Current Antibiotic Susceptibility Test Underestimates Minority Resistance: Implications for High-Risk Infections

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments:

 

         The Commentary is dealing with antibiotic susceptibility testing (AST) of bacteria in the clinical samples. Antibiotic resistance is a growing problem for effective antimicrobial therapy. Consequently, the precise determination of antibiotic-resistant bacteria (ARB) in clinical samples is a great challenge. Authors point out that heteroresistance or polyclonal diversity of ARB in the sample may evade standard low-inoculum protocols. Thus, they propose that AST results should contain annotation saying that resistant sub-populations may not be detected.

 

Specific comments:

 

Section REFERENCES

 

1. Page 3, lines 86 & 87: Reference No.9 does not exists in the text – should be deleted.

 

2. Page 3, lines 92 & 93: Reference No.12 does not exists in the text – should be deleted.

 

3. Page 3, lines 94 & 95: Reference No.13 does not exists in the text – should be deleted.

Author Response

We sincerely thank the Reviewer   for the careful reading of our manuscript and the constructive comments. We appreciate the opportunity to clarify and improve the reference section. Please find our detailed responses below. for identifying these inconsistencies.

All three references are now properly cited within the main text, and therefore have been retained in the reference list. We appreciate the Reviewer’s attention to detail, which helped improve the clarity and accuracy of the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The commentary titled “Disclosing Susceptibility Heterogeneity in AST Reporting – An Ethical Imperative for Laboratory Practice” (labmed-3995966) was authored by Ivan Brukner  and Matthew Oughton for publication with the LabMed Journal. Authors identify a critical limitation in standard Antibiotic Susceptibility Testing (AST): the classification of bacterial isolates as "susceptible" even when undetected resistant subpopulations exist. They define this phenomenon as Susceptibility Heterogeneity (SH), noting that it arises from heteroresistance or polyclonal diversity. They highlight the significant prevalence of SH, pointing out that it occurs in 15–97% of clinical isolates for major pathogens such as E. coli, S. aureus, P. aeruginosa, and K. pneumoniae. They explain that standard low-inoculum protocols often fail to detect these subpopulations, creating a dangerous gap in diagnostics.

The authors warn that ignoring SH can lead to treatment failure, specifically emphasizing the danger to immunocompromised patients and those with bloodstream infections. They argue that the current binary reporting (Susceptible/Resistant) provides a false sense of security in these complex cases. Authors are proposing that Laboratories should append a standardized annotation to all "susceptible" results stating: "Limited susceptibility possible; resistant subpopulations may not be detected." For high-risk clinical scenarios, the authors recommend moving beyond standard protocols to enhanced testing methods, such as utilizing multiple colonies or higher inoculum concentrations to uncover hidden resistance.

This is a well-written commentary paper with a solid argument overall. I have some minor comments:

The authors employed a numbered referencing style that should follow a chronological order for in-text citations. In the second paragraph, specifically line 31, references were cited, but the assigned reference numbers were already allocated prior to their usage. References [1–3, 5, 6, 8, 10] should have been [1-7], as they are the first citations used in the paper.

Abstract: Line 18: Define the abbreviations CLSI and EUCAST

Line 50: The statement references "many laboratories," yet only two references are included. It would be beneficial to add more references to support this claim.

Author Response

Response to Reviewer 2

We sincerely thank Reviewer 2 for the thoughtful evaluation of our commentary and for the constructive suggestions that helped improve the clarity and structure of the manuscript. We appreciate the positive feedback regarding the importance and relevance of the topic. Below we address each comment in detail.

Comment 1:

“The authors employed a numbered referencing style that should follow a chronological order … references [1–3, 5, 6, 8, 10] should have been [1–7].”

Response:
We thank the Reviewer for noting the inconsistency in the citation order.
In the revised manuscript, we carefully reordered the in-text citations to ensure strict chronological progression according to the Vancouver citation style. The references appearing in the second paragraph now follow the correct sequential numbering, and the reference list has been updated accordingly.

 

Comment 2:

“Define the abbreviations CLSI and EUCAST.”

Response:
Thank you for highlighting this omission. In the revised abstract, the following definitions were added at first use:

• CLSI: Clinical and Laboratory Standards Institute
• EUCAST: European Committee on Antimicrobial Susceptibility Testing

This improves accessibility for readers unfamiliar with these bodies.

 

Comment 3:

“The statement references ‘many laboratories,’ yet only two references are included.”

Response:
We agree with this suggestion. Additional supporting references documenting widespread observations of susceptibility heterogeneity across multiple institutions have been added to strengthen the claim.

We are grateful for Reviewer 2’s constructive suggestions. All recommendations have been incorporated, resulting in a clearer and more consistent manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “Disclosing Susceptibility Heterogeneity in AST Reporting  - An Ethical Imperative for Laboratory Practice” have discussed an important issue in clinical microbiology: the ethical and diagnostic implications of susceptibility heterogeneity (SH) in AST reporting. The topic is timely, relevant, and of increasing importance. However, there are various suggestions that need to be considered.

The Prevalence range (15–97%) is extremely broad and may give an impression of imprecision or selective citation. This needs to be contextualized by organism type, method used, and definition of SH.

The methodological variability in SH detection should be discussed further. Some of the highest reported prevalences arise from specialized methods that differ significantly from routine AST.

The manuscript attempts to link SH reporting to ethical principles (non-maleficence, transparency, equity). The argument is not fully justified in clinical risk quantification or actual outcomes. How often does SH truly lead to treatment failure? Are there identifiable risk thresholds?

Moreover, “Equity” as an ethical principle is mentioned but not sufficiently explored. How does SH reporting specifically relate to laboratory resource inequities?

Proposing that every AST “S” result should carry a warning such as “Limited susceptibility possible” may have involuntary consequences. If clinicians receive too many warnings, they start ignoring them, even if some warnings are important. It could undermine trust in AST specificity and reduce compliance with stewardship guidelines? It may encourage unnecessary increase in using broader-spectrum antimicrobial therapy that can worsen AMR?

Some suggestions such as multiple-colony testing, higher inoculum, orthogonal methods are not feasible in many laboratories due to workload, turnaround time constraints, accreditation standards and cost and capacity limitations. The authors need to discuss these as well.

“Susceptibility heterogeneity” is not yet a widely standardized term. Please define it clearly and distinguish it from the hetero-resistance.

Author Response

Response to Reviewer 3

We sincerely thank Reviewer 3 for their thoughtful and highly valuable comments. The suggestions have markedly improved the clarity, balance, and impact of the manuscript. Below we address each point and provide the exact revised text now appearing in the manuscript.

 

Comment 1 — Prevalence range (15–97%) is too broad

Response:
We contextualized the wide range by linking variability to methodological differences.
Exact revised text (lines 54–58):
“Across pathogens such as E. coli, S. aureus, K. pneumoniae, and P. aeruginosa, SH prevalence ranges widely—reported between 15% and 97%—depending on species and detection methods [1,2,3,4,5,7]. Studies using high-inoculum or multi-colony AST, population analysis profiling, and zone inspection report significantly higher detection rates than routine single-colony protocols [1,8].”

 

Comment 2 — More detail needed on methodological variability

Response:
Addressed within the same revised sentences, clarifying differences between routine and high-sensitivity methods.

 

Comment 3 — Clarify clinical impact

Response:
We have substantially expanded the clinical-impact section to incorporate relevant evidence added to the revised manuscript. The updated text reflects the full breadth of clinically documented susceptibility heterogeneity in immunocompromised hosts, fungal infections, Mycobacterium tuberculosis mixed-strain infections, minority-frequency resistance in Gram-negative pathogens, oncology and transplant settings, cystic fibrosis, and pneumococcal genomic heterogeneity.

Exact revised text (updated):

“Susceptibility heterogeneity has demonstrated clinical consequences: heteroresistant subpopulations may expand under therapy, resulting in breakthrough growth, treatment failure, or evolution to full resistance, as shown in colistin-heteroresistant Acinetobacter baumannii and other pathogens [9]. In immunocompromised patients, susceptibility heterogeneity is not just a theoretical concern: vancomycin-heteroresistant coagulase-negative staphylococcal bloodstream infections in children with leukemia, as well as echinocandin-heteroresistant Candida parapsilosis fungemia in allogeneic hematopoietic-cell–transplant recipients, have both been directly associated with treatment or prophylaxis failure [10,11].

The clinical relevance of SH extends far beyond these examples. In Mycobacterium tuberculosis, mixed-strain (polyclonal) infections frequently produce discordant resistance profiles within the same patient sample; diagnostic sputum often contains multiple genetic variants at resistance-associated loci, while standard single-colony culture captures only one subpopulation, potentially yielding a falsely susceptible MIC [16–18].

In Gram-negative pathogens, Weiss and colleagues demonstrated that minority resistant subpopulations at frequencies as low as 10⁻⁶–10⁻⁸ can directly cause treatment failure despite a susceptible population-level MIC [7].

In oncology and transplant settings, polymicrobial and polyclonal infections commonly include coexisting clones with divergent resistance phenotypes, reducing the predictive value of a single MIC in high-risk hosts [19].

In cystic fibrosis and chronic Pseudomonas aeruginosa infection, ceftolozane/tazobactam heteroresistance enables rapid selection of resistant subclones under therapy even when baseline isolates test fully susceptible by routine AST [20].

Finally, genomic epidemiology studies of Streptococcus pneumoniae show that many specimens reported as containing ‘one isolate’ actually represent mixtures of closely related lineages with differing susceptibility and tolerance phenotypes, challenging the traditional assumption that a single MIC reflects the infectious population [21].”

 

Comment 4 — Equity principle underdeveloped

Response:
Expanded to include resource-disparity implications.
Exact revised text (lines 82–84):
“equity (avoiding information gaps between labs with differing resources)”

 

Comment 5 — Concern about “warning fatigue”

Response:
Clarified that this is an informational transparency statement analogous to widely accepted disclaimers.
Exact revised text:
“This is analogous to disclaimers already used for ESBL, inducible clindamycin resistance, and certain agent–organism combinations in EUCAST guidance and has been explicitly recommended for susceptibility heterogeneity [1,14,15].”

 

Comment 6 — Feasibility of enhanced testing

Response:
Recommendations are now stratified into universal and selective approaches.
Exact revised text (lines 100–102):
“To balance feasibility and clinical impact, we divide these recommendations into universal measures and selective ones for high-risk scenarios.”

 

Comment 7 — Clearer definition of SH

Response:
A concise definition has been added early in the manuscript.
Exact revised text (lines 46–49):
“Susceptibility heterogeneity (SH) refers to the presence of subpopulations within a bacterial sample that exhibit differing antibiotic susceptibility. It encompasses both clonal heteroresistance—resistant subpopulations within a genetically identical group—and polyclonal diversity, where distinct strains coexist in a specimen [2,3].”

 

Additional strengthening of the manuscript

We repositioned the new Heyman et al. Lancet Microbe 2025 study as Reference 1, as it independently supports our conclusions and recommends an almost identical cautionary comment for all susceptible results.

We emphasize that although disclaimers exist for specific drug–bug combinations, no international guideline currently mandates systematic transparency for susceptibility heterogeneity across major pathogens. Our commentary remains timely and calls for consistent and equitable global reporting practices.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I appreciate the revisions and the effort invested in strengthening the manuscript.  I am satisfied with the current version and recommend the manuscript for acceptance...