Development and Application of a Novel Nucleic Amplification Kit on Detection of Several Pathogens

2014 ◽  
Vol 618 ◽  
pp. 293-297 ◽  
Author(s):  
Yang Deng ◽  
Li Li Ji ◽  
Lin Li ◽  
Bing Li ◽  
Jian Yu Su
Keyword(s):  
Escherichia Coli ◽  
New England ◽  
Shiga Toxin ◽  
Vibrio Parahaemolyticus ◽  
Lamp Assay ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Three Solutions ◽  
E Coli ◽  
Shiga Toxin 2

Escherichia coliO157,Psuedomonas aeruginosa,Salmonella,Vibrio parahaemolyticusandListeriaare important pathogens for human. With increased awareness in public health, development of a rapid, sensitive, cost-effective and easy-operating bacteriological detection is of the utmost importance and urgent necessity. In this study, we developed and applied a simple amplification kit based on loop-mediated isothermal amplification (LAMP) methods for rapid detection of various pathogens includingEscherichia coliO157,Psuedomonas aeruginosa,Salmonella,Vibrio parahaemolyticusandListeria, as well as related virulence. Nine targets, includingrfbE(E. coli-specific),stx1 (coding for Shiga toxin 1),stx2 (coding for Shiga toxin 2),oprI(P. aeruginosa–specific),invA(Salmonella-specific),hlyA(Listeria-specific),tlh(coding for thermolable haemolysin),tdh(coding for thermostable direct haemolysin) andtrh(coding for TDH-related haemolysin), were selected for identification forEscherichia coliO157,Psuedomonas aeruginosa,Salmonella,Vibrio parahaemolyticusandListeria, as well as related virulence.. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on the targets. Three solutions labeled A, B and C was included in the kit. The experiment were carried out in a total of 25 μl reaction mixture: solution A containing 1.6 μM (each) of the primers FIP and BIP, 0.2 μM (each) of the primers F3 and B3, 0.8 μM (each) of primers LF and LB; solution B containing 1.6 mM of deoxynucleoside triphosphates, 6 mM MgSO4, 1 M betain (Sigma, St. Louis, MO, USA), 1 X thermopol buffer (New England Biolabs, Ipswich, MA, USA); solution C containing BST polymerase. Twenty-two reference strains, including various species of gram-negative and-positive isolates, were included in this study to evaluate and optimize LAMP assays. Application of the optimized LAMP assays was performed on a total of 200 strains (with 40 strains for each species of the pahtogens). The optimal reaction condition was found to be 65°C for 45 min. Application of the kit assays were performed on various types of pathogens, the sensitivity for the 9 targets was found to be 100%; with a 100% specificity and positive predictive value (PPV) for all the 9 targets targets. In conclusion, the isothermal amplification kits were demonstrated to be useful and powerful tools for rapid differentiation of various pathogens (includingEscherichia coliO157,Psuedomonas aeruginosa,Salmonella,Vibrio parahaemolyticusandListeria), and undoubtedly, the rapidness, easiness and cost-effectiveness of LAMP assay will aid in the broad application of bacteriological detection of common pathogens.

scholarly journals Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

2007 ◽  
Vol 74 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Alexander Mellmann ◽  
Shan Lu ◽  
Helge Karch ◽  
Jian-guo Xu ◽  
Dag Harmsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Integration Site ◽  
Hot Spot ◽  
Blot Hybridization ◽  
Enterohemorrhagic Escherichia Coli ◽  
Biologically Active ◽  
E Coli ◽  
Shiga Toxin 2

ABSTRACT Using colony blot hybridization with stx 2 and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx 2-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx 2-positive isolates fliC H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx 2-positive strains by loss of stx 2 during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx 2-positive and stx-negative isolates demonstrated that yecE was occupied in stx 2-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx 2-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx 2-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx 2-harboring phages. The ability to recycle stx 2, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.

scholarly journals Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E. coli (STEC) infections in the Netherlands, January 2008 to December 2011

2014 ◽  
Vol 19 (17) ◽  
Author(s):  
I Friesema ◽  
K van der Zwaluw ◽  
T Schuurman ◽  
M Kooistra-Smid ◽  
E Franz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
The Netherlands ◽  
Shiga Toxin ◽  
Distinct Group ◽  
Shiga Toxins ◽  
E Coli ◽  
Mild Disease ◽  
Shiga Toxin 2 ◽  
Almost All ◽  
Human Infections

The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx2f is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC surveillance in the Netherlands, 198 STEC O157 cases and 351 STEC non-O157 cases, including 87 stx2f STEC isolates, were reported between 2008 and 2011. Most stx2f strains belonged to the serogroups O63:H6 (n=47, 54%), O113:H6 (n=12, 14%) and O125:H6 (n=12, 14%). Of the 87 stx2f isolates, 84 (97%) harboured the E. coli attaching and effacing (eae) gene, but not the enterohaemorrhagic E. coli haemolysin (hly) gene. Stx2f STEC infections show milder symptoms and a less severe clinical course than STEC O157 infections. Almost all infections with stx2f (n=83, 95%) occurred between June and December, compared to 170/198 (86%) of STEC O157 and 173/264 (66%) of other STEC non-O157. Stx2f STEC infections in the Netherlands are more common than anticipated, and form a distinct group within STEC with regard to virulence genes and the relatively mild disease.

scholarly journals Comparative Survival of Free Shiga Toxin 2-Encoding Phages and Escherichia coli Strains outside the Gut

1999 ◽  
Vol 65 (12) ◽  
pp. 5615-5618 ◽  
Author(s):  
Maite Muniesa ◽  
Francisco Lucena ◽  
Juan Jofre
Keyword(s):  
Escherichia Coli ◽  
Heat Treatment ◽  
River Water ◽  
Shiga Toxin ◽  
Water Environment ◽  
Somatic Coliphages ◽  
E Coli ◽  
Naturally Occurring ◽  
Shiga Toxin 2

ABSTRACT The behavior outside the gut of seeded Escherichia coliO157:H7, naturally occurring E. coli, somatic coliphages, bacteriophages infecting O157:H7, and Shiga toxin 2 (Stx2)-encoding bacteriophages was studied to determine whether the last persist in the environment more successfully than their host bacteria. The ratios between the numbers of E. coli and those of the different bacteriophages were clearly lower in river water than in sewage of the area, whereas the ratios between the numbers of the different phages were similar. In addition, the numbers of bacteria decreased between 2 and 3 log units in in situ survival experiments performed in river water, whereas the numbers of phages decreased between 1 and 2 log units. Chlorination and pasteurization treatments that reduced by approximately 4 log units the numbers of bacteria reduced by less than 1 log unit the numbers of bacteriophages. Thus, it can be concluded that Stx2-encoding phages persist longer than their host bacteria in the water environment and are more resistant than their host bacteria to chlorination and heat treatment.

scholarly journals Nonpathogenic Escherichia coli Can Contribute to the Production of Shiga Toxin

2003 ◽  
Vol 71 (6) ◽  
pp. 3107-3115 ◽  
Author(s):  
Shantini D. Gamage ◽  
Jane E. Strasser ◽  
Claudia L. Chalk ◽  
Alison A. Weiss
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Gastrointestinal Disease ◽  
Intestinal Flora ◽  
Gene Promoter ◽  
Toxin Production ◽  
Lytic Cycle ◽  
Resistant Bacteria ◽  
E Coli ◽  
Shiga Toxin 2

ABSTRACT The food-borne pathogen, Escherichia coli O157:H7, has been associated with gastrointestinal disease and the life-threatening sequela hemolytic uremic syndrome. The genes for the virulence factor, Shiga toxin 2 (Stx2), in E. coli O157:H7 are encoded on a temperate bacteriophage under the regulation of the late gene promoter. Induction of the phage lytic cycle is required for toxin synthesis and release. We investigated the hypothesis that nonpathogenic E. coli could amplify Stx2 production if infected with the toxin-encoding phage. Toxin-encoding phage were incubated with E. coli that were either susceptible or resistant to the phage. The addition of phage to phage-susceptible bacteria resulted in up to 40-fold more toxin than a pure culture of lysogens, whereas the addition of phage to phage-resistant bacteria resulted in significantly reduced levels of toxin. Intestinal E. coli isolates incubated with Shiga toxin-encoding phage produced variable amounts of toxin. Of 37 isolates, 3 produced significantly more toxin than was present in the inoculum, and 1 fecal isolate appeared to inactivate the toxin. Toxin production in the intestine was assessed in a murine model. Fecal toxin recovery was significantly reduced when phage-resistant E. coli was present. These results suggest that the susceptibility of the intestinal flora to the Shiga toxin phage could exert either a protective or an antagonistic influence on the severity of disease by pathogens with phage-encoded Shiga toxin. Toxin production by intestinal flora may represent a novel strategy of pathogenesis.

Seasonal Prevalence of Escherichia coli O157:H7 in Beef Cattle Feces†

2006 ◽  
Vol 69 (12) ◽  
pp. 3018-3020 ◽  
Author(s):  
M. J. ALAM ◽  
L. ZUREK
Keyword(s):  
Escherichia Coli ◽  
Beef Cattle ◽  
Shiga Toxin ◽  
Bacterial Colonization ◽  
Feedlot Cattle ◽  
Escherichia Coli O157 ◽  
Human Pathogen ◽  
E Coli ◽  
Cattle Feces ◽  
Shiga Toxin 2

Cattle are an asymptomatic reservoir of Escherichia coli O157:H7, but the bacterial colonization and shedding patterns are poorly understood. The prevalence and shedding of this human pathogen have been reported to be seasonal with rates typically increasing during warm months. The objectives of this study were (i) to assess the prevalence of E. coli O157:H7 in feces of feedlot cattle in Kansas during summer, fall, and winter months, and (ii) to characterize E. coli O157:H7 by screening for virulence factors. Of 891 fecal samples collected, 82 (9.2%) were positive for E. coli O157:H7. No significant differences in prevalence were detected among summer, fall, and winter months. The highest monthly prevalence (18.1%) was detected in February. All tested isolates were positive for stx2 (Shiga toxin 2) and eaeA (intimin) genes; 14 isolates (12.8%) also carried stx1. Our results indicate the prevalence of E. coli O157:H7 in beef cattle feces is not necessarily season dependent.

scholarly journals A New Shiga Toxin 2 Variant (Stx2f) fromEscherichia coli Isolated from Pigeons

2000 ◽  
Vol 66 (3) ◽  
pp. 1205-1208 ◽  
Author(s):  
Herbert Schmidt ◽  
Jürgen Scheef ◽  
Stefano Morabito ◽  
Alfredo Caprioli ◽  
Lothar H. Wieler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Shiga Toxin ◽  
E Coli ◽  
Fromescherichia Coli ◽  
Feral Pigeons ◽  
Pigeon Droppings ◽  
Variant Gene ◽  
Shiga Toxin 2

ABSTRACT We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designatedstx2f . This gene is most similar tosltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx2f gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.

scholarly journals Antibody-Based Protection of Gnotobiotic Piglets Infected with Escherichia coli O157:H7 against Systemic Complications Associated with Shiga Toxin 2

1999 ◽  
Vol 67 (7) ◽  
pp. 3645-3648 ◽  
Author(s):  
Art Donohue-Rolfe ◽  
Ivanela Kondova ◽  
Jean Mukherjee ◽  
Kerry Chios ◽  
David Hutto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Systemic Complications ◽  
E Coli ◽  
Control Serum ◽  
Shiga Toxin 2 ◽  
Uremic Syndrome ◽  
Cerebellar Lesions ◽  
Serious Disease ◽  
Gnotobiotic Piglets

ABSTRACT Hemolytic-uremic syndrome (HUS) is a serious disease in children, attributable in the majority of cases to infection with Shiga toxin (Stx)-producing Escherichia coli. Using gnotobiotic piglets orally infected with E. coli O157:H7, which develop Stx-related cerebellar lesions and fatal neurological symptoms, we show that administration of Stx2-specific antiserum well after challenge protected, in a dose-response fashion, against these symptoms for at least 24 h after bacterial challenge. Twenty-six of 30 piglets given Stx2 antiserum survived the challenge, compared to only 4 of 16 animals given control serum or saline. Given our observations in piglets, Stx antibody of human origin may likewise prevent HUS in children.

scholarly journals Isogenic Lysogens of Diverse Shiga Toxin 2-Encoding Bacteriophages Produce Markedly Different Amounts of Shiga Toxin

1999 ◽  
Vol 67 (12) ◽  
pp. 6710-6714 ◽  
Author(s):  
Patrick L. Wagner ◽  
David W. K. Acheson ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Fragment Length ◽  
Toxin Production ◽  
E Coli ◽  
Length Polymorphisms ◽  
Shiga Toxin 2 ◽  
K 12 ◽  
Host Strains ◽  
Restriction Fragment Length

ABSTRACT We produced isogenic Escherichia coli K-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis.

scholarly journals Escherichia coli Serogroup O107/O117 Lipopolysaccharide Binds and Neutralizes Shiga Toxin 2

2004 ◽  
Vol 186 (16) ◽  
pp. 5506-5512 ◽  
Author(s):  
Shantini D. Gamage ◽  
Colleen M. McGannon ◽  
Alison A. Weiss
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Biochemical Characterization ◽  
Vero Cells ◽  
Systemic Complications ◽  
E Coli ◽  
Major Virulence Factor ◽  
Shiga Toxin 2 ◽  
Type Strains

ABSTRACT The AB5 toxin Shiga toxin 2 (Stx2) has been implicated as a major virulence factor of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains in the progression of intestinal disease to more severe systemic complications. Here, we demonstrate that supernatant from a normal E. coli isolate, FI-29, neutralizes the effect of Stx2, but not the related Stx1, on Vero cells. Biochemical characterization of the neutralizing activity identified the lipopolysaccharide (LPS) of FI-29, a serogroup O107/O117 strain, as the toxin-neutralizing component. LPSs from FI-29 as well as from type strains E. coli O107 and E. coli O117 were able bind Stx2 but not Stx1, indicating that the mechanism of toxin neutralization may involve inhibition of the interaction between Stx2 and the Gb3 receptor on Vero cells.

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