Antibiotic Heteroresistance: What Is It and How Does It Impact Patients?

A bacterial isolate in a pure culture is conventionally thought to comprise a population with shared behaviors and traits. In the context of antibiotic resistance, a population with homogenously low minimum inhibitory concentration (MIC) is designated susceptible by conventional antimicrobial susceptibility testing (AST). Upon acquisition of horizontally acquired antibiotic resistance genes or mutations that confer resistance, the population will homogenously demonstrate high MIC and be designated resistant by AST.

However, it is increasingly appreciated that bacterial populations often harbor subpopulations with distinct traits, termed phenotypic heterogeneity. Although the conventional AST designations of susceptible or resistant accurately describe many clinical isolates, strains of numerous bacterial pathogens exhibit a form of heterogeneity called heteroresistance to many classes of antibiotics.¹ Heteroresistance is a form of phenotypic heterogeneity in which a minority subpopulation of cells has a higher MIC than the majority susceptible population. The resistant subpopulation can be present at a very low frequency (as low as 1 resistant cell per million), making it challenging to detect by AST.

Thus, most heteroresistant isolates are inaccurately designated susceptible. In one large study of carbapenem-resistant Enterobacterales, for example, heteroresistance was a more frequent feature than conventional susceptibility or resistance for many antibiotics,² suggesting that clinical AST likely encounters heteroresistant isolates frequently, and often classifies those isolates as susceptible. A unique feature of heteroresistance is the transient and unstable nature of the resistant subpopulation. When exposed to the antibiotic, the resistant subpopulation is capable of growth and will take over the whole population as the susceptible cells are killed or growth arrested. This enrichment perseveres while the antibiotic is present, but when the selective pressure of the antibiotic is removed, the resistant cells will rapidly return to their baseline frequency. Thus, in patients, the resistant subpopulation may survive and grow in the face of treatment.

MECHANISMS OF HETERORESISTANCE
The features of this unstable heteroresistance are not the product of permanent genetic changes in the resistant cells, but rather more complex and plastic features of the subpopulation. The mechanisms that generate heteroresistance remain an active area of investigation: How do cells in a population derived from a single cell gain levels of resistance much greater than their sister cells? One mechanism is increased copy number of an antibiotic resistance gene in the resistant subpopulation, which results in higher MIC compared with the majority susceptible cells encoding only a single copy of the gene.³

This phenomenon is often generated by a process called gene duplication and amplification and appears especially common for heteroresistance to β-lactams in gram-negative isolates. Gene amplification is a dynamic process that can generate high copy number of a gene as well as reversion to a single copy, allowing for a range of gene copy number in a population capable of changing rapidly. The unstable nature of amplification makes detection of the subpopulation challenging.⁴

Gene amplification is particularly intriguing because as new generations of β-lactams and β-lactamase inhibitors are introduced, the preexisting armamentarium of β-lactamase genes in clinical isolates may include those capable of undergoing amplification. Therefore, clinical isolates never exposed to a novel drug may already contain subpopulations with a β-lactamase in sufficiently high copy number to generate heteroresistance to a novel antibiotic. Other mechanisms beyond gene amplification can result in resistant subpopulations. For example, population heterogeneity in outer membrane modification results in heteroresistance to colistin in Enterobacter spp; cells with more lipopolysaccharide modification have higher colistin MIC.^5-7 For all types of heteroresistance toward various antibiotic classes, the principal source of the population heterogeneity, the advantages conferred by exhibiting heteroresistance, and the consequences of heteroresistance on treatment outcome remain under investigation.

HETERORESISTANCE IN THE CLINIC
Critical to understanding heteroresistance in the context of clinical care is uncovering whether this common form of phenotypic resistance affects treatment outcome. Because heteroresistance is common but challenging to detect, many isolates will be treated with an antibiotic to which a resistant subpopulation exists. If the resistant cells are frequent enough to be present in meaningful numbers at the site of infection, and survive any immune-mediated killing, then the subpopulation is expected to grow in the presence of antibiotic and cause treatment failure. This has been observed in murine models of infection with Klebsiella and Enterobacter isolates.^2,7,8 In patients, instances of heteroresistant isolates designated susceptible by AST and causing treatment failure are not as well documented, but a body of evidence is growing. The recently approved cephalosporin cefiderocol serves as an example. Cefiderocol heteroresistance was described quickly upon clinical introduction of the drug,⁹ and appears often to be the result of β-lactamase gene amplification.^10,11 Most carbapenem-resistant Acinetobacter and Klebsiella are designated susceptible, but rates of heteroresistance for these isolates are high.⁹ In fact, correlations between the rate of cefiderocol heteroresistance in these species and the treatment outcome in clinical trials have been reported recently, suggesting heteroresistance may have been one of the factors contributing to poor outcomes.^9,12,13 In one case report, undetected cefiderocol heteroresistance may have contributed to treatment failure in a patient infected with Klebsiella,^14,15 and the emergence of conventional resistance following treatment of a heteroresistant Pseudomonas isolate with cefiderocol has been reported.¹⁶ Thus, heteroresistance to cefiderocol may be one contributing factor to treatment failure, and may lead to cefiderocol resistance in the long term, as heteroresistance has been proposed as an evolutionary stepping stone for β-lactams.¹⁷ Additional investigations of cefiderocol heteroresistance in the clinic should help clarify the role of heteroresistance in treatment failure. Heteroresistance to an antibiotic is unlikely to always be predictive of treatment failure. Reports for different species and antibiotics have found that heteroresistance negatively affected outcome,¹⁸ did not affect outcome,¹⁹ or the effect varied based on infection site²⁰ (additional reports have been summarized elsewhere1). For certain antibiotics, the frequency of heteroresistant isolates is greater than the frequency of treatment failure, suggesting heteroresistance is not always a cause of failure. Additionally, the complexity of clinical care, the frequent use of combination therapy for difficult-to-treat infections, and the variety of factors that contribute to treatment failure together hinder the ability to easily analyze heteroresistance-related outcomes.

OUTLOOK
In the face of the antimicrobial resistance crisis, infectious disease physicians, clinical microbiologists, and basic research scientists need to coalesce around improving our understanding of mechanisms of resistance, including heteroresistance. Additionally, many of the features of bacterial heteroresistance appear to be true for fungal pathogens as well; and as antifungal resistance climbs, so should investigation into antifungal heteroresistance. Heteroresistance may be an important factor to account for to improve treatment outcome, susceptibility testing, dosing, and pharmacokinetics, as well as novel antimicrobial development.

References

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