Poster: 76Persistent positive cultures for Mycobacterium abscessus in cystic fibrosis does not reflect genomic divergence or enhanced virulence

Abstract

Background

Mycobacterium abscessus (MABS) is a highly drug-resistant pathogen in cystic fibrosis (CF), associated with chronic infection and accelerated lung function decline. A subset of people with CF (pwCF) show persistent positive cultures despite prolonged multidrug therapy, raising the concept of a “persister” phenotype. Yet, whether persistence reflects true bacterial evolution, enhanced virulence, or phenotypic adaptation is poorly defined. We investigated the biological basis of MABS persistence in CF by comparing longitudinal isolates across pre-treatment and persistent (post-treatment) states, integrating genomic, phenotypic, and host-pathogen interaction analyses.

Mycobacterium abscessus (MABS) is a highly drug-resistant pathogen in cystic fibrosis (CF), associated with chronic infection and accelerated lung function decline. A subset of people with CF (pwCF) show persistent positive cultures despite prolonged multidrug therapy, raising the concept of a “persister” phenotype. Yet, whether persistence reflects true bacterial evolution, enhanced virulence, or phenotypic adaptation is poorly defined. We investigated the biological basis of MABS persistence in CF by comparing longitudinal isolates across pre-treatment and persistent (post-treatment) states, integrating genomic, phenotypic, and host-pathogen interaction analyses.

Methods

56 longitudinal CF isolates were categorized as indolent, pre-treatment, or persistent (post-treatment). Whole genome phylogenetic analysis assessed genetic divergence. Phenotypic profiling included morphological classification, growth kinetics, and biofilm formation with antibiotic susceptibility testing. Host-pathogen interactions were evaluated using primary alveolar macrophages and monocyte-derived macrophages from CF & non-CF donors, measuring uptake, intracellular survival, replication, and TNFα. DualRNAseq was performed on infected macrophages from healthy donors at 24hrs post-infection to assess host and bacterial transcriptional responses.

56 longitudinal CF isolates were categorized as indolent, pre-treatment, or persistent (post-treatment). Whole genome phylogenetic analysis assessed genetic divergence. Phenotypic profiling included morphological classification, growth kinetics, and biofilm formation with antibiotic susceptibility testing. Host-pathogen interactions were evaluated using primary alveolar macrophages and monocyte-derived macrophages from CF & non-CF donors, measuring uptake, intracellular survival, replication, and TNFα. DualRNAseq was performed on infected macrophages from healthy donors at 24hrs post-infection to assess host and bacterial transcriptional responses.

Results

Phylogenetic analysis revealed high genomic similarity within longitudinal isolates, with no evidence of genetic divergence in pre-treatment vs. persistent strains. Rough morphotypes were most common but did not correlate closely with treatment status. Growth kinetics were comparable across isolates but rough exhibited significantly reduced growth compared to smooth independent of clinical category. Biofilm and antibiotic tolerance were observed across isolates without distinguishing persistent strains. In alveolar macrophages, no significant differences were observed in phagocytic uptake, intracellular survival, replication, or TNFα between clinical isolate groups. Morphotype, not treatment status, influenced intracellular replication dynamics, with rough exhibiting reduced late intracellular growth. DualRNAseq revealed time-dependent transcriptional changes in both macrophages and MABS across longitudinally collected samples. Differential gene expression profiles were noted between indolent and symptomatic infections. Modest macrophage transcriptional alterations were identified linked with pre- and post-treatment states following antibiotic or phage therapy.

Phylogenetic analysis revealed high genomic similarity within longitudinal isolates, with no evidence of genetic divergence in pre-treatment vs. persistent strains. Rough morphotypes were most common but did not correlate closely with treatment status. Growth kinetics were comparable across isolates but rough exhibited significantly reduced growth compared to smooth independent of clinical category. Biofilm and antibiotic tolerance were observed across isolates without distinguishing persistent strains. In alveolar macrophages, no significant differences were observed in phagocytic uptake, intracellular survival, replication, or TNFα between clinical isolate groups. Morphotype, not treatment status, influenced intracellular replication dynamics, with rough exhibiting reduced late intracellular growth. DualRNAseq revealed time-dependent transcriptional changes in both macrophages and MABS across longitudinally collected samples. Differential gene expression profiles were noted between indolent and symptomatic infections. Modest macrophage transcriptional alterations were identified linked with pre- and post-treatment states following antibiotic or phage therapy.

Conclusions

Persistent MABS culture positivity was not associated with genomic divergence or increased virulence under the conditions studied. Rather, persistence reflected phenotypic heterogeneity, mainly morphotype focused. These findings challenge the concept of a discrete “persister genotype” and suggest host-pathogen interactions and bacterial phenotypic plasticity, rather than genetic evolution, underlie chronic infection. This has key implications for interpreting culture persistence and optimizing treatment strategies in CF-associated MABS infection.

Persistent MABS culture positivity was not associated with genomic divergence or increased virulence under the conditions studied. Rather, persistence reflected phenotypic heterogeneity, mainly morphotype focused. These findings challenge the concept of a discrete “persister genotype” and suggest host-pathogen interactions and bacterial phenotypic plasticity, rather than genetic evolution, underlie chronic infection. This has key implications for interpreting culture persistence and optimizing treatment strategies in CF-associated MABS infection.