Antibiotics Resistance Genes Screening and Comparative Genomics Analysis of Commensal Escherichia coli Isolated from Poultry Farms between China and Sudan.

Escherichia coli (E. coli) strains, from the gut of animals and humans, harbor wide range of drug resistance genes. A comparative study is conducted on the intestinal E. coli from fecal samples of healthy chicken from China and Sudan in order to monitor the antimicrobial sensitivity pattern. A number of 250 E. coli isolates from chicken farms, including 120 from China and 130 from Sudan, were isolated and identified. All isolates were subjected to susceptibility tests against 10 antibiotics and the distribution of antibiotic resistant genes was confirmed by PCR amplification, involving genes such as ampC, tetA, pKD13, acrA, ermA, ermB, ermC, tetB, mphA, aadA14, aadA1, aac3-1, and aac3- III. Many isolates were found to exhibit resistance against more than one antibiotic. However, the Chinese isolates showed more antibiotics resistance and resistance genes compared to the Sudanese isolates. For better understanding of the multidrug resistance factors, we conducted whole genome analyses of E. coli D107 isolated from China, which revealed that the genome possesses multiple resistance genes including tetracycline, erythromycin, and kanamycin. Furthermore, E. coli D4 isolate from Sudan was more sensitive to antibiotics such as erythromycin, tetracycline, and gentamicin. After analysis by RAST and MAUVE, the two strains showed 89% average nucleotide identity. However, the genomes mostly differed at the number of antibiotics-related genes, as the genome of D107 revealed a considerable number of antibiotics resistance genes such as ermA and mphD which were found to be absent in D4 genome. These outcomes provided confirmation that the poultry farms environment in different countries (China and Sudan) may serve as a potential reservoir of antimicrobial resistance genes and also indicated the evolutionary differences of strains in terms of resistant genes expression.

Characterization and virulence clustering analysis of extraintestinal pathogenic Escherichia coli isolated from swine in China.

BACKGROUND: Swine extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that leads to economic and welfare costs in the swine industry worldwide, and is occurring with increasing frequency in China. By far, various virulence factors have been recognized in ExPEC. Here, we investigated the virulence genotypes and clonal structure of collected strains to improve the knowledge of phylogenetic traits of porcine ExPECs in China. RESULTS: We isolated 64 Chinese porcine ExPEC strains from 2013 to 14 in China. By multiplex PCR, the distribution of isolates belonging to phylogenetic groups B1, B2, A and D was 9.4%, 10.9%, 57.8% and 21.9%, respectively. Nineteen virulence-related genes were detected by PCR assay; ompA, fimH, vat, traT and iutA were highly prevalent. Virulence-related genes were remarkably more prevalent in group B2 than in groups A, B1 and D; notably, usp, cnf1, hlyD, papA and ibeA were only found in group B2 strains. Genotyping analysis was performed and four clusters of strains (named I to IV) were identified. Cluster IV contained all isolates from group B2 and Cluster IV isolates had the strongest pathogenicity in a mouse infection model. As phylogenetic group B2 and D ExPEC isolates are generally considered virulent, multilocus sequence typing (MLST) analysis was performed for these isolates to further investigate genetic relationships. Two novel sequence types, ST5170 and ST5171, were discovered. Among the nine clonal complexes identified among our group B2 and D isolates, CC12 and CC95 have been indicated to have high zoonotic pathogenicity. The distinction between group B2 and non-B2 isolates in virulence and genotype accorded with MLST analysis. CONCLUSION: This study reveals significant genetic diversity among ExPEC isolates and helps us to better understand their pathogenesis. Importantly, our data suggest group B2 (Cluster IV) strains have the highest risk of causing animal disease and illustrate the correlation between genotype and virulence.

Functional role of ompF and ompC porins in pathogenesis of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli is an important pathogen causes systemic infections in avian species and large economic losses in poultry industry worldwide. The functional role of porins during the infection and their mechanisms of interaction with host tissues for adhesion to and invasion are poorly understood. However, whether porins play a role in infection remains unclear. In this study we evaluated the potential of ompF and ompC outer membrane porins in the pathogenesis of avian pathogenic E. coli (APEC) strain TW-XM. The ompF and ompC were deleted to generate a series of mutants. We found that, ΔompF and ΔompC reduced significantly the adherence by 41.3% and 46.1% and invasion capabilities of APEC to mouse brain microvascular endothelial cell (BMEC) bEnd.3 cells in vitro by 51.9% and 49.7% respectively, compared with the wild strain TW-XM. In vivo experiment based on the measurement of the LD50 have also shown that, ΔompF and ΔompC reduced the bacterial virulence by 9.8-fold, 12.3-fold in ducklings and 9-fold, 10.2-fold in mouse models. Animal infection experiments further revealed that, loss of ompF and ompC reduced TW-XM colonization and invasion capacity in brains, lungs and blood compared to wild-type strain TW-XM (P > 0.01). These virulence-related phenotypes were partially recoverable by genetic complementation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) indicated that, the loss of ompF and ompC significantly decreased the expression levels of ompA, fimC and iBeA genes in the mutant strains, compared to wild-type strainTW-XM (P < 0.01). Collectively, our data demonstrate that inactivation of these two porins decreased adhesion, invasion, colonization, proliferation capacities, possibly by reduced expression levels of ompA, fimC and iBeA, which may indicate the involvement of ompF and ompC in APEC pathogenesis.

Role of outer membrane protein T in pathogenicity of avian pathogenic Escherichia coli.

An outer membrane protein T (OmpT) could play a vital role in the pathogenesis of the neonatal meningitis Escherichia coli (NMEC) in human and animals. However, whether ompT plays a role in avian pathogenic E. coli (APEC) infection remains unclear. In this study we evaluated the potential of ompT in APEC pathogenesis. An ompT gene was deleted from APEC mutant strain (TW-XM) was constructed and characterized. The inactivation of ompT reduced significantly the adherence and invasion capabilities of APEC to mouse brain microvascular endothelial cell (BMEC) bEnd.3 cells at the rates of 43.8% and 28.8% respectively, compared with the wild strain TW-XM. Further studies showed that deletion of ompT gene reduced the bacterial virulence with 15.2-fold in ducklings and 9.7-fold in mouse models based on the measurement of the LD50. Furthermore, experimental infection of animals revealed that, loss of ompT showed reduced APEC colonization and invasion capacity in brains, lungs and blood by 2-fold, 1.96-fold, and 1.7-fold, respectively, compared with the wild-type strain TW-XM. These virulence-related phenotypes were partially recoverable by genetic complementation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) indicated that the loss of ompT significantly decreased the expression levels of ompA, fimC and tsh in the mutant strain ΔOmpT, when compared with TW-XM (p<0.01). Collectively, our data showed that inactivation of ompT decreased adhesion, invasion, colonization, proliferation capacities, possibly by reduced expression levels of ompA, fimC and tsh, which may justify that, ompT is implicated in APEC pathogenicity.