx1 e Stx2 e caracterizar a resistência antimicrobiana em 106 isolados de Escherichia coli produtoras de toxinas Shiga (STEC) isoladas de fezes de bovinos e ovinos. A PCR foi utilizada para determinar os subtipos e o método de difusão em disco foi utilizado para avaliar a resistência antimicrobiana. Dez antibióticos de cinco classes diferentes foram testados. Entre os isolados de origem bovina, foram identificados dois subtipos de Stx1 (Stx1a e Stx1c) e quatro subtipos de Stx2 (Stx2a, Stx2b, Stx2c e Stx2d). Nos isolados de origem ovina, foram identificados dois subtipos de Stx1 (Stx1a e Stx1c) e quatro subtipos de Stx2 (Stx2a, Stx2b, Stx2c e Stx2g). Os resultados obtidos sugerem a presença de alta variabilidade nos genes Stx1 e Stx2. Além disso, 96,6% (57/59) dos isolados fecais de bovinos e 89,4% (42/47) dos isolados de ovinos mostraram resistência a pelo menos um antibiótico testado. Em ambas as espécies animais, a maioria das cepas foi multirresistente (MDR) (67,8% em bovinos e 59,6% em ovinos), sem diferença significativa entre as espécies animais do reservatório. Os animais adultos tiveram oito vezes mais chances de apresentar STEC com maior potencial patogênico. STEC com o maior potencial patogênico teve três vezes mais chances de ser multirresistente do que o STEC com o menor potencial patogênico. Os dados relatados neste estudo sugerem a ocorrência de cepas com alto potencial de patogenicidade na região estudada. Portanto, os ruminantes dessa região são hospedeiros de isolados que podem causar infecções em humanos.(AU)
Subject(s)
Animals , Cattle , Cattle/microbiology , Sheep/microbiology , Shiga Toxins , Escherichia coli/isolation & purification , Shiga-Toxigenic Escherichia coli , Anti-Infective Agents , Polymerase Chain ReactionABSTRACT
Aim: To investigate the stx1, stx2, hly, cnf2, sfa/foc, afaI, papC, and afaC virulence-associated genes of pathogenic Escherichia coli, isolated from diarrheic and non-diarrheic calves. Materials and Methods: The genes to be investigated were first isolated from the stool samples obtained from 150 diarrheic and non-diarrheic calves, between the ages of one day and six months, during the period 2016 to 2017. Rectal swabs were aseptically and randomly collected from several herds, from different regions in the province of Basra. Results of Research: The polymerase chain reaction (PCR) results showed that pathogenic E. coli were detected in a total of 34 out of 41 (82.9%) distributed as 23 out of 26 (88.5%) and 11 out of 15 (73.3%) of the diarrheagenic and non-diarrheagenic calves, respectively. The results also showed that stx1, hlyA, and stx2genes showed a higher incidence of distribution in both diarrheagenic and non-diarrheagenic calves, in a percentage rate of 69.6%, 65.2%, 56.5% and 63.6%, 63.6%, 45.5%, respectively. Moreover, eight different virulence gene profiles were established in the present study. Most of the isolates analyzed had at least two or three gene arrangements and only four isolates were seen in a combination of four genes stx1, stx2, hlyA, and afaI. Conclusion: It is concluded that E. coli isolates from healthy and diarrheic calves that carried various virulence genes, of which the most frequent were stx1, stx2, and hlyA. A high percentage of these isolates are found in both diarrheic and non-diarrheic calves.
ABSTRACT
This study determined the distribution of stx1 and stx2 genes in Escherichia coli isolated from dairy herds with regard to animal age, season, and farm production-scale, and analyzed the phylogenetic distribution of the groups A, B1, B2, and D of 276 isolates of bovine feces Shiga toxin-producing E. coli (STEC). The stx1 profile was the most common, detected in 20.4% (202/990) of the isolates, followed by stx2 (4.54%, 45/990) and stx1+stx2 (2.92%, 29/990). The stx1 gene was detected more frequently in calves than in adult animals. In the dry season (winter), the presence of stx1+stx2 profile in cattle feces was higher than in the rainy season (summer), while no significant changes were observed between seasons for the stx1 and stx2 profiles. The most predominant phylogenetic groups in adult animals were B1, A, and D, while groups A and B1 prevailed in calves. Our data highlight the importance of identifying STEC reservoirs, since 7.5% of the tested isolates were positive for stx2, the main profile responsible for the hemolytic-uremic syndrome (HUS). Moreover, these microorganisms are adapted to survive even in hostile environments and can contaminate the food production chain, posing a significant risk to consumers of animal products.(AU)
Esse estudo determinou a distribuição dos genes stx1 e stx2 em Escherichia coli isolados de rebanhos leiteiros em relação a idade, estação e produção, e analisaram a distribuição filogenética dos grupos A, B1, B2 e D de 276 E. coli produtoras de toxina Shiga (STEC). O perfil stx1 foi mais comum, detectado em 20,4% (202/990) dos isolados, seguido de stx2 (4,54%, 45/990) e stx1+stx2 (2,92%, 29/990). O gene stx1 foi detectado mais frequentemente em bezerros que animais adultos. No período de seca (inverno), a presença do perfil stx1+stx2 nas fezes dos bovinos foi mais prevalente que no período chuvoso (verão), apesar de não haver diferença significativa entre estações para os perfis stx1 e stx2. Os grupos filogenéticos mais predominantes em animais adultos foram B1, A e D, enquanto grupos A e B2 prevaleceram em bezerros. Nossos dados enfatizam a importância de se detectar reservatórios de STEC já que 7,5% dos isolados testados foram positivos para stx2, o perfil mais prevalente em casos de síndrome hemolítica-urêmica. Ademais, esses microorganismos são adaptados à sobreviver em ambientes hostis e contaminam a cadeia alimentar, levando a risco significativo para consumidores de alimentos de origem animal.(AU)
Subject(s)
Animals , Cattle , Cattle/genetics , Shiga Toxin 1/genetics , Shiga Toxin 2/genetics , Escherichia coli/isolation & purification , Escherichia coli/geneticsABSTRACT
Escherichia coli isolates from different samples of droppings collected from the different farms of broiler and local poultry were characterized to confirm the virulence. E. coli isolates recovered from different farms were examined for presence of genes encoding patho groups such as shiga like toxin producing Escherichia coli (STEC), (stx1/stx2) by Duplex PCR and then analysed the PCR Amplicons by AGE. The three and four isolates of E. coli recovered from local and boiler poultry were STEC because of presence of the stx1 and stx2 genes. Presence of stx1 and stx2 genes clearly indicated these as prime cause of pathogenecity. Further, demonstration of STEC in poultry becomes a public health concern, as poultry are potential reservoir of such agents, which may cause extra intestinal diseases like haemolytic uremic syndrome and thrombocytopenic purpurea by stx1 &2 and these toxins kill vascular endothelial cells.
ABSTRACT
PCR detection of ‘stx 1’ and ‘stx 2’ toxigenic genes in multiple antibiotic resistant Escherichia coli population and Phenotypic detection of ESBL producing Escherichia coli isolated from traditional local variety of poultry was conducted The study revealed that out of 30 samples collected 20 were positive for E.coli All the 20 isolates were subjected for the detection of 2 toxin genes (stx1 & stx2 genes) by PCR. Only 3 isolates (L3, L4 & L5) gave rise to PCR product against its specific primers of stx1. All the isolates were negative for the presence of stx2 gene. Thus 15% of E.coli population under this study is potential isolates and indicates that healthy retail poultry birds are the carrier of E.coli pathogenic strains. The poultry birds may therefore, be considered as a major reservoir of E.coli.
ABSTRACT
This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.