Revisiting Mutational Resistance to Ampicillin and Cefotaxime in Haemophilus influenzae

Infectious Diseases Biochemistry Medicine Genetics Life Sciences Microbiology
Haemophilus influenzae ampicillin resistance cefotaxime resistance BLNAR MIC genome-wide association study phylogenomics

Revisiting Mutational Resistance to Ampicillin and Cefotaxime in Haemophilus influenzae

Background and Motivation for the Study

Haemophilus influenzae is an opportunistic bacterial pathogen that can cause severe respiratory tract infections and invasive infections such as septicemia and meningitis, particularly in infants, the elderly, and immunocompromised individuals. In recent years, with the emergence of β-lactamase-negative ampicillin-resistant (BLNAR) strains and unclear correlations between genotypic and phenotypic resistance, there have been significant challenges in clinical empirical treatments and patient management.

β-lactam antibiotics such as ampicillin used to be the preferred treatment for H. influenzae infections. However, due to the increasing prevalence of ampicillin-resistant strains, treatment protocols in many countries have shifted toward incorporating β-lactamase inhibitors or third-generation cephalosporins. The resistance mechanisms of BLNAR strains primarily involve alterations to the drug target, particularly penicillin-binding protein 3 (PBP3) encoded by the ftsI gene, as well as possible other genetic changes. However, discrepancies between genotypic and phenotypic resistance complicate classification and predictive diagnostics. Additionally, identifying novel mutations and understanding the evolution of these resistance mutations in the population are critical areas of investigation.

This study aims to reevaluate the molecular mechanisms of resistance in H. influenzae using systematic review, population genomics, and genome-wide association studies (GWAS) to explore novel determinants of resistance and mutational patterns, providing further guidance for clinical management and research.

Source and Context of the Study

The study, titled “Revisiting mutational resistance to ampicillin and cefotaxime in Haemophilus influenzae”, was conducted by Margo Diricks and co-researchers from academic institutions across Germany, Portugal, and Europe. It was published in the open-access journal Genome Medicine (Volume 16, 2024) and provides comprehensive data and computational tools for further analysis.

Research Design and Workflow

The study involved three main components:

  1. Systematic Review and Meta-Analysis
    A dataset of 291 β-lactamase-negative H. influenzae isolates was compiled to assess associations between PBP3 mutation groups (classified into I, II, III, and related subgroups) and resistance phenotypes for ampicillin and cefotaxime.

  2. Global Population Genomics
    A total of 555 H. influenzae genome sequences from the PubMLST database, representing isolates from 21 countries, were analyzed to explore evolutionary trajectories and resistome-associated mutations in the ftsI gene.

  3. Microbial GWAS Analysis
    A novel clinical dataset (298 isolates) was analyzed to validate candidate resistance genes and identify additional mutations linked to ampicillin resistance using MIC (Minimum Inhibitory Concentration) data and resistance status.

The MIC data were determined using gradient diffusion strips (such as Etest) or broth microdilution methods and interpreted according to standards set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or the Clinical and Laboratory Standards Institute (CLSI).

Major Findings and Insights

  1. Associations Between PBP3 Groups and Resistance Phenotypes

    • For ampicillin resistance, PBP3 Group II isolates were significantly associated with resistance; however, their classification showed low specificity (<16%) in predicting phenotypic resistance, as some isolates remained phenotypically susceptible.
    • PBP3 Group III-related mutations conferred higher resistance levels to cefotaxime, varying with cumulative substitutions.

  2. Evolutionary Trajectories in Global Genomics
    Analysis of PBP3-associated mutations across unrelated lineages (e.g., different sequence types) revealed patterns of independent emergence (convergent evolution). Furthermore, several mutations under positive selection, such as v547i and n569s, were identified as associated with resistance progression.

  3. Findings from GWAS

    • The GWAS identified the ftsI gene as the locus most significantly associated with ampicillin resistance. Variants such as m377i, a502v, and n526k were the highest effect contributors to resistance status and MIC values.
    • Other candidate resistance genes (e.g., rd_05960, rida, oppA, and ompP2) and transport-related pathways were identified but require further investigation for functional validation.

  4. Haplotype and Resistance Network Construction
    New haplotypes derived from ftsI mutations were discovered. The most common haplotype (H1) included m377i, a502v, n526k, and was frequently associated with ampicillin-resistant isolates.

Study Significance and Key Implications

This study provides new insights and addresses the complexity of BLNAR resistance mechanisms in H. influenzae. It has significant implications for both academic and clinical domains:

  1. Clinical Implications
    Current phenotypic assays (e.g., gradient diffusion or broth microdilution) may fail to consistently classify isolates with MICs near clinical breakpoints. The authors recommend introducing an area of technical uncertainty (ATU) to flag cases where genotypic resistance determinants (e.g., PBP3 mutations) are present alongside phenotypic ambiguity.

  2. Public Health Recommendations
    The findings provide a comprehensive database of resistance-associated mutations in H. influenzae, which could inform global surveillance and infection control strategies.

  3. Diagnostics and Predictive Modeling
    The integration of genotypic data and phenotypic MIC thresholds, possibly through machine learning, could improve diagnostic accuracy and resistance predictions.

Key Highlights and Future Directions

  • Core PBP3 substitutions (m377i, v547i, and n526k) play critical roles in driving MIC elevations for β-lactam resistance.
  • The GWAS approach effectively provided MIC-specific associations but underscores the need for larger datasets, geographic diversity, and data uniformity.
  • Emerging cephalosporin resistance mutations call for expanded monitoring of H. influenzae resistant clones and their potential to cause treatment failures.

Conclusion

The study advances our understanding of BLNAR resistance dynamics in H. influenzae, providing robust analyses of phenotypic-genotypic correlates. Despite significant associations between PBP3 patterns and resistance, limitations in phenotypic tests call for combined genotypic and phenotypic assessment tools. Future large-scale genomic studies combining MIC data and clinical outcomes will further refine our ability to predict resistance mutations and improve clinical decision-making.

The comprehensive research framework outlined in this paper serves as a valuable resource for microbiologists, clinicians, and public health authorities, contributing to the long-term goal of combating antimicrobial resistance.