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Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in Haemophilus influenzae and Haemophilus parainfluenzae during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by H. influenzae for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 H. influenzae and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. H. influenzae isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in H. influenzae P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent H. influenzae strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the

Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly sw

Tracking bacterial evolution during chronic infection provides insights into how host selection pressures shape bacterial genomes. The human-restricted opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) infects the lower airways of patients suffering chronic obstructive pulmonary disease (COPD) and contributes to disease progression. To identify bacterial genetic variation associated with bacterial adaptation to the COPD lung, we sequenced the genomes of 92 isolates collected from the sputum of 13 COPD patients over 1 to 9years. Individuals were colonized by distinct clonal types (CTs) over time, but the same CT was often reisolated at a later time or found in different patients. Although genomes from the same CT were nearly identical, intra-CT variation due to mutation and recombination occurred. Recurrent mutations in several genes were likely involved in COPD lung adaptation. Notably, nearly a third of CTs were polymorphic for null alleles of ompP1 (also called fadL), which encodes a bifunctional membrane protein that both binds the human carcinoembryonic antigen-related cell adhesion molecule 1 (hCEACAM1) receptor and imports long-chain fatty acids (LCFAs). Our computational studies provide plausible three-dimensional models for FadL's interaction with hCEACAM1 and LCFA binding. We show that recurrent fadL mutations are likely a case of antagonistic pleiotropy, since loss of FadL reduces NTHi's ability to infect epithelia but also increases its resistance to bactericidal LCFAs enriched within the COPD lung. Supporting this interpretation, truncated fadL alleles are common in publicly available NTHi genomes isolated from the lower airway tract but rare in others. These results shed light on molecular mechanisms of bacterial pathoadaptation and guide future research toward developing novel COPD therapeutics.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in patients with chronic obstructive pulmonary disease (COPD). To elucidat

Backgrounds The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is encountered in a significant proportion in the lower airways of Chronic Obstructive Pulmonary Disease (COPD) patients. The COPD lung is likely to be a unique niche favouring its adaptation to persistent infection. Objectives To unravel NTHi evolutionary dynamics in the lung of the COPD patient over time, and to identify microbial patho-adaptive features. Methods Ninety-two NTHi isolates from sputum samples of 13 COPD patients at exacerbation, recovered in a prospective longitudinal study (2000-2014), were genome sequenced: (a) Pacific Biosciences longread sequencing was used to generate complete assemblies for one representative of each clonal type; (b) Illumina short-read sequencing was applied to all isolates. Genome assembly used a customized pipeline and raw reads were also aligned to the PacBio reference genomes to generate variant tables for each set of clonal type isolates. Functional characterization of parallel evolutionrelated allelic variation was analyzed for selected genes. Conclusions Ninety-two NTHi COPD isolates have been (i) phylogenomically analyzed in the context of all publicly available NTHi genome sequences, (ii) organized in 33 clonal types, (iii) used to establish variation in NTHi genome architecture, (iv) used to identify traits of parallel evolution by fixing at least one single nucleotide variant in more than one clonal type. Detailed analysis of the fadL-ompP1 gene parallel evolution and allelic variation will be discussed, in terms of its role as an exogenous long-chain fatty acids transporter, as a ligand of the human receptor CEACAM-1 and as a source of antigenic variation.

The PLOS Pathogens Staff. Notice of Republication.This article was republished on May 13, 2016, to correct the title which was incorrectly published as “Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza”. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.

[Abstract] Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2Aby hmw1Aresulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1Awas confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1Amight be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1Abacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation., [Author Summary] Many bacteria are naturally competent, actively taking up DNA from their surroundings and incorporating it into their genomes by homologous recombination. This cellular process has had a large impact on the evolution of these species, for example by enabling pathogens to acquire virulence factors and antibiotic resistances from their relatives. But natural competence can also be exploited by researchers to identify the underlying genetic variation responsible for naturally varying phenotypic traits, similar to how eukaryotic geneticists use meiotic recombination during sexual reproduction to create genetically admixed populations. Here we exploited natural competence, phenotypic selection, and deep sequencing to rapidly identify the hmw1 locus as a major contributor to intracellular invasion of airway epithelial cells by the human pathogen Haemophilus influenzae, a trait that likely allows bacterial cells to evade the immune system and therapeutic interventions during chronic infections. Genetic variation in this locus can strongly modulate bacterial intracellular invasion rates, and possession of a certain allele favors adhesion and self-aggregation, which appear to prompt bacteria to invade airway cells as groups, rather than as individuals. Overall, our findings indicate that targeting HMW1 could block the ability of H. influenzae to invade airway cells, which would make antibiotic therapy to treat chronic lung infections more effective. Furthermore, our new approach to identifying the genetic basis of natural phenotypic variation is applicable to a wide-range of phenotypically selectable traits within the widely distributed naturally competent bacterial species, including pathogenesis traits in many human pathogens.