Abstract
BackgroundThe Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens.ResultsWe developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes.ConclusionsOur results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.
Highlights
The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies
Concept of microarray design Our strategy to design a diagnostic microarray based on a new set of pathogroup-specific determinants is structured according to clinically distinct enterobacterial pathogroups
The comparisons were split into three main levels of organisation within the pathogroup tree: (I) the genus level, (II) the distinction between Shigella, pathogenic and non-pathogenic E. coli as well as (III) the diversity among intestinal and extraintestinal E. coli pathotypes
Summary
The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. Enterobacteriaceae are frequent causes of human infectious diseases. This family comprises a broad variety of non-pathogenic and commensal variants. E. coli K-12 strains such as strain MG1655 are well-known model organisms in genetics diarrhoea with high incidence in developing countries. EIEC are highly similar to Shigella isolates, which are clinically associated with varying degrees of dysentery. Various types of so-called extraintestinal pathogenic E. coli (ExPEC) have been described to cause infections outside of the gastrointestinal tract, i.e. urinary tract infection, newborn meningitis or sepsis. Uropathogenic E. coli (UPEC), newborn meningitisassociated E. coli (MNEC) as well as sepsis-associated E. coli (SEPEC) differ in their repertoire of virulenceassociated genes from IPEC [1]
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