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The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates.

ABSTRACT: Escherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were bla_TEM-1 (56%) and bla_CTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.

SUBMITTER: Bernreiter-Hofer T

PROVIDER: S-EPMC8400056 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Based on 32 Escherichia coli and Shigella genome sequences, we have developed an E. coli pan-genome microarray. Publicly available genomes were annotated in a consistent manor to define all currently known genes potentially present in the species. The chip design was evaluated by hybridization of DNA from two sequenced E. coli strains, K-12 MG1655 (a commensal) and O157:H7 EDL933 (an enterotoxigenic E. coli). A dual channel and single channel analysis approach was compared for the comparative genomic hybridization experiments. Moreover, the microarray was used to characterize four unsequenced probiotic E. coli strains, currently marketed for beneficial effects on the human gut flora. Results: Based on the genomes included in this study, we were able to group together 2,041 genes that were present in all 32 genomes. Furthermore, we predict that the size of the E. coli core genome will approach ~1,560 essential genes, considerably less than previous estimates. Although any individual E. coli genome contains between 4,000 and 5,000 genes, we identified more than twice as many (11,872) distinct gene groups in the total gene pool (“pan-genome”) examined for microarray design. Benchmarking of the design based on sequenced control strain samples demonstrated a high sensitivity and relatively low false positive rate. Moreover, the array was highly sufficient to investigate the gene content of apathogenic isolates, despite the strong bias towards pathogenic E. coli strains that have been sequenced so far. Our analysis of four probiotic E. coli strains demonstrate that they share a gene pool very similar to the E. coli K-12 strains but also show significant similarity with enteropathogenic strains. Nonetheless, virulence genes were largely absent. Strain-specific genes found in probiotic E. coli but absent in E. coli K12 were most frequently phage-related genes, transposases and other genes related to mobile DNA, and metabolic enzymes or factors that may offer colonization fitness, which together with their asymptomatic nature may explain their nature. Conclusion: This high-density microarray provides an excellent tool for characterizing either DNA content or gene expression from unknown E. coli strains. Keywords: Comparative genomic hybridizations

Project description:Bacterial biofilm formation is a widespread phenomenon and a complex process requiring a set of genes facilitating the initial adhesion, maturation, and production of the extracellular polymeric matrix and subsequent dispersal of bacteria. Most studies on Escherichia coli biofilm formation have investigated nonpathogenic E. coli K-12 strains. Due to the extensive focus on laboratory strains in most studies, there is poor information regarding biofilm formation by pathogenic E. coli isolates. In this study, we genotypically and phenotypically characterized 187 human clinical E. coli isolates representing various pathotypes (e.g., uropathogenic, enteropathogenic, and enteroaggregative E. coli). We investigated the presence of biofilm-associated genes ("genotype") and phenotypically analyzed the isolates for motility and curli and cellulose production ("phenotype"). We developed a new screening method to examine the in vitro biofilm formation ability. In summary, we found a high prevalence of biofilm-associated genes. However, we could not detect a biofilm-associated gene or specific phenotype correlating with the biofilm formation ability. In contrast, we did identify an association of increased biofilm formation with a specific E. coli pathotype. Enteroaggregative E. coli (EAEC) was found to exhibit the highest capacity for biofilm formation. Using our image-based technology for the screening of biofilm formation, we demonstrated the characteristic biofilm formation pattern of EAEC, consisting of thick bacterial aggregates. In summary, our results highlight the fact that biofilm-promoting factors shown to be critical for biofilm formation in nonpathogenic strains do not reflect their impact in clinical isolates and that the ability of biofilm formation is a defined characteristic of EAEC.IMPORTANCE Bacterial biofilms are ubiquitous and consist of sessile bacterial cells surrounded by a self-produced extracellular polymeric matrix. They cause chronic and device-related infections due to their high resistance to antibiotics and the host immune system. In nonpathogenic Escherichia coli, cell surface components playing a pivotal role in biofilm formation are well known. In contrast, there is poor information for their role in biofilm formation of pathogenic isolates. Our study provides insights into the correlation of biofilm-associated genes or specific phenotypes with the biofilm formation ability of commensal and pathogenic E. coli Additionally, we describe a newly developed method enabling qualitative biofilm analysis by automated image analysis, which is beneficial for high-throughput screenings. Our results help to establish a better understanding of E. coli biofilm formation.

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The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates.

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