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Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

ABSTRACT: Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

SUBMITTER: Desloges I

PROVIDER: S-EPMC6854850 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Desloges Isabelle I Taylor James A JA Leclerc Jean-Mathieu JM Brannon John R JR Portt Andrea A Spencer John D JD Dewar Ken K Marczynski Gregory T GT Manges Amee A Gruenheid Samantha S Le Moual Hervé H Thomassin Jenny-Lee JL

MicrobiologyOpen 20190908 11

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and ina ...[more]

PMID: 31496120

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Project description:BackgroundUropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infection (UTI); however, treatment of UTI has been challenging due to increased antimicrobial resistance (AMR). One of the most important types of AMR is carbapenem resistance (CR). CR bacteria are known as an important threat to global public health today. Class B metallo-beta-lactamases (MBLs) are one of the major factors for resistance against carbapenems. We aimed to investigate the characteristics of UPEC isolates producing MBL.MethodsA cross-sectional study was conducted from October 2018 to December 2019 in Ahvaz; Iran. UPEC isolates were identified by biochemical and molecular methods. Metallo-beta-lactamase-producing isolates were detected using modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) tests. MBL genes, phylogenetic group, and virulence genes profile of carbapenem resistant isolates were determined. Conjugation assay and plasmid profiling were conducted to evaluate the ability of transferring of CR to other E. coli isolates. Clonal similarity of isolates were assessed using Enterobacterial intergenic repetitive element sequence (ERIC)-PCR.ResultsAmong 406 UPEC isolates, 12 (2.95%) carbapenem-resistant were detected of which 11 were phenotypically MBL-producing strains. Four isolates were resistant to all investigated antimicrobial agents and were considered possible pandrug-resistant (PDR). blaNDM, blaOXA-48, blaIMP-1, and blaIMP-2 genes were found in 9, 5, 1, and 1 isolates, respectively. Among 30 virulence genes investigated, the traT, fyuA followed by fimH, and iutA with the frequency of 8 (66.7%), 8 (66.7%), 7 (58.3%), and 7 (58.3%) were the most identified genes, respectively. Siderophore production was the main virulence trait among carbapenem-resistant UPEC isolates. Except for two, all other isolates showed weak to moderate virulence index. In all recovered isolates, CR was readily transmitted via plasmids to other isolates during conjugation experiments.ConclusionMBL and carbapenemase genes, especially blaNDM and blaOXA-48 are spreading rapidly among bacteria, which can be a threat to global public health. Therefore monitoring the emergence and dissemination of new AMR is necessary to continuously refine guidelines for empiric antimicrobial therapy. Understanding the mechanisms of resistance and virulence in this group of bacteria can play an effective role in providing new therapeutic methods.

Project description:INTRODUCTION:Uropathogenic E. coli is the leading cause of Urinary tract infections (UTIs), contributing to 80-90% of all community-acquired and 30-50% of all hospital-acquired UTIs. Biofilm forming Uropathogenic E. coli are associated with persistent and chronic inflammation leading to complicated and or recurrent UTIs. Biofilms provide an environment for poor antibiotic penetration and horizontal transfer of virulence genes which favors the development of Multidrug-resistant organisms (MDRO). Understanding biofilm formation and antimicrobial resistance determinants of Uropathogenic E. coli strains will provide insight into the development of treatment options for biofilm-associated UTIs. The aim of this study was to determine the biofilm forming capability, presence of virulence genes and antimicrobial susceptibility pattern of Uropathogenic E. coli isolates in Uganda. METHODS:This was a cross-sectional study carried in the Clinical Microbiology and Molecular biology laboratories at the Department of Medical Microbiology, Makerere University College of Health Sciences. We randomly selected 200 Uropathogenic E. coli clinical isolates among the stored isolates collected between January 2018 and December 2018 that had significant bacteriuria (>?105?CFU). All isolates were subjected to biofilm detection using the Congo Red Agar method and Antimicrobial susceptibility testing was performed using the Kirby disk diffusion method. The isolates were later subjected PCR for the detection of Urovirulence genes namely; Pap, Fim, Sfa, Afa, Hly and Cnf, using commercially designed primers. RESULTS:In this study, 62.5% (125/200) were positive biofilm formers and 78% (156/200) of these were multi-drug resistant (MDR). The isolates were most resistant to Trimethoprim sulphamethoxazole and Amoxicillin (93%) followed by gentamycin (87%) and the least was imipenem (0.5%). Fim was the most prevalent Urovirulence gene (53.5%) followed by Pap (21%), Sfa (13%), Afa (8%), Cnf (5.5%) and Hyl (0%). CONCLUSIONS:We demonstrate a high prevalence of biofilm-forming Uropathogenic E. coli strains that are highly associated with the MDR phenotype. We recommend routine surveillance of antimicrobial resistance and biofilm formation to understand the antibiotics suitable in the management of biofilm-associated UTIs.

Project description:UnlabelledThe vacuolating autotransporter toxin (Vat) contributes to uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator; we termed this gene vatX The vat-vatX genes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, the hns gene was mutated in CFT073 (to generate CFT073 hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073 hns compared to that in wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein of Enterobacteriaceae (SPATE) secreted by UPEC during infection.ImportanceUropathogenic Escherichia coli (UPEC) is the major cause of hospital- and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation of vat was demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream gene vatX (encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected with vat-positive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor.

Dataset Information

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Publications

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

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