Heteroresistance to first-line β-lactams among Gram-negative bloodstream isolates reported susceptible by standard-of-care testing: a prospective two-centre study across a high- and a low-resistance setting
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Background
Antibiotic heteroresistance (HR) — a resistant subpopulation within a clonal isolate reported susceptible by routine testing — can drive unexplained treatment failure and on-treatment emergence of resistance, yet prospective prevalence data for first-line β-lactams in Gram-negative bloodstream infection are lacking. We assessed the prevalence and genomic basis of HR to piperacillin, piperacillin/tazobactam, or meropenem among bloodstream isolates reported susceptible by standard-of-care testing.
Methods
In this prospective two-centre prevalence study we screened consecutive monomicrobial Gram-negative positive blood cultures at the University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany, and the Fondazione Policlinico Universitario A. Gemelli IRCCS (FPG), Rome, Italy. Enterobacterales and Pseudomonadales reported susceptible by standard-of-care AST were allocated to one investigated beta-lactam (piperacillin, piperacillin/tazobactam or meropenem) according to their routine susceptibility phenotype (Vitek2), and entered a two-tier phenotypic workflow: an agar filter at 0.5x the EUCAST breakpoint in triplicate, followed - if positive - by population analysis profiling (PAP) up to 4x the breakpoint. Heteroresistance was defined as a clonal resistant subpopulation at a frequency of at least 0.5×10 -6 surviving in the 4x breakpoint zone. Prevalence was estimated with Wilson-score 95% CIs and compared between centres with Fisher’s exact test. Confirmed heteroresistant isolates and their paired susceptible bulk populations underwent short- and long-read whole-genome sequencing, and each resistant/susceptible pair was tested for tandem gene amplification, differential subclonal point variants and structural variation.
Findings
279 isolates were screened (93 at UKE, 186 at Gemelli). The 0.5x breakpoint filter was positive in 22 of 93 isolates (23.7%) at UKE and 66 of 186 (35.5%) at Gemelli. PAP confirmed heteroresistance in 6 of 93 isolates at UKE (6.5%, 95% CI 3.0-13.4) and 27 of 186 at Gemelli (14.5%, 10.2-20.3), a pooled prevalence of 33 of 279 (11.8%, 8.5-16.1); the between-centre difference did not reach significance (Fisher’s exact test p=0.051). Heteroresistance was essentially confined to piperacillin/tazobactam: 32 of 150 isolates screened against piperacillin/tazobactam were heteroresistant (21.3%, 15.5-28.6), versus 1 of 71 for piperacillin (1.4%) and 0 of 58 for meropenem. Escherichia coli accounted for 17 of the 33 heteroresistant isolates. In the resistant/susceptible pairs sequenced to date a candidate mechanism was resolved in 18: 13 carried a tandem amplification of a beta-lactamase locus (copy-number ratio 2.4-33.0; amplified units 5-136 kb, most often plasmid-borne and centred on blaTEM-1, blaCTX-M or blaOXA-1 ), and 5 carried point variants in the AmpC circuit ( ampD, ampR, dacB/PBP4 ) or in efflux components, three of them fixed loss- or gain-of-function changes and three subclonal.
Interpretation
Roughly one in eight Gram-negative bloodstream isolates called susceptible to a first-line beta-lactam harboured a resistant subpopulation, and the burden fell almost entirely on piperacillin/tazobactam - the agent most often used empirically in this setting. The dominant genomic substrate was unstable amplification of an existing beta-lactamase gene rather than acquisition of a new resistance determinant, which explains why the phenotype is reversible, invisible to AST and, in principle, quantifiable by copy-number assays at the point of care.
Funding
Part of these data were presented at ESCMID Global 2026, and D.S. received an ESCMID travel grant in support of that presentation.
Research in context
Evidence before this study
Beta-lactam heteroresistance in Gram-negative bacteria has been characterised mainly in reference collections and in single-species surveys, where unstable tandem amplification of resistance genes was identified as the principal mechanism and shown to be reversible and frequently missed by routine AST. HR has been linked to unexplained treatment failure, shown to cause discrepant susceptibility results, and — in a recent report — demonstrated to underlie within-patient conversion from susceptible to resistant during therapy. However, prospective, multicentre prevalence data for HR to the first-line β-lactams used empirically in Gram-negative bloodstream infection (piperacillin, piperacillin/tazobactam, meropenem), restricted to isolates that routine AST reports as susceptible, and paired with genome-resolved mechanisms, were lacking.
Added value of this study
To our knowledge this is the first prospective, multicentre study to quantify PAP-confirmed HR to first-line β-lactams specifically among Gram-negative bloodstream isolates categorised as susceptible by standard-of-care AST, and to pair that prevalence with complete (short-plus long-read) genome analysis of clonal resistant/susceptible pairs. We show that HR is common (6.45% at UKE, 14.97% at FPG; pooled 12.1%, 34/280), that it is almost entirely confined to piperacillin/tazobactam (33 of 34 HR isolates; 21.9% of piperacillin/tazobactam-screened isolates versus ≤1.4% for piperacillin and none for meropenem), and that among 33 evaluable clonal pairs a genomic lesion is identifiable in 42·4%, dominated by unstable tandem amplification of β-lactamase-bearing blocks (11/33; 3·5–36×), with de-repressing mutations in efflux and AmpC regulators accounting for the remainder. Two further contributions are methodological. First, nearly half the pairs (45·5%) are negative on both genomic arms, placing non-genetic heteroresistance on the same footing as amplification rather than treating it as an exception. Second, resequencing the same isolates reproduced every amplification call but not its magnitude (+20% to −55%), showing that amplicon copy number is a snapshot of subclone frequency rather than a strain attribute — a constraint that any copy-number-based diagnostic must accommodate. Resistant subpopulations carried no evident fitness cost.
Implications of all the available evidence
AST-susceptible Gram-negative bloodstream isolates not infrequently harbour resistant subpopulations that current diagnostics cannot see but that can expand under first-line β-lactam therapy. Together with evidence that pre-existing HR can drive on-treatment resistance, our findings argue for the development of rapid, target-specific molecular assays (e.g. copy-number digital PCR) to detect HR in the diagnostic laboratory — read as quantitative, condition-dependent measurements rather than as fixed strain attributes — and for a prospective clinical study to determine whether breakpoint-crossing HR predicts therapeutic failure in Gram-negative bloodstream infection.
