scholarly journals Genetic Analysis of the Cronobacter sakazakii O4 to O7 O-Antigen Gene Clusters and Development of a PCR Assay for Identification of All C. sakazakii O Serotypes

2012 ◽  
Vol 78 (11) ◽  
pp. 3966-3974 ◽  
Author(s):  
Yamin Sun ◽  
Min Wang ◽  
Quan Wang ◽  
Boyang Cao ◽  
Xin He ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Cronobacter Sakazakii ◽  
Pcr Assay ◽  
Gram Negative ◽  
Content Type ◽  
Antigen Gene ◽  
O Antigen ◽  
Multiplex Pcr Assay ◽  
Food Borne ◽  
Invasive Infections

ABSTRACTThe Gram-negative bacteriumCronobacter sakazakiiis an emerging food-borne pathogen that causes severe invasive infections in neonates. Variation in the O-antigen lipopolysaccharide in the outer membrane provides the basis for Gram-negative bacteria serotyping. The O-antigen serotyping scheme forC. sakazakii, which includes seven serotypes (O1 to O7), has been recently established, and the O-antigen gene clusters and specific primers for threeC. sakazakiiserotypes (O1, O2, and O3) have been characterized. In this study, theC. sakazakiiO4, O5, O6, and O7 O-antigen gene clusters were sequenced, and gene functions were predicted on the basis of homology.C. sakazakiiO4 shared a similar O-antigen gene cluster withEscherichia coliO103. The general features and anomalies of all sevenC. sakazakiiO-antigen gene clusters were evaluated and the relationship between O-antigen structures and their gene clusters were investigated. Serotype-specific genes for O4 to O7 were identified, and a molecular serotyping method for allC. sakazakiiO serotypes, a multiplex PCR assay, was developed by screening against 136 strains ofC. sakazakiiand closely related species. The sensitivity of PCR-based serotyping method was determined to be 0.01 ng of genomic DNA and 103CFU of each strain/ml. This study completes the elucidation ofC. sakazakiiO-antigen genetics and provides a molecular method suitable for the identification ofC. sakazakiiO1 to O7 strains.

scholarly journals Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene,cgcA, To Differentiate Species within the Genus Cronobacter

2012 ◽  
Vol 79 (2) ◽  
pp. 734-737 ◽  
Author(s):  
L. Carter ◽  
L. A. Lindsey ◽  
C. J. Grim ◽  
V. Sathyamoorthy ◽  
K. G. Jarvis ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Diguanylate Cyclase ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplexed Pcr ◽  
Multiplex Pcr Assay ◽  
Food Borne ◽  
Encoding Gene

ABSTRACTIn a comparison to the widely usedCronobacter rpoBPCR assay, a highly specific multiplexed PCR assay based oncgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305Cronobacterisolates was designed. This assay will be a valuable tool for identifying suspectedCronobacterisolates from food-borne investigations.

scholarly journals Molecular analysis ofShigella boydiiO1 O-antigen gene cluster and its PCR typing

2005 ◽  
Vol 51 (5) ◽  
pp. 387-392 ◽  
Author(s):  
Jiang Tao ◽  
Lei Wang ◽  
Dan Liu ◽  
Yue Li ◽  
David A Bastin ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gene Clusters ◽  
Pcr Assay ◽  
Pcr Screening ◽  
E Coli ◽  
Antigen Gene ◽  
O Antigen ◽  
Genes Encoding ◽  
Type Strains ◽  
Overnight Culture

Shigella is an important human pathogen and is closely related to Escherichia coli. O-antigen is the most variable part of the lipopolysaccharide on the cell surface of Gram-negative bacteria and plays an important role in pathogenicity. The O-antigen gene cluster of S. boydii O1 was sequenced. The putative genes encoding enzymes for rhamnose synthesis, transferases, O-unit flippase, and O-unit polymerase were identified on the basis of homology. The O-antigen gene clusters of S. boydii O1 and E. coli O149, which share the same O-antigen form, were found to have the same genes and organization by adjacent gene PCR assay. Two genes specific for S. boydii O1 and E. coli O149 were identified by PCR screening against E. coli- and Shigella-type strains of the 186 known O-antigen forms and 39 E. coli clinical isolates. A PCR sensitivity of 103to 104CFU/mL overnight culture of S. boydii O1 and E. coli O149 was obtained. S. boydii O1 and E. coli O149 were differentiated by PCR using lacZ- and cadA-based primers.Key words: O-antigen gene cluster, S. boydii O1, E. coli O149, molecular typing.

scholarly journals Molecular Characterization of Cronobacter Lipopolysaccharide O-Antigen Gene Clusters and Development of Serotype-Specific PCR Assays

2011 ◽  
Vol 77 (12) ◽  
pp. 4017-4026 ◽  
Author(s):  
K. G. Jarvis ◽  
C. J. Grim ◽  
A. A. Franco ◽  
G. Gopinath ◽  
V. Sathyamoorthy ◽  
...  
Keyword(s):  
Fragment Length Polymorphism ◽  
Gene Clusters ◽  
Enterobacter Sakazakii ◽  
Polymerase Gene ◽  
Content Type ◽  
Antigen Gene ◽  
O Antigen ◽  
Specific Pcr ◽  
Pcr Assays

ABSTRACTCronobacter(formerlyEnterobacter sakazakii) is a recently defined genus consisting of six species,C. sakazakii,C. malonaticus,C. dublinensis,C. muytjensii,C. turicensis, andCronobactergenomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located betweengalFandgnd, were used to identify serotypes inCronobacterspp. Seven O-antigen RFLP clusters were generated, including threeC. sakazakiiclusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including twoC. sakazakiistrains, twoC. malonaticusstrains, oneC. turicensisstrain, and oneC. muytjensiistrain, revealed three O-antigen gene clusters shared amongCronobacterspecies. PCR assays were developed, targeting thewzxO-antigen polymerase gene, and used to screen 231Cronobacterstrains to determine the frequency of these newly identified serotypes.

scholarly journals Organization of Escherichia coli O157 O Antigen Gene Cluster and Identification of Its Specific Genes

1998 ◽  
Vol 66 (8) ◽  
pp. 3545-3551 ◽  
Author(s):  
Lei Wang ◽  
Peter R. Reeves
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Repeat Unit ◽  
Gene Clusters ◽  
The United States ◽  
Fast Method ◽  
Gram Negative ◽  
E Coli ◽  
Antigen Gene ◽  
O Antigen

ABSTRACT The O157:H7 clone of Escherichia coli, which causes major, often prolonged outbreaks of gastroenteritis with hemolytic-uremic syndrome (HUS) such as those in Japan, Scotland, and the United States recently, is thought to be resident normally in cattle or other domestic animals. This clone is of major significance for public health and the food industry. We have developed a fast method for sequencing a given O antigen gene cluster and applied it to O157. The O157 O antigen gene cluster is 14 kb in length, comprising 12 genes and a remnant H-repeat unit. Based on sequence similarity, we have identified all the necessary O antigen genes, including five sugar biosynthetic pathway genes, four transferase genes, the O unit flippase gene, and the O antigen polymerase gene. By PCR testing against all 166 E. coli O serogroups and a range of gram-negative bacterial strains, including some that cross-react serologically with E. coli O157 antisera, we have found that certain O antigen genes are highly specific to O157 E. coli. This work provides the basis for a sensitive test for rapid detection of O157 E. coli. This is important both for decisions on patient care, since early treatment may reduce the risk of life-threatening complications, and for detection of sources of contamination. The method for fast sequencing of O antigen gene clusters plus an ability to predict which genes will be O antigen specific will enable PCR tests to be developed as needed for other clones of E. coli or, once flanking genes are identified, clones of any gram-negative bacterium.

scholarly journals Relationships among the O-Antigen Gene Clusters ofSalmonella enterica Groups B, D1, D2, and D3

1998 ◽  
Vol 180 (4) ◽  
pp. 1002-1007 ◽  
Author(s):  
Heather Curd ◽  
Dan Liu ◽  
Peter R. Reeves
Keyword(s):  
Cell Wall ◽  
Gene Cluster ◽  
Salmonella Enterica ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Gram Negative Bacteria ◽  
Polymerase Gene ◽  
Gram Negative ◽  
Antigen Gene ◽  
O Antigen

ABSTRACT The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of Salmonella enterica group D3. This segment includes a novel O-antigen polymerase gene (wzy D3). The polymerase gives α(1→6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-likewzy gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original wzy gene of these O-antigen gene clusters.

scholarly journals A Novel Method of Serum Resistance by Escherichia coli That Causes Urosepsis

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Carrie F. Coggon ◽  
Andrew Jiang ◽  
Kelvin G. K. Goh ◽  
Ian R. Henderson ◽  
Mark A. Schembri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Serum Resistance ◽  
Gram Negative ◽  
Content Type ◽  
Bacterial Surface ◽  
O Antigen ◽  
High Prevalence ◽  
Novel Method ◽  
Inhibitory Antibodies

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection, which in some patients can develop into life-threatening urosepsis. Serum resistance is a key virulence trait of strains that cause urosepsis. Recently, we identified a novel method of serum resistance in patients with Pseudomonas aeruginosa lung infections, where patients possessed antibodies that inhibited complement-mediated killing (instead of protecting against infection). These inhibitory antibodies were of the IgG2 subtype, specific to the O-antigen component of lipopolysaccharide (LPS) and coated the bacterial surface, preventing bacterial lysis by complement. As this mechanism could apply to any Gram-negative bacterial infection, we hypothesized that inhibitory antibodies may represent an uncharacterized mechanism of serum resistance in UPEC. To test this, 45 urosepsis patients with paired blood culture UPEC isolates were screened for serum titers of IgG2 specific for their cognate strain’s LPS. Eleven patients had sufficiently high titers of the antibody to inhibit serum-mediated killing of UPEC isolates by pooled healthy control sera. Depletion of IgG or removal of O-antigen restored sensitivity of the isolates to the cognate patient serum. Importantly, the isolates from these 11 patients were more sensitive to killing by serum than isolates from patients with no inhibitory antibodies. This suggests the presence of inhibitory antibodies may have allowed these strains to infect the bloodstream. The high prevalence of patients with inhibitory antibodies (24%) suggests that this phenomenon is an important mechanism of UPEC serum resistance. LPS-specific inhibitory antibodies have now been identified against three Gram-negative pathogens that cause disparate diseases. IMPORTANCE Despite improvements in the early detection and management of sepsis, morbidity and mortality are still high. Infections of the urinary tract are one of the most frequent sources of sepsis with Escherichia coli the main causative agent. Serum resistance is vital for bacteria to infect the bloodstream. Here we report a novel method of serum resistance found in patients with UPEC-mediated sepsis. Antibodies in sera usually protect against infection, but here we found that 24% of patients expressed “inhibitory antibodies” capable of preventing serum-mediated killing of their infecting isolate. Our data suggest that these antibodies would allow otherwise serum-sensitive UPEC strains to cause sepsis. The high prevalence of patients with inhibitory antibodies in this cohort suggests that this is a widespread mechanism of resistance to complement-mediated killing in urosepsis patients, invoking the potential for the application of new methods to prevent and treat sepsis.

scholarly journals Structure and genetics of Escherichia coli O antigens

2019 ◽  
Vol 44 (6) ◽  
pp. 655-683 ◽  
Author(s):  
Bin Liu ◽  
Axel Furevi ◽  
Andrei V Perepelov ◽  
Xi Guo ◽  
Hengchun Cao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Basis ◽  
Structural Diversity ◽  
Gene Clusters ◽  
Current Standard ◽  
E Coli ◽  
Antigen Gene ◽  
O Antigen ◽  
Or Groups ◽  
O Antigens

ABSTRACT Escherichia coli includes clonal groups of both commensal and pathogenic strains, with some of the latter causing serious infectious diseases. O antigen variation is current standard in defining strains for taxonomy and epidemiology, providing the basis for many serotyping schemes for Gram-negative bacteria. This review covers the diversity in E. coli O antigen structures and gene clusters, and the genetic basis for the structural diversity. Of the 187 formally defined O antigens, six (O31, O47, O67, O72, O94 and O122) have since been removed and three (O34, O89 and O144) strains do not produce any O antigen. Therefore, structures are presented for 176 of the 181 E. coli O antigens, some of which include subgroups. Most (93%) of these O antigens are synthesized via the Wzx/Wzy pathway, 11 via the ABC transporter pathway, with O20, O57 and O60 still uncharacterized due to failure to find their O antigen gene clusters. Biosynthetic pathways are given for 38 of the 49 sugars found in E. coli O antigens, and several pairs or groups of the E. coli antigens that have related structures show close relationships of the O antigen gene clusters within clades, thereby highlighting the genetic basis of the evolution of diversity.

scholarly journals Additional Og-Typing PCR Techniques Targeting Escherichia coli-Novel and Shigella-Unique O-Antigen Biosynthesis Gene Clusters

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
Atsushi Iguchi ◽  
Hironobu Nishii ◽  
Kazuko Seto ◽  
Jiro Mitobe ◽  
Kenichi Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Strong Association ◽  
Gene Clusters ◽  
Epidemiological Studies ◽  
Content Type ◽  
E Coli ◽  
O Antigen ◽  
Wide Range ◽  
Biosynthesis Gene ◽  
Almost All

ABSTRACT The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.

Sign in / Sign up

Export Citation Format

Share Document