Biology subjects

Bhatti, M. M.

Publications and source records attributed to Bhatti, M. M..

5 recordsLinked to original sources

bioRxiv2024

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure

BackgroundIt remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant. MethodsCarbapenem mutation frequency was measured in multiple subclades of extended-spectrum {beta}-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/H30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. ResultsAll thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P-value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing blaCTX-M-15 copy numbers via modular, insertion sequence 26 (IS26) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ConclusionsESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/H30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

microbiology↗

bioRxiv2023

Dynamic Periodicity of Extended-Spectrum Cephalosporin Resistant Bloodstream Escherichia coli Infections Driven by Non-CC131 Strains

Extended-spectrum cephalosporin resistant Escherichia coli (ESC-R-Ec) is an urgent public health threat with sequence type clonal complex 131 (STc131), phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R-Ec infections. To address the paucity of recent ESC-R-Ec molecular epidemiology data in the United States, we used whole genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R-Ec at a tertiary care cancer center in Houston, Texas collected from 2016-2020. During the study timeframe, there were 1154 index E. coli bloodstream infections (BSIs) of which 389 (33.7%) were ESC-R-Ec. Using time series analyses, we identified a temporal dynamic of ESC-R-Ec distinct from ESC-susceptible E. coli (ESC-S-Ec), with cases peaking in the last six months of the calendar year. WGS of 297 ESC-R-Ec strains revealed that while STc131 strains accounted for [~]45% of total BSIs, the proportion of STc131 strains remained stable across the study time frame with infection peaks driven by genetically heterogeneous ESC-R-Ec clonal complexes. BlaCTX-M variants accounted for most {beta}-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R-Ec), and amplification of blaCTX-M genes was widely detected in ESC-R-Ec strains, particularly in carbapenem non-susceptible, recurrent BSI strains. BlaCTX-M-55 was significantly enriched within phylogroup A strains, and we identified blaCTX-M-55 plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R-Ec infections at a large tertiary care cancer center and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens. IMPORTANCEGiven that E. coli is the leading cause of worldwide ESC-R Enterobacterales infections, we sought to assess the current molecular epidemiology of ESC-R-Ec using a WGS analysis of many BSIs over a five-year period. We identified fluctuating temporal dynamics of ESC-R-Ec infections, which has also recently been identified in other geographical regions such as Israel. Our WGS data allowed us to visualize the stable nature of STc131 over the study period and demonstrate a limited, but genetically diverse group of ESC-R-Ec clonal complexes are detected during infection peaks. Additionally, we provide a widespread assessment of {beta}-lactamase gene copy number in ESC-R-Ec infections and delineate mechanisms by which such amplifications are achieved in a diverse array of ESC-R-Ec strains. These data suggest that serious ESC-R-Ec infections are driven by a diverse array of strains in our cohort and impacted by environmental factors suggesting that community-based monitoring could inform novel preventative measures.

microbiology↗

bioRxiv2023

High-level ceftazidime-avibactam resistance in Escherichia coli conferred by the novel plasmid-mediated beta-lactamase CMY-185 variant

ObjectivesTo characterize a blaCMY variant associated with ceftazidime-avibactam (CZA) resistance from a serially collected Escherichia coli isolate. MethodsA patient with an intra-abdominal infection due to recurrent E. coli was treated with CZA. On day 48 of CZA therapy, E. coli with a CZA MIC of >256 mg/L was identified from abdominal drainage. Illumina WGS was performed on all isolates to identify potential resistance mechanisms. Site-directed mutants of CMY {beta}-lactamase were constructed to identify amino acid residues responsible for CZA resistance. ResultsWGS revealed that all three isolates were E. coli ST410. The CZA-resistant strain uniquely acquired a novel CMY {beta}-lactamase gene, herein called blaCMY-185, harbored on an IncI{gamma}-type conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2 including A114E, Q120K, V211S, and N346Y and conferred high-level CZA resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced CZA susceptibility. However, double and triple mutants containing N346Y previously associated with CZA resistance in other AmpC enzymes, conferred CZA MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to the steric hindrance between the side chain of Y346 and the sulfate group of avibactam. ConclusionWe identified CZA resistance in E. coli associated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer CZA resistance.

microbiology↗

bioRxiv2022

Systematic Analysis of Mobile Genetic Elements Mediating β-lactamase Gene Amplification in Non-Carbapenemase-Producing Carbapenem Resistant Enterobacterales Bloodstream Infections

Non-carbapenemase-producing carbapenem resistant Enterobacterales (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem resistant Enterobacterales (CRE) infections. However, there is limited understanding of mechanisms underlying non-CP-CRE causing invasive disease. Long- and short-read whole genome sequencing (WGS) was used to elucidate carbapenem non-susceptibility determinants in Enterobacterales bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem non-susceptible Enterobacterales (CNSE) mechanisms through a combination of phylogenetic analysis, antimicrobial resistant (AMR) gene detection/copy number quantification, porin assessment, and mobile genetic element (MGE) characterization. Most CNSE isolates sequenced were non-CP-CRE (41/79; 51.9%) whereas 25.3% (20/79) were carbapenem intermediate Enterobacterales (CIE) and 22.8% (18/79) were carbapenemase producing Enterobacterales (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum {beta}-lactamase (ESBL) genes (Wilcoxon Test; p-value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6X; n= 17) and K. pneumoniae (median CNV = 3.2X, n = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., ompC/ompF or ompK36/ompK35). Completely resolved CNSE genomes revealed that IS26 and ISEcp1 structures harboring blaCTX-M variants along with other AMR elements were the primary drivers of gene amplification, occurring in mostly IncFIB/IncFII plasmid contexts. MGE mediated {beta}-lactamase gene amplifications resulted in either tandem arrays, primarily mediated by IS26 translocatable units, or segmental duplication, typically due to ISEcp1 transposition units. Non-CP-CRE strains were the most prevalent cause of CRE bacteremia with carbapenem non-susceptibility driven by concurrent porin loss and MGE-mediated amplification of blaCTX-M genes. IMPORTANCECarbapenem resistant Enterobacterales (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase producing Enterobacterales (CPE) even though non-carbapenemase-producing CRE (non-CP-CRE) comprise 50% or more of isolates in some surveillance studies. Thus, carbapenem resistance mechanisms in non-CP-CRE remain poorly characterized. To address this problem, we applied a combination of short- and long-read sequencing technologies to a cohort of CRE bacteremia isolates and used these data to unravel complex mobile genetic element structures mediating {beta}- lactamase gene amplification. By generating complete genomes of 65 carbapenem non-susceptible Enterobacterales (CNSE) covering a genetically diverse array of isolates, our findings both generate novel insights into how non-CP-CRE overcome carbapenem treatments and provide researchers scaffolds for characterization of their own non-CP-CRE isolates. Improved recognition of mechanisms driving development of non-CP-CRE could assist with design and implementation of future strategies to mitigate the impact of these increasingly recognized AMR pathogens.

genomics↗

bioRxiv2019

β-lactamase Amplification and Porin Loss Drive Progressive β-lactam Resistance in Recurrent ESBL Enterobacteriaceae Bacteremia

BackgroundCarbapenem resistant Enterobacterales (CRE) remain urgent antimicrobial resistance threats. Approximately half of CRE clinical isolates lack carbapenem hydrolyzing enzymes and develop carbapenem resistance through alternative mechanisms. The purpose of this study was to elucidate the development of carbapenem resistance mechanisms from clonal, recurrent extended-spectrum {beta}-lactamase positive Enterobacterales (ESBL-E) bacteremia isolates in a vulnerable patient population. MethodsThis study investigated a historical, retrospective cohort of ESBL-E bacteremia cases in the University of Texas MD Anderson Cancer Center (MDACC) from January 2015 to July 2016. Phylogenetic and comparative genomic analyses were performed to identify clonal, recurrent ESBL-E isolates developing carbapenem resistance. Oxford Nanopore Technology (ONT) long-read and Illumina short-read sequencing data were used to generate consensus assemblies and to identify signatures of mobile genetic element mediated amplification and transposition of antimicrobial resistance genes. Serial passaging experiments were performed on a set of clinical ST131 ESBL-E isolates to recapitulate in vivo observations. qPCR and qRT-PCR were used to determine respective copy number and transcript levels of {beta}-lactamase genes. Results116 ESBL-E bacteremia cases were identified, 16 of which had documented recurrent infections. Four serial, recurrent isolates displayed a carbapenem resistant phenotype, three without the acquisition of a known carbapenemase. These three isolates had non-carbapenemase-producing CRE (non-CP-CRE) mechanisms driven by IS26- and ISEcp1-mediated amplification of respective translocatable units (TU) and transposition units (TPU) harboring both blaOXA-1 and blaCTX-M variants with concomitant outer membrane porin disruption. The TU and TPU structures inserted into the open reading frames of outer membrane porin genes in a subset of non-CP-CRE isolates. Serial passage of an index ST131 ESBL-E isolate under selective carbapenem exposure resulted in chromosomal amplification of modular, TUs harboring {beta}-lactamase genes with concomitant porin inactivation, recapitulating the in vivo carbapenem resistance progression. Long-read sequencing of two additional MDACC bacteremia strains identified similar non-CP-CRE mechanisms observed in the serial isolates. ConclusionsNon-CP-CRE de novo mechanisms were the primary driver of CRE development in recurrent bacteremia cases within this vulnerable patient population. The incorporation of long-read ONT data into AMR surveillance platforms is critical to identify high-risk CRE isolates that are difficult to identify with low-resolution phenotypic and molecular characterization methods.

genomics↗