scholarly journals Development of three multiplex-PCR assays for virulence profiling of different iron acquisition systems in Escherichia coli

2020 ◽  
Author(s):  
Hamideh Kalateh Rahmani ◽  
Gholamreza Hashemi Tabar ◽  
Mahdi Askari Badouei ◽  
Babak Khoramian
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Virulence Genes ◽  
Current Knowledge ◽  
Iron Acquisition ◽  
E Coli ◽  
Essential Nutrient ◽  
Pcr Assays ◽  
Virulence Typing ◽  
Different Origin

Background and Objectives: Escherichia coli is responsible for various enteric and extraintestinal infections in animals and humans. Iron as an essential nutrient, has a proven role in pathogenicity of E. coli. Pathogenic E. coli benefits of having complicated systems for iron acquisition but our current knowledge is limited because of complexity of these systems. In the present study, three multiplex-PCR assays were developed to screen nine different virulence genes related to diverse iron acquisition systems in E. coli. Materials and Methods: The multiplex-PCR systems were designed and optimized in three panels. Each panel includes a triplex-PCR cocktail. The panels are as follow: panel 1: iroN, iutA and fecA; panel 2: fyuA, sitA and irp2; and panel 3: iucD, chuA and tonB. A total of 39 pathogenic E. coli was screened according to the designed multiplex-PCR. Results: In total, the top three frequent genes were tonB (100%), fecA (66.6%) and sitA (58.9%). With the exception of fecA and tonB, comparing the prevalence of genes among different origin of isolates (human, cattle, poultry and pigeon) showed significant associations (P < 0.05). Moreover, the iroN, sitA and iucD genes were significantly prevalent (P < 0.05) among members of extraintestinal pathogenic E. coli in comparison with the group of diarrheagenic E. coli. Conclusion: The current multiplex-PCR assays could be a valuable, rapid and economic tool to investigate diverse iron acquisition systems in E. coli for more precise virulence typing of pathogenic or commensal strains.

Rapid and Reliable Detection of Shiga Toxin–Producing Escherichia coli by Real-Time Multiplex PCR

2012 ◽  
Vol 75 (4) ◽  
pp. 643-650 ◽  
Author(s):  
KELLY S. ANKLAM ◽  
KAUSHI S. T. KANANKEGE ◽  
TINA K. GONZALES ◽  
CHARLES W. KASPAR ◽  
DÖRTE DÖPFER
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Real Time ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Health Concern ◽  
E Coli ◽  
Reliable Detection ◽  
Pcr Assays

Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin–producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx1 and stx2), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non–E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100% specificity. The detection limits of the assays were 103 or 104 CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 100 CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.

Primers with 5′ Flaps Improve the Efficiency and Sensitivity of Multiplex PCR Assays for the Detection of Salmonella and Escherichia coli O157:H7

2013 ◽  
Vol 76 (4) ◽  
pp. 668-673 ◽  
Author(s):  
CHRIS TIMMONS ◽  
SHEFALI DOBHAL ◽  
JACQUELINE FLETCHER ◽  
LI MARIA MA
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Fold Increase ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Robust Detection ◽  
E Coli ◽  
Pcr Multiplex ◽  
Pcr Assays

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5′ end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5′ flap (5′-AATAAATCATAA-3′). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

scholarly journals Detection of virulence genes, phylogenetic group and antibiotic resistance of uropathogenic Escherichia coli in Mongolia

2017 ◽  
Vol 11 (01) ◽  
pp. 51-57 ◽  
Author(s):  
Yandag Munkhdelger ◽  
Nyamaa Gunregjav ◽  
Altantsetseg Dorjpurev ◽  
Nishi Juniichiro ◽  
Jav Sarantuya
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Virulence Genes ◽  
Wide Spectrum ◽  
Iron Acquisition ◽  
Uropathogenic Escherichia Coli ◽  
Phylogenetic Group ◽  
Diffusion Method ◽  
Phylogenetic Groups ◽  
E Coli

Introduction: The severity of urinary tract infection (UTI) produced by uropathogenic Escherichia coli (UPEC) is due to the expression of a wide spectrum of virulence genes. E. coli strains were divided into four phylogenetic groups (A, B1, B2 and D) based on their virulence genes. The present study aimed to assess the relationship between virulence genes, phylogenetic groups, and antibiotic resistance of UPEC. Methodology: A total of 148 E. coli were tested for antimicrobial resistance against 10 drugs using the disk diffusion method. The isolates were screened by polymerase chain reaction (PCR) for detection of virulence genes and categorized into the four major phylogenetic groups. Results: Phylogenetic group B2 was predominant (33.8%), followed by D (28.4%), A (19.6), and B1 (18.2%). A higher prevalence of fimH (89.9%), fyuA (70.3%), traT (66.2%), iutA (62.2%), kpsMTII (58.8%), and aer (56.1%) genes were found in UPEC, indicating a putative role of adhesins, iron acquisition systems, and protectins that are main cause of UTIs. The most common antibiotic resistance was to cephalotin (85.1%), ampicillin (78.4%) and the least to nitrofurantoin (5.4%) and imipenem (2%). In total, 93.9% of isolates were multidrug resistant (MDR). Conclusions: This study showed that group B2 and D were the predominant phylogenetic groups and virulence-associated genes were mostly distributed in these groups. The virulence genes encoding components of adhesins, iron acquisition systems, and protectins were highly prevalent among antibiotic-resistant UPEC. Although the majority of strains are MDR, nitrofurantoin is the drug of choice for treatment of UTI patients in Ulaanbaatar.

scholarly journals Virulence and antibiotic resistance profile of avian Escherichia coli strains isolated from colibacillosis lesions in central of Algeria

2019 ◽  
Vol 12 (11) ◽  
pp. 1840-1848 ◽  
Author(s):  
Nacima Meguenni ◽  
Nathalie Chanteloup ◽  
Angelina Tourtereau ◽  
Chafika Ali Ahmed ◽  
Saliha Bounar-Kechih ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Virulence Factors ◽  
Virulence Genes ◽  
Iron Acquisition ◽  
Virulence Gene ◽  
Resistance Profile ◽  
E Coli ◽  
Antibiotic Resistance Profile ◽  
Embryo Lethality

Background and Aim: Avian pathogenic Escherichia coli cause extensive mortality in poultry flocks, leading to extensive economic losses. To date, in Algeria, little information has been available on virulence potential and antibiotics resistance of avian E. coli isolates. Therefore, the aim of this study was the characterization of virulence genes and antibiotic resistance profile of Algerian E. coli strains isolated from diseased broilers. Materials and Methods: In this study, 43 avian E. coli strains isolated from chicken colibacillosis lesions at different years were analyzed to determine their contents in 10 virulence factors by polymerase chain reaction, antimicrobial susceptibility to 22 antibiotics belonging to six different chemical classes and genomic diversity by pulsed-field gel electrophoresis (PFGE). Results: Mainly E. coli isolates (58.1%) carried two at six virulence genes and the most frequent virulence gene association detected were ompT (protectin), hlyF (hemolysin) with 55.8% (p<0.001), and iroN, sitA (iron acquisition/uptake systems), and iss (protectin) with 41.8% (p<0.001). Some strains were diagnosed as virulent according to their virulence gene profile. Indeed, 23.25% of the isolates harbored iroN, ompT, hlyF, iss, and sitA combination, 14% ompT, hlyF, and frzorf4 (sugar metabolism), and 11,6% iroN, hlyF, ompT, iss, iutA (iron acquisition/uptake systems), and frzorf4. The chicken embryo lethality assay performed on five isolates confirmed the potential virulence of these strains. All isolates submitted to PFGE analysis yielded different genetic profiles, which revealed their diversity. Overall, 97.2% of the isolates were resistant to at least one antibiotic and 53.5% demonstrated multi-antimicrobial resistance to three different antimicrobial classes. The highest resistance levels were against nalidixic acid (83.4%), amoxicillin and ampicillin (83.3%), ticarcillin (80.5%), pipemidic acid (75%), and triméthoprim-sulfamethoxazole (66.6%). For beta-lactam class, the main phenotype observed belonged to broad-spectrum beta-lactamases. However, extended-spectrum beta-lactamase associated with three at six virulence factors was also detected in 13 isolates. Two of them were attested virulent as demonstrated in the embryo lethality test which constitutes a real public threat. Conclusion: It would be imperative in avian production to discourage misuse while maintaining constant vigilance guidelines and regulations, to limit and rationalize antimicrobial use.

IDENTIFICATION OF THE AVIAN PATHOTYPE OF ESCHERICHIA COLI ON LAYER FLOCKS BY MULTIPLEX PCR

2019 ◽  
Vol 7 ◽  
pp. 905-911
Author(s):  
Marilena Burtan ◽  
Virgilia Popa ◽  
Maria Rodica Gurau ◽  
Doina Danes
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Economic Losses ◽  
Ompa Gene ◽  
Avian Pathogenic Escherichia Coli ◽  
E Coli ◽  
Pathogenic Escherichia Coli

Introduction: Colibacillosis in poultry is determined by avian pathogenic Escherichia coli (APEC) and represents an important source of economic losses  in the poultry industry. APEC’s pathogenicity relies on the presence and expression of different virulence factors. The genes ompA , iss  and  fimH, encoding the outer membrane protein, the protein inducing resistance to complement and the synthesis of type 1 fimbria are present in APEC strains. Objective: Escherichia coli strains isolated from layers were analysed to assess the pathotype they belong to. Methods: In order to detect the three genes associated with APEC strains, 16 E. coli isolates were investigated for virulence associated genes ompA, iss and fimH, using multiplex PCR. Results: From the 16 E.coli strains submitted, multiplex PCR assessment revealed that 14 (87.5%) of the E. coli strains isolated contained at least one virulence gene, while 2 (12.5%) strains did not harbour any of the virulence genes tested. The fimH gene was noted in 13 (81.25%) of the strains tested, the ompA gene has been present in 12 (75%) strains and the iss gene was present in 9 (56.25%) strains. Eight (50%) strains were found to present all three investigated genes. Conclusion: Presence of these genes is a strong indicatory to consider those strains as belonging to the APEC pathotype.

scholarly journals Verotoxins in commensal Escherichia coli in cattle: the effect of injectable subcutaneous oxytetracycline in addition to in-feed chlortetracycline on prevalence

2004 ◽  
Vol 132 (1) ◽  
pp. 77-85 ◽  
Author(s):  
A. M. O'CONNOR ◽  
K. A. ZIEBELL ◽  
C. POPPE ◽  
S. A. McEWEN
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Observational Study ◽  
Multiplex Pcr ◽  
Virulence Genes ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

Using a self-paired observational study, the association between therapeutic oxytetracycline use and the prevalence of virulence genes in commensal Escherichia coli (E. coli) from cattle was examined. Faeces were collected from 39 yearling bulls prior to and after treatment with oxytetracycline and from 44 untreated animals. Between samplings all animals received in-feed chlortetracycline for 16 days. Five E. coli were isolated from each sample and tested by a polymerase chain reaction (PCR) capable of detecting all verotoxin (vt) genes. Positive isolates were further tested with a multiplex PCR to detect vt1, vt2, eaeA and hlyA. For vt, 23 animals were positive at both samplings, 26 negative at both samplings, 22 negative animals became positive and 12 positive animals became negative. Sixty-eight per cent of the discordant pairs changed from vt-negative to vt-positive (95% CI 48–80) suggesting pressure toward becoming vt-positive perhaps due to the transfer of genes due to mixing of cattle in the months between samplings or an effect of chlortetracycline.

scholarly journals Detection and Characterization of Shiga ToxigenicEscherichia coli by Using Multiplex PCR Assays forstx 1, stx 2,eaeA, Enterohemorrhagic E. coli hlyA,rfb O111, andrfb O157

1998 ◽  
Vol 36 (2) ◽  
pp. 598-602 ◽  
Author(s):  
Adrienne W. Paton ◽  
James C. Paton
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Genetic Characterization ◽  
Gastrointestinal Disease ◽  
Diverse Group ◽  
E Coli ◽  
Pcr Products ◽  
Uremic Syndrome ◽  
Pcr Assays

Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of stx 1,stx 2 (including variants ofstx 2), eaeA, and enterohemorrhagicE. coli hlyA, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the rfb (O-antigen-encoding) regions of E. coli serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively. The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic E. colistrains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.

Escherichia coliserogroup O2 and O28ac O-antigen gene cluster sequences and detection of pathogenicE. coliO2 and O28ac by PCR

2010 ◽  
Vol 56 (4) ◽  
pp. 308-316 ◽  
Author(s):  
Pina M. Fratamico ◽  
Xianghe Yan ◽  
Yanhong Liu ◽  
Chitrita DebRoy ◽  
Brian Byrne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Rapid Identification ◽  
Open Reading Frames ◽  
E Coli ◽  
Antigen Gene ◽  
O Antigen ◽  
Pcr Assays

The O-antigen gene clusters of Escherichia coli serogroups O2 and O28ac were sequenced, and PCR assays were developed to identify strains belonging to these 2 serogroups. Sixteen and 8 open reading frames were mapped to these loci in E. coli O2:H4 U 9-41 and E. coli O28ac:H25 96-3286, respectively. The wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the E. coli O2 and O28ac O-antigen gene clusters were selected as targets for PCR assays for their identification. PCR assays targeting the wzx and wzy genes were specific for these serogroups, with one exception. Escherichia coli serogroup O42 strains gave positive results with wzx and wzy PCR assays targeting E. coli O28ac, and antiserum raised against O42 cross-reacted with serogroup O28ac strains. The O-antigen gene cluster of a strain of E. coli serogroup O42 was sequenced, and there were only 3 nt differences between the O-antigen gene clusters of the O28ac and O42 strains. Multiplex PCR assays targeting the O2 wzx gene, the stx1, stx2, hly, eae, and saa genes, and the O28ac wzx, ial, ipaC, and ipaH genes were developed for detecting Shiga toxin-producing E. coli O2 strains and enteroinvasive E. coli O28ac strains, respectively. The O2 and O28ac wzx and wzy genes can be used as diagnostic markers in PCR assays for rapid identification of these serogroups as an alternative to serotyping, and the multiplex PCR assays targeting serogroup-specific genes in combination with virulence genes can be used to identify and to detect pathogenic serogroup O2 and O28ac strains.

scholarly journals Molecular Identification Virulence Genes Of Escherichia coli Isolated From Bovine Clinical Mastitis

2014 ◽  
Vol 8 (2) ◽  
pp. 1-3 ◽  
Author(s):  
Gholam Ali Moradli ◽  
Tagi Zahraei Salehii ◽  
Mahmod Jamshidian ◽  
Farhad Mosakhani
Keyword(s):  
Escherichia Coli ◽  
Molecular Identification ◽  
Virulence Genes ◽  
Life Sciences ◽  
Bovine Mastitis ◽  
Virulence Gene ◽  
Clinical Mastitis ◽  
Common Gene ◽  
E Coli ◽  
Pcr Assays

The aims of this study were molecular identification some of virulence genes in Escherichia coli isolated from milk of bovines with clinical mastitis. (n = 60) E. coli isolates from acute clinical mastitis were examined for detect the presence of the genes encodingshigatoxin1 (stx1), intimin (eaeA), cytotoxicnecrotizingfactor 2 (cnf2), aerobactin (iucD) and P fimberiae (pap). The majors finding in the PCR assays were: 8 isolates (13.33%) had at least one virulence gene, None of isolates contained the genes for stx1, eaeA , the most common gene in the examined isolate was iucD which was positive in 6 isolates (10%). One isolate (1.66%) was positive for both iucD and pap genes and one isolate (1.66%) for cnf2 gene. In this study similar to previous investigations indicated that prevalence virulence genes in E. coli isolated of bovine mastitis is deferent. The results of this investigate similar to previous studies indicated none of the potential virulence genes or specific pathotype was observed in E. coli isolates from bovine clinical mastitis. DOI: http://dx.doi.org/10.3126/ijls.v8i2.10219   International Journal of Life Sciences Vol.8(2): 2014; 1-3 

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