scholarly journals New Monoclonal Antibodies against a Novel Subtype of Shiga Toxin 1 Produced by Enterobacter cloacae and Their Use in Analysis of Human Serum

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Craig Skinner ◽  
Stephanie Patfield ◽  
Rowaida Khalil ◽  
Qiulian Kong ◽  
Xiaohua He
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Human Serum ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Enterobacter Cloacae ◽  
Clinical Value ◽  
Content Type ◽  
Clinical Detection ◽  
Detection And Diagnosis

ABSTRACT Stxs are among the most clinically important virulence factors of Shigella and enterohemorrhagic Escherichia coli. There are many varieties of Stx, and although Stx1a and Stx2a are the most common and widely distributed types of Stx, new variants of Stx are continually emerging. These new variants of Stx can be challenging to detect, since most Stx detection kits are optimized for the detection of Stx1a and Stx2a. Stx1e, recently discovered in an atypical host (Enterobacter cloacae), is undetectable by many Stx assays. To formulate new assays for the detection of Stx1e, we generated four new MAbs that recognize this Stx subtype. Using these antibodies, we generated an assay capable of detecting Stx1e at low picogram-per-milliliter concentrations. This assay is also compatible with a human serum matrix, suggesting that it may have utility for the clinical detection and diagnosis of Stx1e-associated infections. Shiga toxin (Stx) is a major virulence factor of several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges for the clinical diagnosis of infections caused by Stx-producing organisms. Here, we report the development of four new monoclonal antibodies (MAbs) against Stx1e, a novel subtype of Stx1 that was produced by an Enterobacter cloacae strain and had limited reactivity with existing anti-Stx1 antibodies. Western blot analysis indicates that these MAbs were Stx1 specific, bound to the A subunit, and had distinct preferences for subtypes of Stx1. Of the four MAbs, Stx1e-2 was capable of partially neutralizing cytotoxicities derived from Stx1e in Vero cells. Enzyme-linked immunosorbent assays assembled with these high-affinity MAbs detected Stx1e at concentrations as low as 4.8 pg/ml in phosphate-buffered saline and 53.6 pg/ml in spiked human serum samples and were also capable of distinguishing Stx1e-producing strains in enriched cultures. These assays may therefore have clinical value in diagnosing Stx1e-producing bacterial infection. Additionally, characteristics of Stx1e, such as the origin of stx1e genes, conditions for toxin expression, receptor binding, and cytotoxicity, were investigated with the new antibodies developed in this study. This information should be useful for further understanding the clinical significance and prevalence of Stx1e-harboring E. cloacae and other organisms. IMPORTANCE Stxs are among the most clinically important virulence factors of Shigella and enterohemorrhagic Escherichia coli. There are many varieties of Stx, and although Stx1a and Stx2a are the most common and widely distributed types of Stx, new variants of Stx are continually emerging. These new variants of Stx can be challenging to detect, since most Stx detection kits are optimized for the detection of Stx1a and Stx2a. Stx1e, recently discovered in an atypical host (Enterobacter cloacae), is undetectable by many Stx assays. To formulate new assays for the detection of Stx1e, we generated four new MAbs that recognize this Stx subtype. Using these antibodies, we generated an assay capable of detecting Stx1e at low picogram-per-milliliter concentrations. This assay is also compatible with a human serum matrix, suggesting that it may have utility for the clinical detection and diagnosis of Stx1e-associated infections.

scholarly journals Prevalences of Shiga Toxin Subtypes and Selected Other Virulence Factors among Shiga-Toxigenic Escherichia coli Strains Isolated from Fresh Produce

2013 ◽  
Vol 79 (22) ◽  
pp. 6917-6923 ◽  
Author(s):  
Peter C. H. Feng ◽  
Shanker Reddy
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Shiga Toxin ◽  
Fresh Produce ◽  
Putative Virulence Factor ◽  
Content Type ◽  
Human Illness ◽  
Subtilase Cytotoxin ◽  
Virulence Factor Genes

ABSTRACTShiga-toxigenicEscherichia coli(STEC) strains were isolated from a variety of fresh produce, but mostly from spinach, with an estimated prevalence rate of 0.5%. A panel of 132 produce STEC strains were characterized for the presence of virulence and putative virulence factor genes and for Shiga toxin subtypes. About 9% of the isolates were found to have theeaegene, which encodes the intimin binding protein, and most of these belonged to known pathogenic STEC serotypes, such as O157:H7 and O26:H11, or to serotypes that reportedly have caused human illness. Among theeae-negative strains, there were three O113:H21 strains and one O91:H21 strain, which historically have been implicated in illness and therefore may be of concern as well. TheehxAgene, which encodes enterohemolysin, was found in ∼60% of the isolates, and thesaaandsubABgenes, which encode STEC agglutinating adhesin and subtilase cytotoxin, respectively, were found in ∼30% of the isolates. However, the precise roles of these three putative virulence factors in STEC pathogenesis have not yet been fully established. Thestx1aandstx2asubtypes were present in 22% and 56%, respectively, of the strains overall and were the most common subtypes among produce STEC strains. Thestx2dsubtype was the second most common subtype (28% overall), followed bystx2c(7.5%), and only 2 to 3% of the produce STEC strains had thestx2eandstx2gsubtypes. Almost half of the produce STEC strains had only partial serotypes or were untyped, and most of those that were identified belonged to unremarkable serotypes. Considering the uncertainties of some of these Stx subtypes and putative virulence factors in causing human illness, it is difficult to determine the health risk of many of these produce STEC strains.

scholarly journals Prevalence of Hemolysin Genes and Comparison ofehxASubtype Patterns in Shiga Toxin-Producing Escherichia coli (STEC) and Non-STEC Strains from Clinical, Food, and Animal Sources

2013 ◽  
Vol 79 (20) ◽  
pp. 6301-6311 ◽  
Author(s):  
Sandra C. Lorenz ◽  
Insook Son ◽  
Anna Maounounen-Laasri ◽  
Andrew Lin ◽  
Markus Fischer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Phenotypic Expression ◽  
Food Sources ◽  
Polymorphism Analysis ◽  
Subtype C ◽  
Content Type ◽  
E Coli ◽  
Subtype A

ABSTRACTShiga toxin-producingEscherichia coli(STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, likestxandeae, have been well studied, little is known about the prevalence of theE. colihemolysin genes (hlyA,ehxA,e-hlyA, andsheA) in association with these factors. Hemolysins are potential virulence factors, andehxAandhlyAhave been associated with human illness, but the significance ofsheAis unknown. Hence, 435E. colistrains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigatedehxAsubtype patterns inE. coliisolates from clinical, animal, and food sources. WhilesheAandehxAwere widely distributed,e-hlyAandhlyAwere rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carriedstxand/oreae(P< 0.0001).ehxAsubtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (>94.2%), with subtypes A/D beingeae-negative STECs and subtypes B, C, E, and Feaepositive. Unexpectedly,ehxAsubtype patterns differed significantly between isolates collected from different sources (P< 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particularehxAsubtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.

scholarly journals Virulence Profiling of Shiga Toxin-Producing Escherichia coli O111:NM Isolates from Cattle

2013 ◽  
Vol 79 (13) ◽  
pp. 4164-4165 ◽  
Author(s):  
Musafiri Karama ◽  
Carlton L. Gyles
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Disease Outbreaks ◽  
The United States ◽  
Full Spectrum ◽  
Gene Markers ◽  
Content Type

ABSTRACTShiga toxin-producingEscherichia coli(STEC) O111:NM is an important serotype that has been incriminated in disease outbreaks in the United States. This study characterized cattle STEC O111:NM for virulence factors and markers by PCR. Major conclusions are that STEC O111:NM characterized in this study lacksstx2and the full spectrum ofnlegene markers, and it has an incomplete OI-122.

scholarly journals Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens

2015 ◽  
Vol 60 (3) ◽  
pp. 1874-1877 ◽  
Author(s):  
S. Baron ◽  
S. Delannoy ◽  
S. Bougeard ◽  
E. Larvor ◽  
E. Jouy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Phylogenetic Groups ◽  
Content Type ◽  
E Coli ◽  
Heat Stable ◽  
Intestinal Infections

This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups ofEscherichia coliisolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producingE. colior enteropathogenicE. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA,cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

scholarly journals Complete Genome Sequence of a Shiga Toxin-Converting Bacteriophage, Escherichia Phage Lys12581Vzw, Induced from an Outbreak Shiga Toxin-Producing Escherichia coli Strain

2019 ◽  
Vol 8 (36) ◽  
Author(s):  
Yujie Zhang ◽  
Yen-Te Liao ◽  
Alexandra Salvador ◽  
Xiaohong Sun ◽  
Vivian C. H. Wu
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Genome Sequence ◽  
Shiga Toxin ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Coli Strain ◽  
Genomic Information ◽  
Content Type

Although numerous Shiga toxin (Stx)-producing Escherichia coli (STEC) strains have been sequenced, genomic information on Stx-converting phages, highly related to the primary virulence factors of STEC, is scarce. Here, we report the complete genome sequence of a Stx-converting phage induced from an outbreak STEC O145 strain.

scholarly journals Bactericidal Monoclonal Antibodies Specific to the Lipopolysaccharide O Antigen from Multidrug-Resistant Escherichia coli Clone ST131-O25b:H4 Elicit Protection in Mice

2015 ◽  
Vol 59 (6) ◽  
pp. 3109-3116 ◽  
Author(s):  
Valéria Szijártó ◽  
Luis M. Guachalla ◽  
Zehra C. Visram ◽  
Katharina Hartl ◽  
Cecília Varga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Human Serum ◽  
Significant Proportion ◽  
Multidrug Resistant ◽  
Bacterial Killing ◽  
Content Type ◽  
E Coli

ABSTRACTTheEscherichia colisequence type 131 (ST131)-O25b:H4 clone has spread worldwide and become responsible for a significant proportion of multidrug-resistant extraintestinal infections. We generated humanized monoclonal antibodies (MAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These MAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assayin vitro, MAbs induced >95% bacterial killing in the presence of human serum as the complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of actionin vivowas investigated by using aglycosylated derivatives of the protective MAbs. The significant binding to liveE. colicells and thein vitroandin vivoefficacy were corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multidrug-resistant Gram-negative pathogens, passive immunization with bactericidal antibodies offers a therapeutic alternative to control infections caused byE. coliST131-O25b:H4.

scholarly journals Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia sp. Cryptic Lineage 1 Strain 7v Harbors a Hybrid Plasmid

2016 ◽  
Vol 82 (14) ◽  
pp. 4309-4319 ◽  
Author(s):  
Susan R. Leonard ◽  
Mark K. Mammel ◽  
David A. Rasko ◽  
David W. Lacher
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Clinical Samples ◽  
Hybrid Strain ◽  
Content Type ◽  
E Coli ◽  
Cryptic Lineage

ABSTRACTHybrid isolates of Shiga toxin-producingEscherichia coli(STEC) and enterotoxigenicE. coli(ETEC) encoding heat-stable enterotoxin (ST) are being reported with increasing frequency from a variety of sources. However, information regarding the plasmids that these strains harbor is scarce. In this study, we sequence and characterize a plasmid, p7v, from the STEC/ETEC hybrid strain 7v. Whole-genome phylogenetic analyses of STEC/ETEC hybrid strains and prototypeE. coliisolates of other pathotypes placed 7v in theEscherichiasp. cryptic lineage 1 (CL1) clade. The complete plasmid, p7v, was determined to be 229,275 bp and encodes putative virulence factors that are typically carried on STEC plasmids as well as those often carried on ETEC plasmids, indicating that the hybrid nature of the strain extends beyond merely encoding the two toxins. Plasmid p7v carries two copies ofstawith identical sequences, which were discovered to be divergent from thestasequences found in the prototype human ETEC strains. Using a nomenclature scheme based on a phylogeny constructed fromstaandstbsequences, thestaencoded on p7v is designated STa4.In silicoanalysis determined that p7v also encodes the K88 fimbria, a colonization factor usually associated with porcine ETEC plasmids. The p7v sequence and the presence of plasmid-encoded virulence factors are compared to those of other STEC/ETEC CL1 hybrid genomes and reveal gene acquisition/loss at the strain level. In addition, the interrogation of 24 STEC/ETEC hybrid genomes for identification of plasmid replicons, colonization factors, Stx and ST subtypes, and other plasmid-encoded virulence genes highlights the diversity of these hybrid strains.IMPORTANCEHybrid Shiga toxin-producingEscherichia coli/enterotoxigenicEscherichia coli(STEC/ETEC) strains, which have been isolated from environmental, animal, and human clinical samples, may represent an emerging threat as food-borne pathogens. Characterization of these strains is important for assessing virulence potential, aiding in the development of pathogen detection methods, and understanding how the hybrid strains evolve to potentially have a greater impact on public health. This study represents, to our knowledge, both the first characterization of a closed plasmid sequence from a STEC/ETEC hybrid strain and the most comprehensive phylogenetic analysis of available STEC/ETEC hybrid genomes to date. The results demonstrate how the mobility of plasmid-associated virulence genes has resulted in the creation of a diverse plasmid repertoire within the STEC/ETEC hybrid strains.

scholarly journals Molecular Epidemiology of NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Isolates from Pakistan

2014 ◽  
Vol 58 (9) ◽  
pp. 5589-5593 ◽  
Author(s):  
Anna L. Sartor ◽  
Muhammad W. Raza ◽  
Shahid A. Abbasi ◽  
Kathryn M. Day ◽  
John D. Perry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Epidemiology ◽  
Genetic Relatedness ◽  
Antibiotic Resistance Genes ◽  
Enterobacter Cloacae ◽  
Content Type ◽  
Plasmid Replicon ◽  
Genes Encoding ◽  
Clonal Spread ◽  
Encoding Genes

ABSTRACTThe molecular epidemiology of 66 NDM-producing isolates from 2 Pakistani hospitals was investigated, with their genetic relatedness determined using repetitive sequence-based PCR (Rep-PCR). PCR-based replicon typing and screening for antibiotic resistance genes encoding carbapenemases, other β-lactamases, and 16S methylases were also performed. Rep-PCR suggested a clonal spread ofEnterobacter cloacaeandEscherichia coli. A number of plasmid replicon types were identified, with the incompatibility A/C group (IncA/C) being the most common (78%). 16S methylase-encoding genes were coharbored in 81% of NDM-producingEnterobacteriaceae.

scholarly journals Detection of virulence factors and antimicrobial resistance patterns in shiga toxin-producing Escherichia coli isolates from sheep

2015 ◽  
Vol 35 (9) ◽  
pp. 775-780 ◽  
Author(s):  
Marcos R.A. Ferreira ◽  
Talícia dos S. Silva ◽  
Ariel E. Stella ◽  
Fabricio R. Conceição ◽  
Edésio F. dos Reis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Resistance Profile ◽  
Antimicrobial Resistance Profile ◽  
Healthy Sheep ◽  
Resistance Patterns ◽  
Group A ◽  
Stx1 Gene

Abstract: In order to detect virulence factors in Shiga toxin-producing Escherichia coli (STEC) isolates and investigate the antimicrobial resistance profile, rectal swabs were collected from healthy sheep of the races Santa Inês and Dorper. Of the 115 E. coli isolates obtained, 78.3% (90/115) were characterized as STEC, of which 52.2% (47/90) carried stx1 gene, 33.3% (30/90) stx2 and 14.5% (13/90) both genes. In search of virulence factors, 47.7% and 32.2% of the isolates carried the genes saa and cnf1. According to the analysis of the antimicrobial resistance profile, 83.3% (75/90) were resistant to at least one of the antibiotics tested. In phylogenetic classification grouped 24.4% (22/90) in group D (pathogenic), 32.2% (29/90) in group B1 (commensal) and 43.3% (39/90) in group A (commensal). The presence of several virulence factors as well as the high number of multiresistant isolates found in this study support the statement that sheep are potential carriers of pathogens threatening public health.

scholarly journals Comparison of the Superpolymyxin and ChromID Colistin R Screening Media for the Detection of Colistin-Resistant Enterobacteriaceae from Spiked Rectal Swabs

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Delphine Girlich ◽  
Thierry Naas ◽  
Laurent Dortet
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Confidence Interval ◽  
Enterobacter Cloacae ◽  
Bacterial Isolates ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Content Type ◽  
Rectal Swabs ◽  
Stool Samples

ABSTRACT The dissemination of carbapenemase-producing Enterobacteriaceae (CPE) has led to the increased use of colistin, which has resulted in the emergence of colistin-resistant Enterobacteriaceae worldwide. One of the most threatening scenarios is the dissemination of colistin resistance in CPE, particularly the plasmid-encoded resistance element MCR. Thus, it has now become mandatory to possess reliable media to screen for colistin-resistant Gram-negative bacterial isolates, especially Enterobacteriaceae. In this study, we evaluated the performances of the Superpolymyxin medium (ELITechGroup) and the ChromID Colistin R medium (bioMérieux) to screen for colistin-resistant Enterobacteriaceae from spiked rectal swabs. Stool samples were spiked with a total of 94 enterobacterial isolates (Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Enterobacter cloacae), including 53 colistin-resistant isolates. ESwabs (Copan Diagnostics) were then inoculated with those spiked fecal suspensions, and culture proceeded as recommended by both manufacturers. The sensitivity of detection of colistin-resistant Enterobacteriaceae was 86.8% (95% confidence interval [95% CI] = 74.0% to 94.0%) using both the Superpolymyxin medium and the ChromID Colistin R plates. Surprisingly, the isolates that were not detected were not the same for both media. The specificities were high for both media, at 97.9% (95% CI = 87.3% to 99.9%) for the Superpolymyxin medium and 100% (95% CI = 90.4% to 100%) for the ChromID Colistin R medium. Both commercially available media, ChromID Colistin R and Superpolymyxin, provide useful tools to screen for colistin-resistant Enterobacteriaceae from patient samples (rectal swabs) regardless of the level and mechanism of colistin resistance.

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