Instituto Valenciano de Microbiología

Masía El Romeral
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Escherichia coli producing shigatoxin (STEC) - Determination of serogroup-O and Serotype-H: Molecular identification (PCR).

Information 28-07-2019.

Escherichia coli shiga toxin (STEC) bacteria, particularly O157 strains, are important foodborne pathogens that generate numerous outbreaks worldwide. Other STEC no O157 strains, particularly strains O26, O45, O103, O111, O121 and O145, are also recognized as a major public health problem. They are responsible for infections and gastrointestinal diseases, such as hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Many foods have been linked to these outbreaks, highlighting meat, meat derivatives or undercooked meats.

Bacteria belonging to the species E. coli include both pathogens and commensals, which are currently identified by the combination of their antigens O (lipopolysaccharide) and H (flagellum protein). More than 200 different STEC O:H serotypes have been associated with human diseases. O antigens on the surface of E. coli are important virulence factors that are targets of the innate and adaptive immune system and play an important role in pathogenicity. The O antigens, responsible for the antigenic specificity of the strain, determine serogroup O.

E. coli strains producing Shiga toxin, Stx (STEC) has become an important foodborne pathogen and is a serious public health problem. However, the majority of clinical STEC infections, particularly those associated with severe outbreaks and clinical conditions in patients, are attributable to strains belonging to a subset of STEC serotypes called enterohemorrhagic E. coli (EHEC). This term was originally coined for the strains that cause HC and HUS, synthesize Stx and produce adhesion lesions and flattening of the mucosa (A/F), and possess the virulence plasmid 60-MDa. Specifically, STEC strains that produce Stx and A/F lesions and that possess plasmid 60-MDa are called "typical EHECs", which include serotypes O157:H7, O26:H11, O103:H2, O111:H8, O121:H19, and O145:H28. STEC strains associated with the disease that do not produce A/F lesions and/or do not possess the 60-MDa plasmid, less frequently involved in hemorrhagic diseases than typical EHEC but, nevertheless, a frequent cause of diarrhea, are they call "atypical EHEC", and includes serotypes O91:H21, O113:H21 and O104:H21, as well as O76:H19, O128:H2, O146:H28 and even O104:H4. In addition, in recent years, new EHEC serotypes have emerged as an important cause of foodborne infections in humans, including serotypes O5: H, O15:H2, O45:H2, O55:H7, O103:H25/H11, O118:H16, O123:H11, O165:H25, O172:H25, or O177:H, which have been referred to as "emerging EHEC".

Regardless of this classification, the forms of the O antigen: O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172 and O177 constitute the 21 most clinically important O-serogroups of E. coli producing Shiga toxin (STEC). Although serotype O157: H7 has been implicated in most outbreaks and in most cases of HUS, there is growing concern about the risk to human health associated with STEC serotypes not O157 STEC, which may also be responsible for major outbreaks, such as the famous German outbreak of 2011, caused by the STEC strain O104:H4. Serogroups O26, O45, O103, O111, O121 and O145 have been identified as the "big six" STEC no O157 by the Centers for Disease Control and Prevention as causative agents of human disease outbreaks worldwide. Cattle and dairy cattle are known reservoirs of STEC strains that can potentially contaminate meat carcasses during processing.

Traditionally, the determination of E. coli serotypes is performed by agglutination reactions using antisera against the different standard reference strains of O and H. However, traditional serotyping is laborious, time-consuming and often generates equivocal results due to cross-reaction between different serogroups. In addition, results may not be obtained when testing rough strains, which are refractory to typing. The reference technique requires the prior thermal inactivation, at different temperatures, of the capsule to expose the O antigens, and also the use of a wide collection of antisera, which is too expensive for most laboratories and can only be generated by specialized laboratories with facilities for animals. Therefore, rapid, less expensive and more specific molecular methods have been developed to identify different serotypes of E. coli. Much of the variation of the O antigen in E. coli is a consequence of the extensive genetic diversity within the rfb region gene group, which encodes many of the enzymes involved in the biosynthesis and assembly of the O antigen. The rbf region it generally includes three different types of genes: (i) genes that encode enzymes involved in the synthesis of the sugars that form the O subunit; (ii) genes encoding transferases, which assemble sugar substituents in the O subunit; and (iii) genes that encode proteins involved in the processing and assembly stages to construct the O-antigen from the O subunit, such as the wzx gene (which surrounds the O-antigen or flipase transporter) and the wzy gene (which encompasses the polymerase of the O antigen). Several genes in the O antigen gene group, particularly wzx and wzy, show a relatively low similarity between the different serogroups of E. coli and, therefore, by designing specific serogroup primers targeting the wzx and wzy serogroup-specific PCR assays can be developed, which allows the presence of each of the O-serogroups of E.coli to be detected by PCR.

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