In Vivo dynamic Evolution of Drug Resistance of fatal Salmonella infection in a Leukemia Patient

Background Salmonella 's development of carbapenem resistance is a serious public health concern, especially for immunocompromised hosts. However, the evolutionary mechanisms driving carbapenem resistance during clinical therapy remain poorly understood. Objective This study aims to investigate the dynamic evolution of carbapenem resistance in Salmonella isolated from a leukemia patient during treatment, as well as the molecular mechanisms involved. Methods Four Salmonella strains (D1415, D1969, D2053, D2054) were consecutively isolated from different anatomical sites of a leukemia patient eceiving multiple antibiotics, including carbapenems, quinolones, piperacillin-tazobactam, cefoperazone/sulbactam, and omadacycline. Antibiotic susceptibility testing was performed to determine bacterial resistance profiles. Growth curve analysis and biofilm formation assays were conducted to evaluate the adaptive cost of resistance. Whole-genome sequencing (Illumina), serotype prediction (SeqSero2), core-genome SNP(cgSNP) analysis (Snippy) and serum agglutination tests were used for molecular typing, serotype identification and phylogenetic relationship. Nanopore long-read sequencing was employed to analyze plasmid structures and gene copy number variations. Quantitative PCR (qPCR) was used to validate the expression levels of the bla _CTX−M , efflux pump gene tolC , and porin genes ompC / ompF. Results: The four isolates exhibited minimal cgSNP divergence, indicating a shared clonal origin. All isolates were assigned to sequence type ST198 and were identified as Salmonella serovar Kentucky. Among them, the D1969 isolate exhibited carbapenem resistance and demonstrated a lower growth rate compared to the other isolates. Nanopore sequencing revealed that all strains carried the IncFIB(K) plasmid. Notably, the bla _CTX-M gene copy number was increased to three in the pD1969 plasmid, while only a single copy was found in pD1415, and the gene was absent in pD2053 and pD2054. qPCR analysis confirmed an elevated expression of bla _CTX-M in D1969, along with upregulation of the efflux pump gene tolC , and downregulation of the porin genes ompC and ompF . Conclusion Under prolonged carbapenem selective pressure, Salmonella acquired carbapenem resistance through two co-evolving mechanisms: plasmid remodeing (manifested as bla _CTX-M gene amplification and the loss of non-essential genes) and chromosomal adaptation (including overexpression of efflux pumps and porin deficiencies). This study elucidates a non-carbapenemase-mediated mechanism of Salmonella resistance, highlighting the risk of resistance evolution in immunocompromised hosts during antibiotic treatment.