Systematic Analysis of Mobile Genetic Elements Mediating β-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant Enterobacterales Bloodstream Infections.

Noncarbapenemase-producing carbapenem-resistant (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem-resistant (CRE) infections. However, there is limited understanding of mechanisms underlying non-CP-CRE causing invasive disease. Long- and short-read whole-genome sequencing was used to elucidate carbapenem nonsusceptibility determinants in bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem nonsusceptible (CNSE) mechanisms (i.e., isolates with carbapenem intermediate resistance phenotypes or greater) through a combination of phylogenetic analysis, antimicrobial resistance 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 with intermediate susceptibility to carbapenems (CIE), and 22.8% (18/79) were carbapenemase-producing (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum β-lactamase (ESBL) genes (Wilcoxon Test; -value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6×; = 17) and K. pneumoniae (median CNV = 3.2×,  = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., or ). Completely resolved CNSE genomes revealed that IS and IS structures harboring variants along with other antimicrobial resistance elements were associated with gene amplification, occurring in mostly IncFIB/IncFII plasmid contexts. MGE-mediated β-lactamase gene amplifications resulted in either tandem arrays, primarily mediated by IS translocatable units, or segmental duplication, typically due to IS transposition units. Non-CP-CRE strains were the most common cause of CRE bacteremia with carbapenem nonsusceptibility driven by concurrent porin loss and MGE-mediated amplification of genes. Carbapenem-resistant (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase-producing (CPE) even though noncarbapenemase-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 β-lactamase gene amplification. By generating complete genomes of 65 carbapenem nonsusceptible (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.

Shropshire WC ，Konovalova A ，McDaneld P ，Gohel M ，Strope B ，Sahasrabhojane P ，Tran CN ，Greenberg D ，Kim J ，Zhan X ，Aitken S ，Bhatti M ，Savidge TC ，Treangen TJ ，Hanson BM ，Arias CA ，Shelburne SA ... -  《mSystems》

IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort.

Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. 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. 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. 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.

Shropshire WC ，Aitken SL ，Pifer R ，Kim J ，Bhatti MM ，Li X ，Kalia A ，Galloway-Peña J ，Sahasrabhojane P ，Arias CA ，Greenberg DE ，Hanson BM ，Shelburne SA ... -  《-》

Clinical and molecular characteristics and risk factors for patients acquiring carbapenemase-producing and non-carbapenemase-producing carbapenem-nonsusceptible-Enterobacterales bacteremia.

Carbapenem-nonsusceptible Enterobacterales (CNSE) are a growing global threat. Carbapenemases are often produced by plasmids, which allow rapid transmission. This study aimed to investigate (1) the bacterial type (2) resistant genes (3) antimicrobial susceptibility and (4) risk factors for acquisition of carbapenemase-producing carbapenem-nonsusceptible Enterobacterales (CP-CNSE) and non-carbapenemase-producing carbapenem-nonsusceptible Enterobacterales (non-CP-CNSE) bacteremia. There were a total of 113 isolates of Enterobacterales from 2013 to 2018. After excluding nonblood isolates and including only one sample from each patient, 99 isolates were analyzed and the medical charts of these patients were reviewed. Carbapenemase genes, β-lactamase genes and antimicrobial susceptibility of the isolates were determined. Multilocus sequence typing (MLST) was performed on CP-CNSE isolates. CP-CNSE carried more blaSHV (P = 0.004) and were more resistant to imipenem than non-CP-CNSE (P < 0.001). In the univariate analyses, we found that CP-CNSE bloodstream infection was associated with patient <65 years of age (odds ratio, 3.90; 95% confidence interval [CI], 1.16 to 13.10; P = 0.027), mechanical ventilation at the time of bloodstream infection (BSI) (odds ratio, 3.85; 95% CI, 1.16-12.78; P = 0.028) and exposure to piperacillin/tazobactam (odds ratio, 3.96; 95% CI, 1.09-14.38; P = 0.037). However, on multivariate analyses, no independent predictor for CP-CNSE was identified in this study. CP-CNSE carried more blaSHV and were more resistant to imipenem when compared to non-CP-CNSE. No independent predictor for CP-CNSE was identified after multivariate analysis. This is the first study conducted in Taiwan comparing risk factors between CP-CNSE and non-CP-CNSE from both clinical and molecular aspects.

Wu AY ，Chang H ，Wang NY ，Sun FJ ，Liu CP ... -  《-》

Phenotypic and molecular characterization of multidrug-resistant Enterobacterales isolated from clinical samples in Palestine: a focus on extended-spectrum β-lactamase- and carbapenemase-producing isolates.

Infections resulting from multidrug-resistant Enterobacterales (MDR-E) pose a growing global threat, presenting challenges in treatment and contributing significantly to morbidity and mortality rates. The main objective of this study was to characterize phenotypically and genetically extended-spectrum β-lactamase- and carbapenemase- producing Enterobacterales (ESBLE and CPE respectively) isolated from clinical samples in the West Bank, Palestine. A cross sectional study was conducted in October 2023 on clinical bacterial isolates collected from five governmental hospitals in the West Bank, Palestine. The isolates obtained from the microbiology laboratories of the participating hospitals, underwent identification and antibiotic susceptibility testing (AST) using the VITEK® 2 Compact system. ESBL production was determined by the Vitek2 Compact system. A modified carbapenem inactivation method (mCIM) was employed to identify carbapenemase-producing Enterobacterales (CPE). Resistance genes were detected by real-time PCR. Out of the total 1380 collected isolates, we randomly selected 600 isolates for analysis. Our analysis indicated that 287 (47.83%) were extended-spectrum beta-lactamase producers (ESBLE), and 102 (17%) as carbapenem-resistant Enterobacterales (CRE) isolates. A total of 424 isolates (70.67%) were identified as multidrug-resistant Enterobacterales (MDRE). The most prevalent ESBL species were K. pneumoniae (n = 124; 43.2%), E. coli (n = 119; 41.5%) and E. cloacae (n = 31; 10.8%). Among the CRE isolates, 85 (83.33%) were carbapenemase-producing Enterobacterales (CPE). The most frequent CRE species were K. pneumoniae (n = 63; 61.7%), E. coli (n = 25; 24.5%) and E. cloacae (n = 13; 12.8%). Additionally, 47 (7.83%) isolates exhibited resistance to colistin (CT), with 38 (37.62%) being CT-resistant CRE and 9 (3.14%) being CT-resistant ESBLE while sensitive to carbapenems. We noticed that 11 isolates (6 Klebsiella pneumoniae and 5 Enterobacter cloacae complex) demonstrated sensitivity to carbapenems by phenotype but carried silent CPE genes (1 blaOXA48, and 6 blaNDM, 4 blaOXA48, blaNDM). ESBL-producing Enterobacterales strains exhibited varied resistance patterns across different antibiotic classes. E. coli isolates showed notable 48% resistance to trimethoprim/sulfamethoxazole. K. pneumoniae isolates displayed a significant resistance to trimethoprim/sulfamethoxazole, nitrofurantoin, and fosfomycin (54%, 90%, and 70% respectively). E. cloacae isolates showed complete resistance to nitrofurantoin and fosfomycin. P. mirabilis isolates exhibited high resistance against fluoroquinolones (83%), and complete resistance to trimethoprim/sulfamethoxazole, nitrofurantoin and fosfomycin. This study showed the high burden of the ESBLE and CRE among the samples collected from the participating hospitals. The most common species were K. pneumoniae and E. coli. There was a high prevalence of blaCTXm. Adopting both conventional and molecular techniques is essential for better surveillance of the emergence and spread of antimicrobial-resistant Enterobacterales infections in Palestine.

Ibaideya MA ，Taha AA ，Qadi M 《BMC INFECTIOUS DISEASES》

In vivo selection of carbapenem resistance during persistent Klebsiella pneumoniae sequence type 395 bloodstream infection due to OmpK36 deletion.

Strahilevitz J ，Motro Y ，Temper V ，Merezhko D ，Ayalon O ，Bar Moshe Y ，Lam MMC ，Holt KE ，Moran-Gilad J ... -  《-》