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Hence, the present study aimed to elucidate the potential mechanisms of CZA resistance and heteroresistance in clinical carbapenemase (KPC)-producing\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      . A total of 25 KPC-producing\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      were collected and identified 19 CZA-resistant and 3 CZA-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Whole-genome sequencing and comparative genomic analysis of the heteroresistant parental strain and its resistant subpopulation showed mutations in\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      <jats:sub>KPC-2<\/jats:sub>\n                      (KPC-71 variant) and tandem amplification of the lipoprotein diacylglyceryl transferase\n                      <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                      allele in the resistant subpopulation. Reverse transcription-quantitative PCR (qRT-PCR) results demonstrated that the copy number of\n                      <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                      gene is associated with resistance to CZA.\n                      <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                      overexpression was induced in\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      strains involved in CZA heteroresistance and failed CZA therapy in infected mice. Flow cytometry and parallel reaction monitoring (PRM) assays were performed to investigate the heteroresistance mechanisms in\n                      <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                      overexpression strains. Flow cytometry analysis showed that overexpression of the\n                      <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                      gene decreased propidium iodide incorporation in the cells of this strain. PRM assays showed that the peptidoglycan-associated lipoprotein Pal and the outer membrane (OM) protein OmpA were significantly upregulated in the\n                      <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                      -overexpressing\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      strain. In conclusion, we demonstrated a new mechanism for CZA heteroresistance wherein tandem\n                      <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                      amplification generated population heterogeneity and failed CZA therapy\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      experimentally infected mice. Lipoprotein biosynthetic pathways are considered a potential target for CZA resistance.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        The novel synthetic \u03b2-lactamase inhibitor combination ceftazidime-avibactam has been available clinically for a few years. However, evidence that resistance and heteroresistance to ceftazidime-avibactam are responsible for treatment failure in clinical settings is increasing, and the underlying molecular mechanisms involved in developing heteroresistance have not been elucidated in detail. This study highlights that the lipoprotein diacylglyceryl transferase\n                        <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                        gene amplification conferred heteroresistance to\n                        <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                        strains. Moreover, we found that\n                        <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                        gene amplification-driven ceftazidime-avibactam heteroresistance might have resulted\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        treatment failure during\n                        <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                        infection in mice. We also observed that the\n                        <jats:italic toggle=\"yes\">umpA<\/jats:italic>\n                        gene amplification upregulated the expression levels of lipoproteins and outer membrane proteins and decreased outer membrane permeability in\n                        <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                        strains. These findings suggest that ceftazidime-avibactam heteroresistance is a major clinical concern in eradicating bacterial infections. Furthermore, lipoprotein biosynthetic pathways are considered a promising target for overcoming ceftazidime-avibactam (CZA) resistance.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01722-25","type":"journal-article","created":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T13:00:28Z","timestamp":1776085228000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Tandem amplification of the\n                    <i>umpA<\/i>\n                    allele contributes to ceftazidime-avibactam heteroresistance in clinical carbapenem-resistant\n                    <i>Klebsiella pneumoniae<\/i>\n                    isolates"],"prefix":"10.1128","volume":"14","author":[{"given":"Mengyao","family":"Wang","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04c8eg608","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biotechnology, College of Basic Medical Science, Dalian Medical University","place":["Dalian, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3671-5572","authenticated-orcid":true,"given":"Ying","family":"Li","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/012f2cn18","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University","place":["Dalian, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia","family":"Hu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04c8eg608","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biotechnology, 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