An Improved Multiplex-Polymerase Chain Reaction Method for Simultaneous Identification and Discrimination of Emerging Escherichia and Shigella Species

Abstract

<strong data-sider-select-id="79723bd5-071a-46fc-8584-72cacba5c920">Background: Several prominent bacterial species known to induce diarrhea in human hosts encompass <em data-sider-select-id="0237854c-707e-407c-9a39-3cceb0366cd9">Escherichia coli, Escherichia albertii, Escherichia fergusonii, and various <em data-sider-select-id="2aabf174-2d34-4b85-82e3-38a1c8e284c1">Shigella spp. Given that these organisms contribute to the burden of food-borne illness, it is essential to rapidly and correctly identify them in a clinical laboratory or food microbiology unit to prevent their transmission and spread. These pathogens are often mistakenly identified because of their genetic and phenotypic similarities. Phenotypic tests are not highly discriminatory and are time-consuming. Whole-genome sequencing is expensive and unavailable in most clinical laboratories. <strong data-sider-select-id="29a111ec-f6b2-4778-a5cb-825c3f710d75">Materials and Methods: To simplify their rapid detection, we improved an available multiplex polymerase chain reaction (PCR) assay targeting three species-specific primers, including Eco (the main target for<em data-sider-select-id="2f1039f6-4b1a-4d0f-bff1-e805f2d02a5a"> E. coli identification), <em data-sider-select-id="015006c6-9c09-4f9c-9bff-bb43ad4edefb">Ealb (specific for <em data-sider-select-id="ca3ebbc2-9c3e-46ee-b1a8-7c04924a9df0">E. albertii), and <em data-sider-select-id="382a1307-9323-484f-97a8-2d8e26d064eb">Efer (specific for <em data-sider-select-id="b9f751ba-c4c0-41a1-9239-a2e12d3b2471">E. fergusonii), by adding ipaH and lacY to additionally discriminate between the highly similar Shigella spp. and enteroinvasive<em data-sider-select-id="8bdd04a9-449c-4075-b3ca-83e5c8dffcb0"> E. coli (EIEC) organisms. Primers were tested on 65 defined isolates, including<em data-sider-select-id="ce98464c-60dc-4b3a-8a92-d7b0c78f6dd2"> E. coli (n=29), <em data-sider-select-id="077d8e3e-3e50-465b-b23d-e2c2423f872d">Shigella spp. (n=26), <em data-sider-select-id="2226e62c-88ba-4c07-975f-8fbc7d74696c">E. fergusonii (n=1),<em data-sider-select-id="2ef30808-e4e0-48c8-9ae2-afef7183ca8c"> E. albertii (n=1), and other Enterobacterales (n=8). <strong data-sider-select-id="75e9dc30-27c2-49ae-ba33-c0d603d59b9e">Results: All examined E. coli yielded two amplicons of the expected size (Eco and lacY), except for EIEC, which had three bands (Eco, lacY, and ipaH). All <em data-sider-select-id="e24956d8-edb3-446e-9b6b-50666552e2a5">Shigella spp. yielded two amplicons (Eco and ipaH). <em data-sider-select-id="44d5bff7-e975-4b70-861a-0e51eebc3f32">E. fergusonii had only one band (Efer), and <em data-sider-select-id="f1d803e9-a101-473e-99bd-2abde9821a7e">E. albertii also yielded one band (Ealb). Other Enterobacterales that were tested for validation did not demonstrate a product, except for <em data-sider-select-id="948acbeb-7b44-4ebc-8ac9-65bd0cbd301b">Klebsiella pneumoniae and Klebsiella oxytoca (both lacY). <strong data-sider-select-id="583e71de-857e-4675-99ad-812c2722a2dc">Conclusion: The assay was shown to be a way forward for rapid, specific, and cost-effective primary discrimination of these important or emerging enteropathogens that can be used in clinical and research laboratories.