William C Shropshire1,2, Samuel L Aitken2,3, Reed Pifer4, Jiwoong Kim5, Micah M Bhatti6, Xiqi Li7, Awdhesh Kalia8, Jessica Galloway-Peña2,7,9, Pranoti Sahasrabhojane7, Cesar A Arias1,2,10,11, David E Greenberg2,12,13, Blake M Hanson1,2, Samuel A Shelburne2,7,9. 1. Center for Infectious Diseases, School of Public Health, University of Texas Health Science Center, Houston, TX 77030, USA. 2. Center for Antimicrobial Resistance and Microbial Genomics, Division of Infectious Diseases, University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX 77030, USA. 3. Division of Pharmacy, MD Anderson Cancer Center, Houston, TX 77030, USA. 4. Division of Infectious Diseases, Department of Internal Medicine, University of Texas Health Science Center at Houston, McGovern Medical School at Houston, Houston, TX 77030, USA. 5. Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX 75390, USA. 6. Department of Laboratory Medicine, MD Anderson Cancer Center, Houston, TX 77030, USA. 7. Department of Infectious Diseases, MD Anderson Cancer Center, Houston, TX 77030, USA. 8. Graduate Program in Diagnostic Genetics, School of Health Professions, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 9. Department of Genomic Medicine, MD Anderson Cancer Center, Houston, TX 77030, USA. 10. Department of Microbiology and Molecular Genetics, University of Texas McGovern Medical School at Houston, Houston, TX 77030, USA. 11. Molecular Genetics and Antimicrobial Resistance Unit, International Center for Microbial Genomics, Universidad El Bosque, Bogotá, Ak. 9#131a2, Colombia. 12. Department of Internal Medicine, UT Southwestern, Dallas, TX 75390, USA. 13. Department of Microbiology, UT Southwestern, Dallas, TX 75390, USA.
Abstract
BACKGROUND: Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. OBJECTIVES: To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. METHODS: This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively. RESULTS: Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. CONCLUSIONS: These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.
BACKGROUND: Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. OBJECTIVES: To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. METHODS: This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively. RESULTS: Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. CONCLUSIONS: These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.
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