Rapid Genomic Change and Intraclonal Variation in the Bacterial Opportunistic Pathogens Haemophilus influenzae and Acinetobacter baumannii
Authors:   , Raible Kevin Michael , Ehrlich Garth D , Mell Joshua Chang 
Published: October 2023

The immense genetic diversity observed within many human-associated bacterial species is an important consideration when studying pathogenesis. The studies described here explore the phenotypic impact of genetic diversity across natural populations of nontypeable Haemophilus influenzae (NTHi), as well as clinical samples of Acinetobacter baumannii. NTHi is a prevalent human-restricted bacterium that colonizes the upper respiratory tract. Although usually NTHi behave as commensals, they also frequently cause acute and chronic infections, particularly middle ear infections (otitis media, OM) in children and in chronic lung diseases. Many host-associated bacteria exhibit highly variable serum resistance (SR), or the ability to resist host complement-mediated killing, and variation in this virulence factor has been associated with increased morbidity in NTHi infections. Here we identify potential causal genetic mechanisms behind SR in diverse NTHi strains using comparative and functional genomic approaches on a well-curated collection of clinical NTHi isolates from children with or without OM. In vitro quantitative SR assays revealed considerable complexity in the phenotype among the strains examined from our collection, and comparative genomics was used to determine that an isolate's phylogenetic lineage was a strong predictor of its SR level. SR differences among closely related isolates were largely associated with recombination tracts carrying extensive genetic variation. This analysis and a pan-genome-wide association study identified known SR-related genes as just a subset of the genes needed to explain differences in SR. These included the lipooligosaccharide (LOS) modification gene lex2A. Finally, we performed in vitro laboratory evolution experiments on diverse serum sensitive NTHi isolates. All sensitive isolates rapidly became significantly and substantially more SR upon serial passaging in serum. Genomic analysis of the evolved isolates identified novel mutations associated with SR that directly or indirectly affected outer membrane LOS structure, sometimes revealing "cryptic" resistance mechanisms. Furthermore, while we observed some recurrent mutations among different isolates, most evolved clones showed unique variant sequences associated with SR. Collectively, these results help dissect the natural history and genomic architecture of SR in the NTHi in the context of pediatric ear infections, revealing both lineage-specific effects and the rapid emergence of resistance in sensitive strains under selection. Additionally, this study sheds light on the complex task of mapping genomic changes with measured phenotypes, where these comparative methods are sensitive to artifacts with gene annotation and variant calling. In the final study the model organism is A. baumannii which is a nosocomial pathogen that has emerged as a significant cause of morbidity and mortality within the healthcare facilities. The success of this pathogen is largely due to its ability to rapidly gain resistance to antimicrobial therapies and its capability to persist in abiotic environments through biofilm production. In this study, we explore both genotypic and phenotypic properties of 26 carbapenem-resistant A. baumannii (CRAB) isolates in our tertiary-care hospital (16 isolates were collected between January 2010 and March 2011, and 10 were collected between February and May 2015). We determined that all 26 CRAB isolates possessed multiple [beta]-lactamase genes, including genes from Groups A, C, and D. Specifically, 42% of the isolates possess the potentially plasmid-borne genes of OXA-23-like or OXA-40-like [beta]-lactamase. The presence of mobile gene element integron cassettes and/or integrases in 88% of the isolates suggests a possible mechanism of dissemination of antibiotic resistance genes. Additionally, the presence of the insertion sequence (IS) ISAba1 upstream of the innate [beta]-lactamase genes oxa-51-like, adc-7, and ampC can result in overexpression of these clinically relevant enzymes. Multilocus sequence typing revealed that all 26 CRAB isolates were either sequence type (ST)-229 or ST-2. Interestingly, ST-2 went from being the minority CRAB ST in the 2010-2011 isolates to being the predominant ST in the 2015 isolates (from 32% to 90%). We then went on to show ST-2 isolates have an significantly enhanced ability to produce biofilms in comparison to ST-229 isolates, potentially leading to more successful colonization of the clinical environment over time. These studies demonstrate both the complexity and importance genomic variation plays in diseases.