scholarly journals Comparison of a PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Assay to Pulsed-Field Gel Electrophoresis To Determine the Effect of Repeated Subculture and Prolonged Storage on RFLP Patterns of Shiga Toxin-Producing Escherichia coli O157:H7

2006 ◽  
Vol 44 (11) ◽  
pp. 3963-3968 ◽  
Author(s):  
K. Shima ◽  
Y. Wu ◽  
N. Sugimoto ◽  
M. Asakura ◽  
K. Nishimura ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Gel Electrophoresis ◽  
Fragment Length Polymorphism ◽  
Pulsed Field Gel Electrophoresis ◽  
Prolonged Storage ◽  
Escherichia Coli O157 ◽  
Pcr Rflp ◽  
Rflp Assay ◽  
Repeated Subculture ◽  
Restriction Fragment Length

scholarly journals Development of a PCR-restriction fragment length polymorphism assay for detection and subtyping of cholix toxin variant genes of Vibrio cholerae

2014 ◽  
Vol 63 (5) ◽  
pp. 667-673 ◽  
Author(s):  
Sharda Prasad Awasthi ◽  
Masahiro Asakura ◽  
Sucharit Basu Neogi ◽  
Atsushi Hinenoya ◽  
T. Ramamurthy ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Vibrio Cholerae ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Pcr Rflp ◽  
Cholix Toxin ◽  
Rflp Assay ◽  
Restriction Fragment Length

Cholix toxin (ChxA) is an exotoxin reported in Vibrio cholerae non-O1/non-O139. Apart from its prototype (ChxA I) we have recently identified two novel variants of this toxin, ChxA II and ChxA III. Our previous investigations indicated that the first two variants may instigate extra-intestinal infections and ChxA II can be more lethal than ChxA I in mice. However, all three cholix toxins (ChxA I to III) failed to show any enterotoxicity in rabbit ileal loops. In this study we developed a PCR-restriction fragment length polymorphism (RFLP) assay to differentiate all three chxA variants to further understand the importance of each subtype. By using 53 V. cholerae non-O1/non-O139 strains harbouring chxA genes, which were previously categorized by sequencing, and various other strains as negative controls, the PCR-RFLP assay showed 100 % typability and specificity. Furthermore, when applied to differentiate additional V. cholerae strains, which were also screened for the chxA gene by colony hybridization, this assay identified chxA I and chxA II genes among 18.5 % and 4.5 % of non-O1/non-O139 strains (n = 178), respectively. One non-O1/non-O139 strain was untypable due to the insertion of an IS911-like element. Interestingly, the chxA I gene was detected in 10 out of 137 cholera toxin gene-negative V. cholerae O1 strains. These results suggest that the PCR-RFLP assay developed in this study can be a rapid and simple method to differentiate the chxA subtypes.

Pulsed-Field Gel Electrophoresis Characterization of Shiga Toxin–Producing Escherichia coli O157 from Hides of Cattle at Slaughter

2002 ◽  
Vol 65 (7) ◽  
pp. 1172-1176 ◽  
Author(s):  
S. M. AVERY ◽  
A. SMALL ◽  
C.-A. REID ◽  
S. BUNCIC
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Gel Electrophoresis ◽  
Immunomagnetic Separation ◽  
Pulsed Field Gel Electrophoresis ◽  
Escherichia Coli O157 ◽  
Adult Cattle ◽  
Pulsed Field ◽  
E Coli ◽  
High Prevalence

Contamination of the brisket areas of the hides of healthy adult cattle with Shiga toxin–producing Escherichia coli O157 at slaughter in England was studied. In total, 73 cattle consignments comprising 584 animals delivered to one abattoir over 3 days during 1 week in July 2001 were studied: 26 cattle consignments arriving on Monday, 32 consignments arriving on Wednesday, and 15 consignments arriving on Friday. Consignment sizes ranged from 1 to 23 animals, with a mean consignment size of 8. The hide of the first animal to be slaughtered in each consignment was sampled by using a sponge swab moistened with 0.85% saline to rub an unmeasured brisket (ventral) area (ca. 30 by 30 cm). The process of isolating E. coli O157 from the swabs consisted of enrichment, screening with immunoprecipitation assay kits, and immunomagnetic separation. E. coli O157 was found on 24 of 73 (32.9%) cattle hides examined, and 21 of these 24 isolates produced Shiga toxins. The 24 E. coli O157 isolates produced six different pulsed-field gel electrophoresis profiles, and 18 (75%) of the isolates were of one prevalent clone. The high prevalence of one E. coli O157 clone on the hides of cattle at slaughter could be due to a high prevalence of that clone on the 18 farms involved (not investigated in the current study), in the postfarm transport or lairage environments, or both. Since the lairage environment, but not the farm of origin or the postfarm transport vehicle, was a factor common to all 18 cattle consignments, it could have played an important role in spreading the prevalent E. coli O157 clone to the cattle hides. Lairage pen floors and the stunning box floor were identified as the most probable sites along the unloading-to-slaughter route at which the brisket areas of cattle hides could become contaminated.

scholarly journals Molecular Epidemiology of Penicillin-ResistantStreptococcus pneumoniae Isolates Recovered in Italy from 1993 to 1996

1998 ◽  
Vol 36 (10) ◽  
pp. 2944-2949 ◽  
Author(s):  
Anna Marchese ◽  
Mario Ramirez ◽  
Gian Carlo Schito ◽  
Alexander Tomasz
Keyword(s):  
Gel Electrophoresis ◽  
Fragment Length Polymorphism ◽  
Antibiotic Resistance Genes ◽  
Northern Italy ◽  
Pfge Pattern ◽  
Pulsed Field Gel Electrophoresis ◽  
Pfge Type ◽  
Penicillin Binding Protein ◽  
Additional Resistance ◽  
Restriction Fragment Length

Thirty-nine penicillin-resistant Streptococcus pneumoniae isolates recovered among the approximately 700 pneumococcal strains collected from 1993 to 1996 in central and northern Italy were analyzed for several characteristics, including serotype, antibiotic susceptibility profile, chromosomal relatedness (by using pulsed-field gel electrophoresis [PFGE]), restriction fragment length polymorphism (RFLP) of the penicillin-binding protein (PBP) genes 1A, 2X, and 2B, and the presence of a variety of antibiotic resistance genes (determined by hybridization with appropriate DNA probes). The MICs of penicillin for most of the isolates (30 of 39) were high, in the range of 1 μg/ml or higher, and these 30 isolates carried additional resistance traits to two or more drugs (erythromycin, chloramphenicol, co-trimoxazole, and tetracycline) and expressed serotypes 9, 19, and 23 and three distinct PFGE patterns. More than half (22 of 30) of the isolates for which MICs were high were identified as representatives of two widespread international epidemic clones of S. pneumoniae. The first one of these clones (seven isolates) expressed serotype 23F and possessed all properties characteristic of the widespread Spanish/USA international clone. Seven additional strains with serotype 19 also had the same PFGE pattern, PBP gene, and RFLP polymorphisms, and other properties typical of the serotype 23 Spanish/USA clone, suggesting that these strains were the products of a capsular transformation event (from serotype 23F to serotype 19) in which the Spanish/USA clone was the recipient. The second international clone was represented by eight serotype 9 isolates which were resistant to penicillin and trimethoprim-sulfamethoxazole and had the molecular properties of the French/Spanish epidemic clone. The remaining eight isolates for which penicillin MICs were high appeared to represent a hitherto-undescribed “Italian” clone; they had a novel PFGE type, unique RFLPs for the PBP genes, and resistance to tetracycline, trimethoprim-sulfamethoxazole, and erythromycin, and the penicillin MICs for these isolates were 2 to 4 μg/ml.

Prevalence and Characterization of Escherichia coli O157:H7 on Pork Carcasses and in Swine Colon Contents from Provincially Licensed Abattoirs in Alberta, Canada

2020 ◽  
Vol 83 (11) ◽  
pp. 1909-1917
Author(s):  
SAIDA ESSENDOUBI ◽  
XIANQIN YANG ◽  
ROBIN KING ◽  
JULIA KEENLISIDE ◽  
JAVIER BAHAMON ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Gel Electrophoresis ◽  
Mean Value ◽  
Pulsed Field Gel Electrophoresis ◽  
Escherichia Coli O157 ◽  
Pulsed Field ◽  
E Coli ◽  
Swine Farms

ABSTRACT The objective of this study was to determine the prevalence of Shiga toxin–producing Escherichia coli (STEC) O157:H7 in colon contents and on carcasses from pigs slaughtered at provincially licensed abattoirs (PLAs) in Alberta, Canada. In 2017, carcass sponge samples and colon content samples were collected from 504 healthy market hogs at 39 PLAs and analyzed for E. coli O157:H7. Carcass samples were also analyzed for E. coli and aerobic colony count (ACC). Nine (1.8%) of 504 carcass samples were confirmed positive for E. coli O157:H7. Seven (1.4%) of 504 colon content samples were confirmed positive for E. coli O157:H7. These positives were found in 5 (12.8%) of 39 PLAs from hogs originating from eight farms. The E. coli O157:H7 isolates recovered from the positive samples (n = 1 isolate per sample) were clonal, as determined by pulsed-field gel electrophoresis. Six E. coli O157:H7 isolates obtained over 8 months from one PLA that only processed hogs and sourced hogs from one farm had indistinguishable pulsed-field gel electrophoresis patterns. All 16 E. coli O157:H7 isolates harbored eae and ehxA and were of stx2a subtype, suggesting that swine can carry E. coli O157:H7 of importance to human health. All carcass sponge swabs (100%) were positive for ACC. E. coli was present in 72% of carcass swabs. Carcasses from PLAs slaughtering both beef and hogs had a numerically higher ACC mean value but not statistically different compared with the carcasses from PLAs slaughtering only swine (2,799 and 610 CFU/cm2, respectively). E. coli showed a similar trend with a mean value of 0.88 CFU/cm2 in PLAs slaughtering both species and 0.26 CFU/cm2 in PLAs slaughtering only swine (P ≤ 0.05). This study provides evidence that healthy market hogs from different producers and farms in Alberta can carry E. coli O157:H7, and some strains of the organism may be able to establish persistence on some swine farms. HIGHLIGHTS

scholarly journals Effect of Incubation Temperature on Isolation of Campylobacter jejuni Genotypes from Foodstuffs Enriched in Preston Broth

2003 ◽  
Vol 69 (8) ◽  
pp. 4658-4661 ◽  
Author(s):  
Pam Scates ◽  
Lynn Moran ◽  
Robert H. Madden
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Campylobacter Jejuni ◽  
Length Polymorphism ◽  
Gel Electrophoresis ◽  
Fragment Length Polymorphism ◽  
Incubation Temperature ◽  
Pulsed Field Gel Electrophoresis ◽  
Random Amplification ◽  
Campylobacter Lari ◽  
Restriction Fragment Length

ABSTRACT Preston broth and agar incubated at either 37 or 42°C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42°C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37°C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37°C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42°C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42°C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.

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