BUSCADOR RECOLECTA

La tesis doctoral aborda tres aspectos de la interacción entre el patógeno oportunista colonizador Haemophilus influenzae no tipificable (HiNT) y el sistema respiratorio humano, considerando la regulación patoadaptativa por variación de fase (Capítulo 1), la importancia del mantenimiento de la integridad superficial bacteriana (Capítulo 2), y el potencial terapéutico de moléculas xenohorméticas (Capítulo 3). En conjunto, este trabajo amplía nuestro conocimiento sobre los mecanismos moleculares de patoadaptación respiratoria de HiNT, proporciona evidencias sobre el papel de VacJ/MlaA en la modulación de la supervivencia bacteriana en las vías respiratorias, y señala el potencial terapéutico de moléculas xenohorméticas., This PhD Thesis work tackled three aspects of the interaction between the colonizing opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) and the human airways, by considering the concepts of phase-variable regulation of pathoadaptive traits (Chapter 1), the importance of molecular systems involved in maintaining the bacterial surface integrity (Chapter 2), and the therapeutic potential of xenohormetic molecules (Chapter 3).
Altogether, this work contributes expanding our understanding on molecular mechanisms of NTHi pathoadaptation regulated by phase variation, provides evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules, and highlights the therapeutic potential of xenohormetic molecules against NTHi infection., Este trabajo de Tesis Doctoral se ha desarrollado mediante el disfrute de contratos adscritos a los proyectos Departamento de Innovación, Empresa y Empleo IIQ14064.RI1 (Gobierno de Navarra); SAF2012-311666 (Ministerio deEconomía y Competitividad), SAF2015-66520-R (Ministerio de Economía y Competitividad), Departamento de Salud 3/2016 (Gobierno de Navarra) y RTI2018-096369-B-I00 (Ministerio de Ciencia, Innovación y Universidades)., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Antibiotic resistance is a major Public Health challenge worldwide. Mechanisms other than resistance are described as contributors to therapeutic failure. These include heteroresistance and tolerance, which escape the standardized procedures used for antibiotic treatment decision-making as they do not involve changes in minimal inhibitory concentration (MIC). Haemophilus influenzae causes chronic respiratory infection and is associated with exacerbations suffered by chronic obstructive pulmonary disease (COPD) patients. Although resistance to imipenem is rare in this bacterial species, heteroresistance has been reported, and antibiotic tolerance cannot be excluded. Moreover, development of antibiotic heteroresistance or tolerance during within-host H. influenzae pathoadaptive evolution is currently unknown. In this study, we assessed imipenem resistance, heteroresistance and tolerance in a previously sequenced longitudinal collection of H. influenzae COPD respiratory isolates. The use of Etest, disc diffusion, population analysis profiling, tolerance disc (TD)-test methods, and susceptibility breakpoint criteria when available, showed a significant proportion of imipenem heteroresistance with differences in terms of degree among strains, absence of imipenem tolerance, and no specific trends among serial and clonally related strains could be established. Analysis of allelic variation in the ftsI, acrA, acrB, and acrR genes rendered a panel of polymorphisms only found in heteroresistant strains, but gene expression and genome-wide analyses did not show clear genetic traits linked to heteroresistance. In summary, a significant proportion of imipenem heteroresistance was observed among H. influenzae strains isolated from COPD respiratory samples over time. These data should be useful for making more accurate clinical recommendations to COPD patients., CG-C is funded by a PhD studentship from AEI, PRE2019-088382. SM is supported by Miguel Servet contract (CP19/00096) (ISCIII). This work has been funded by grants from MICIU RTI2018-096369-B-I00 and PID2021-125947OB-I00, 875/2019 from SEPAR, PC150 and PC136 from Gobierno de Navarra, to JG; by grant from Fondo de Investigaciones Sanitarias PI22/00257, to SM. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

El tutor de la tesis es Jon Veramendi Charola, Este trabajo de Tesis Doctoral aborda tres aspectos clave en el estudio de la infección respiratoria por el patógeno oportunista Haemophilus influenzae, centrados en mecanismos alternativos a la resistencia antibiótica asociados al fallo terapeútico (Capítulo 1), la regulación epigenética de la expresión génica (Capítulo 2), y el desarrollo de herramientas de ingeniería genética innovadoras para estudios funcionales de genes bacterianos a escala genómica (Capítulo 3). H. influenzae está incluido en la Lista de Patógenos Prioritarios de la Organización Mundial de la Salud para los que el desarrollo de nuevos antimicrobianos se considera una prioridad sanitaria, en este caso debido a su resistencia creciente a ampicilina. Además de la resistencia, la heterorresistencia, tolerancia y persistencia antibiótica contribuyen al fallo terapéutico, y no son detectados mediante procedimientos estandarizados en la práctica clínica. El imipenem es un antibiótico carbapenémico útil en el tratamiento inicial-empírico de infecciones graves debido a su baja toxicidad y baja resistencia. La resistencia de H. influenzae a imipenen es poco frecuente, si bien la evidencia disponible sugiere que los niveles de heterorresistencia están infraestimados. En el Capítulo 1 de este Trabajo, evaluamos la resistencia, heterorresistencia y tolerancia a imipenem en una colección de cepas clínicas de H. influenzae aisladas de muestras respiratorias de pacientes que sufren Enfermedad Pulmonar Obstructiva Crónica (EPOC), y cuyos genomas habían sido previamente secuenciados. Mediante ensayos de tipo difusión en disco, Etest, TD-test y population analysis profiling identificamos un bajo nivel de resistencia, ausencia de tolerancia, y una proporción significativa de heterorresistencia a imipenem. Si bien buscamos rasgos genómicos responsables de la heterorresistencia observada mediante: i) análisis de la variación alélica de los genes ftsI, acrA, acrB y acrR, previamente asociados con heterorresistencia; ii) análisis de variación alélica a nivel genómico entre cepas pertenecientes al mismo tipo clonal pero fenotípicamente heterogéneas; y iii) análisis comparativo de distribución génica a nivel genómico en cepas susceptibles y heterorresistentes, no detectamos asociaciones significativas entre rasgos genéticos concretos y los fenotipos observados. En conjunto, mostramos que la heterorresistencia es un fenómeno con alta prevalencia, complejo y multifactorial, y destacamos la necesidad de implementar protocolos fáciles y rápidos para su identificación en la práctica clínica.
Por otra parte, H. influenzae es una bacteria patobionte bien adaptada al ser humano, que provoca infecciones respiratorias en pacientes inmunocomprometidos que sufren, entre otras, EPOC. Los elementos reguladores que dictan la supervivencia y adaptación de H. influenzae en el pulmón de pacientes que sufren EPOC son poco conocidos. En γ-proteobacterias, el estado de metilación de motivos GATC localizados en regiones reguladoras modula la unión de la ARN polimerasa y de factores de transcripción, afectando la transcripción. En los genomas bacterianos, la mayoría de los motivos GATC se encuentran metilados por la metiltransferasa Dam, si bien existen sitios GATC que pueden permanecer no metilados o hemi-metilados si la actividad de Dam se encuentra bloqueada por la unión de proteínas al ADN. La combinación de motivos GATC metilados y hemi- o no metilados en regiones reguladoras o promotoras puede estar asociada a eventos de regulación epigenética de la expresión génica. En el Capítulo 2 de este Trabajo, analizamos la supervivencia de H. influenzae en un modelo pulmonar murino mediante mutagénesis por transposición a escala genómica acoplada a secuenciación profunda (Tn-seq), identificando la contribución de la metiltransferasa Dam en la supervivencia pulmonar de esta bacteria. El estudio del patrón de metilación por Dam a escala genómica mediante secuenciación en tiempo real de moléculas individuales (SMRT-sequencing), mostró motivos GATC no metilados o hemi-metilados en regiones reguladoras, lo que nos llevó a identificar el primer caso de variación fenotípica controlada por metilación Dam en una población de células isogénicas de H. influenzae. Además, la inactivación de dam y el estudio de su efecto a nivel transcriptómico, mediante secuenciación de ARN (RNA-seq) y análisis de expresión génica diferencial, reveló una nueva red regulatoria donde la metilación por Dam y los reguladores transcripcionales FNR y Fur regulan de manera coordinada la expresión de un panel de genes implicados en la defensa anaerobia de H. influenzae frente a especies reactivas de nitrógeno. Estos resultados tienen valor pato-adaptativo en nichos con estrés nitrosativo y baja oxigenación como son las vías respiratorias bajas de pacientes que sufren EPOC. Por último, H. influenzae fue el primer organismo de vida libre cuyo genoma completo fue secuenciado, siendo pionero en el desarrollo y empleo de técnologías -ómicas. Los métodos de secuenciación de inserción de transposones han sido útiles para realizar estudios de función génica e identificar genes esenciales en H. influenzae, si bien en su mayoría los genes que son considerados esenciales tienen funciones desconocidas dada la imposibilidad de generar mutantes. La tecnología de interferencia génica mediante CRISPR (clustered, regularly interspaced, short palindromic repeat) acoplada a secuenciación profunda (CRISPRi-seq), solventa esta limitación permitiendo el escrutinio y análisis de genes esenciales a escala genómica mediante silenciamiento génico. En el Capítulo 3 de este Trabajo, desarrollamos una plataforma CRISPRi-seq inducible por anhidrotetraciclina para silenciamiento génico programable en H. influenzae a escala genómica, y validamos su utilidad y potencial, abriendo nuevas vías para la comprensión funcional del genoma de esta bacteria. En conjunto, el Trabajo realizado durante esta Tesis Doctoral supone un avance significativo en el conocimiento de las bases moleculares de la infección por H. influenzae y presenta herramientas de ingeniería genética aplicables a la búsqueda de dianas terapeúticas para el desarrollo de estrategias que mejoren el manejo clínico de infecciones asociadas a este patógeno., This PhD Thesis addresses three key aspects on the study of respiratory infections caused by the opportunistic pathogen Haemophilus influenzae, by focusing on mechanisms different to antibiotic resistance but associated to therapeutic failure (Chapter 1), on the epigenetic regulation of gene expression (Chapter 2), and on the development of innovative genetic engineering tools for genome-wide gene functional studies in H. influenzae (Chapter 3). H. influenzae, due to its increasing resistance to ampicillin, is included in the World Health Organization’s List of Priority Pathogens for which the development of new antimicrobials is a health priority worldwide. In addition to resistance, there are other mechanisms, such as antibiotic heteroresistance, tolerance or persistence, which are underdetected through standardized procedures in the clinical practice but contribute to therapeutic failure. Carbapenem antibiotics, such as imipenem, are useful for initial empirical treatment of severe infections due to their low toxicity and low resistance rates. H. influenzae resistance to imipenem is rare, although available evidence suggests that heteroresistance levels are underestimated. In Chapter 1, we evaluated the resistance, heteroresistance, and tolerance to imipenem in a collection of clinical strains of H. influenzae isolated from respiratory samples of patients suffering Chronic Obstructive Pulmonary Disease (COPD), and whose genomes had been previously sequenced. Through disc diffusion, Etest, TD-test and population analysis profiling assays, we identified a low level of resistance, absence of tolerance, and a significant proportion of imipenem heteroresistance. Although we sought genomic traits responsible for the observed heteroresistance through: i) analysis of allelic variation in the ftsI, acrA, acrB, and acrR genes, previously associated with heteroresistance; ii) analysis of allelic variation at the genomic level between strains belonging to the same clonal type but phenotypically heterogeneous; and iii) comparative analysis of gene distribution at the genomic level in susceptible and heteroresistant strains, we did not detect significant links between specific genetic traits and the observed heteroresistance. Overall, we show that heteroresistance is a highly prevalent, complex, and multifactorial phenomenon, highlighting the need to implement easy and rapid protocols for its identification in clinical practice.
Furthermore, H. influenzae is a well-adapted bacterial pathobiont in humans, causing respiratory infections in immunocompromised patients, including those suffering COPD. The regulatory elements that dictate the survival and adaptation of H. influenzae in the lungs of COPD patients are poorly understood. In γ-proteobacteria, the methylation status of GATC motifs located in regulatory regions modulates RNA polymerase and transcription factor binding, therefore affecting transcription. In bacterial genomes, most GATC motifs are methylated by the Dam methyltransferase, although some GATC sites can remain unmethylated or hemi-methylated if Dam activity is blocked by proteins binding to the DNA. The combination of methylated and hemi- or non-methylated GATC motifs in regulatory or promoter regions may be indicative of epigenetic regulation of gene expression. In Chapter 2, we analyzed the survival of a panel of H. influenzae mutants in a murine model of lung infection through transposon mutagenesis coupled with deep sequencing (Tn-seq), identifying the contribution of the Dam methyltransferase to H. influenzae lung survival. The study of the genome-wide Dam methylation pattern through single-molecule real-time (SMRT) sequencing revealed unmethylated or hemi-methylated GATC motifs in regulatory regions, leading us to identify the first case of phenotypic variation controlled by Dam methylation in an isogenic population of H. influenzae cells. Moreover, inactivation of the dam gene and the study of its effect at the transcriptomic level through RNA sequencing (RNA-seq) and differential gene expression analysis, revealed a new regulatory network where Dam methylation and the transcriptional regulators FNR and Fur coordinately regulate the expression of a panel of genes involved in H. influenzae anaerobic defense against reactive nitrogen species. These results have patho-adaptive value in niches with nitrosative stress and low oxygenation, such as the lower airways of patients suffering COPD. Lastly, H. influenzae was the first free-living organism to have its complete genome sequenced, pioneering the development and use of -omic technologies. Transposon insertion sequencing methods have been useful in conducting gene function studies and identifying essential genes in H. influenzae, although most of them have unknown functions due to the inability to generate mutants. Bacterial gene interference based on clustered, regularly interspaced, short palindromic repeat-CRISPR technology coupled to deep sequencing (CRISPRi-seq), overcomes this limitation by allowing the screening and analysis of essential genes at the genomic scale through gene silencing. In Chapter 3, we developed and validated the utility of an anhydrotetracycline-inducible CRISPRi-seq platform for genome-wide programmable gene silencing in H. influenzae, opening new avenues for the functional understanding of its genome. Together, this PhD Thesis work significantly contributes to our understanding of the molecular mechanisms associated to H. influenzae infection, and presents novel bacterial genomic engineering tools applicable to therapeutic target screening in the development of strategies to improve the clinical management of this pathogen., Contrato de Formación de Personal Investigador en el marco de la Convocatoria de Ayudas para la Formación de Doctores del Ministerio de Ciencia, Innovación y Universidades, referencia PRE2019-088382, vinculado al proyecto RTI2018-096369-B-100, Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

La tutora de la tesis es Inmaculada Farrán Blanch, Este trabajo de Tesis Doctoral aborda el papel de tres aspectos del metabolismo bacteriano
(síntesis de purinas, catabolismo de glucosa, síntesis de ácidos grasos) en la interacción entre el
patógeno Haemophilus influenzae no tipificable (HiNT) y el sistema respiratorio humano. Mediante
análisis de expresión génica global, inactivación génica y caracterización fenotípica in vitro e in vivo,
modelado computacional, química médica, y evaluación antimicrobiana a nivel preclínico,
estudiamos los perfiles transcripcionales de patógeno y hospedador durante la infección respiratoria
(Capítulo 2), la contribución del catabolismo de glucosa en la patogénesis de H.
influenzae (Capítulo 3), y el potencial antimicrobiano de la inhibición de la ruta de biosíntesis de
ácidos grasos de esta bacteria (Capítulo 4).
H. influenzae fue el primer organismo de vida libre cuyo genoma completo fue secuenciado,
haciéndolo pionero en el desarrollo y empleo de técnicas -ómicas. El Capítulo 1 de este trabajo ha
revisado la contribución de abordajes -ómicos incluyendo genómica, transcriptómica, proteómica y
metabolómica, al estudio de la interacción entre HiNT y el sistema respiratorio humano.
En el Capítulo 2 de este trabajo realizamos un estudio multi-ómico in vivo, consistente en la
utilización de RNA-seq dual y Tn-seq durante el proceso de infección respiratoria murina por HiNT.
El perfil de expresión génica diferencial entre bacterias cultivadas in vitro y bacterias recuperadas de
lavado broncoalveolar murino mostró la sobre-expresión de genes que codifican enzimas implicadas
en la síntesis de purinas y aminoácidos, así como de genes que codifican parte de la maquinaria de
competencia natural de la bacteria.
El aumento de los niveles de glucosa en las vías respiratorias de pacientes que sufren
enfermedades respiratorias crónicas facilita la proliferación de patógenos que metabolizan este
azúcar. HiNT cataboliza glucosa mediante una fermentación asistida por respiración que conlleva la
excreción de acetato, formato y succinato. En el Capítulo 3 de este trabajo, diseñamos, generamos
y caracterizamos un panel de cepas mutantes que no producen acetato, formato o succinato mediante
la inactivación de los genes ackA, pflA y frdA, respectivamente. La inactivación de ackA limitó la
producción de acetato y el crecimiento bacteriano, y estimuló tanto la producción de lactato en
anaerobiosis como la atenuación bacteriana in vivo. El acetato excretado estimuló la expresión de
genes pro-inflamatorios en células de epitelio respiratorio en cultivo, lo que sugiere que el
catabolismo de glucosa contribuye no sólo al crecimiento de HiNT sino también a la
inmunomodulación del sistema respiratorio humano.
La resistencia de H. influenzae a antibióticos β-lactámicos ha llevado a su inclusión en la lista de
patógenos bacterianos para los que la OMS considera prioritaria la búsqueda y desarrollo de nuevos
antimicrobianos. En el Capítulo 4 de este trabajo, desarrollamos y validamos un modelo metabólico
de H. influenzae a escala genómica, que utilizamos como herramienta de escrutinio in silico de genes
esenciales de este patógeno, para su explotación como dianas terapéuticas. Este modelo predijo la esencialidad de un gran número de genes implicados en la síntesis de lípidos. Nos centramos en la
enzima FabH, que cataliza la condensación descarboxilativa de malonil-ACP y acil-CoA en la
iniciación de la biosíntesis de ácidos grasos. Nuestro modelado computacional mostró la idoneidad
de la interacción de la molécula ácido 1- (5- (2-fluoro-5- (hidroximetil) fenil) piridin-2-il) piperidin-
4-acético y la proteína FabH. Este inhibidor redujo la viabilidad bacteriana de forma dosisdependiente.
El efecto inhibitorio observado fue variable entre aislados clínicos portadores de
distintas variantes alélicas del gen fabH, e independiente de su expresión. El inhibidor empleado no
generó sinergias, no favoreció el desarrollo de resistencias, y no alteró la dinámica de infección
epitelial por HiNT, mostrado además un efecto protector frente a la infección por HiNT in vivo.
En conjunto, este trabajo de Tesis Doctoral proporciona conocimiento nuevo sobre el papel del
metabolismo bacteriano en la interacción HiNT-sistema respiratorio humano, que esperamos sea de
utilidad en el desarrollo de estrategias anti-infectivas que mejoren el manejo clínico de las
enfermedades infecciosas asociadas a este patógeno., This PhD Thesis work addresses the role of three aspects of bacterial metabolism (purine
synthesis, glucose catabolism, fatty acid synthesis) in the interaction between nontypeable
Haemophilus influenzae (NTHi) and the human airways. Global gene expression analysis, gene
inactivation and phenotypic characterization in vitro and in vivo, computational modeling, medical
chemistry, and antimicrobial evaluation at the preclinical level, led us to study pathogen and host
transcriptional profiles during respiratory infection (Chapter 2), the contribution of glucose
catabolism to H. influenzae pathogenesis (Chapter 3), and the antimicrobial potential of inhibiting
this bacterial fatty acid biosynthesis pathway (Chapter 4).
H. influenzae was the first free-living organism whose genome was fully sequenced, thus
pioneering in the development and use of -omics techniques. Chapter 1 of this work reviewed the
contribution of -omic approaches including genomics, transcriptomics, proteomics and
metabolomics, to the study of this host-pathogen interplay.
In Chapter 2, we carried out an in vivo multi-omic study, using dual RNA-seq and Tn-seq during
murine respiratory infection by NTHi. Differential gene expression profiling of bacteria grown in
vitro compared to those recovered from murine bronchoalveolar lavage fluid samples showed overexpression
of genes that encode enzymes involved in purine and amino acids synthesis, as well as of
genes encoding part of the bacterial natural competence machinery.
The increase of glucose levels in the respiratory tract of patients suffering chronic respiratory
diseases facilitates the proliferation of pathogens able to metabolize this sugar. NTHi catabolizes
glucose through respiration-assisted fermentation involving the excretion of acetate, formate, and
succinate. In Chapter 3 of this work, we designed, generated, and characterized a panel of mutant
strains that did not produce acetate, formate, or succinate by inactivating the ackA, pflA, and frdA
genes, respectively. Inactivation of the ackA gene limited acetate production and bacterial growth,
and stimulated both anaerobic lactate production and bacterial attenuation in vivo. The excreted
acetate stimulated the expression of pro-inflammatory genes by cultured respiratory epithelial cells,
which suggests that glucose catabolism contributes not only to the growth of NTHi but also to
immunomodulation within the human respiratory system.
The H. influenzae resistance to β-lactam antibiotics led to its inclusion in the list of bacterial
pathogens for which the WHO considers a priority the search and development of new
antimicrobials. In Chapter 4 of this work, we developed and validated a H. influenzae genome-scale
metabolic model, which we used as an in silico screening tool to identify bacterial essential genes
suitable as therapeutic targets. This model predicted the essentiality of a large number of genes
involved in lipid synthesis. We focused on the enzyme FabH, which catalyzes the decarboxylative
condensation of malonyl-ACP and acyl-CoA in the initiation of fatty acid biosynthesis.
Computational modeling showed the suitability of the interaction of the chemical inhibitor 1- (5- (2-Fluoro-5- (hydroxymethyl) phenyl) pyridin-2-yl) piperidine-4-acetic acid with FabH. Likewise, this
inhibitor reduced bacterial viability in a dose-dependent manner. The inhibitory effect observed was
variable among clinical isolates carrying different allelic variants of the fabH gene, and independent
of this gene expression. The inhibitor did not generate synergies, did not favor the development of
resistance, and did not alter the dynamics of epithelial infection by NTHi. Notably, this chemical
inhibitor showed a protective effect against NTHi infection in vivo.
Altogether, this PhD Thesis work provides novel knowledge on the role of bacterial metabolism
in the NTHi-human respiratory system interplay, intended to be useful in the development of antiinfective
strategies that will improve the clinical management of infectious diseases associated to
this pathogen., Gobierno de Navarra, convocatoria de ayudas “Doctorados industriales 2018-2020”, referencia 0011-1408-2017-000000. Contrato con cargo a proyecto Retos Investigación de la Agencia Estatal de Investigación, referencia RTI2018-096369-B-I00., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Standardized clinical procedures for antibiotic administration rely on pathogen identification and antibiotic susceptibility testing, often performed on single-colony bacterial isolates. For respiratory pathogens, this could be questionable, as chronic patients may be persistently colonized by multiple clones or lineages from the same bacterial pathogen species. Indeed, multiple strains of nontypeable Haemophilus influenzae, with different antibiotic susceptibility profiles, can be co-isolated from cystic fibrosis and chronic obstructive pulmonary disease sputum specimens. Despite this clinical evidence, we lack information about the dynamics of H. influenzae polyclonal infections, which limits the optimization of therapeutics. Here, we present the engineering and validation of a plasmid toolkit (pTBH, toolbox for Haemophilus), with standardized modules consisting of six reporter genes for fluorescent or bioluminescent labeling of H. influenzae. This plasmid set was independently introduced in a panel of genomically and phenotypically different H. influenzae strains, and two of them were used as a proof of principle to analyze mixed biofilm growth architecture and antibiotic efficacy, and to visualize the dynamics of alveolar epithelial co-infection. The mixed biofilms showed a bilayer architecture, and antibiotic efficacy correlated with the antibiotic susceptibility of the respective single-species strains. Furthermore, differential kinetics of bacterial intracellular location within subcellular acidic compartments were quantified upon co-infection of cultured airway epithelial cells. Overall, we present a panel of novel plasmid tools and quantitative image analysis methods with the potential to be used in a whole range of bacterial host species, assay types, and¿or conditions and generate meaningful information for clinically relevant settings., J.A.-L. is funded by a PhD studentship from Regional Navarra Government, Spain, reference 0011-1408-2020-000007. C.S.M. is funded by a Formación de Profesorado Universitario PhD studentship from the Spanish Ministry of Science and Innovation (MCINN), Spain, reference FPU20/06252. This work has been funded by grants from Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación (MCIU/AEI/10.13039/50110011033) and FEDER funds EU, RTI2018-094494-BC22, PDI2021-122409OB-C22 (C.O.S.), and RTI2018-096369-B-I00, PID2021-125947OB-I00 (J.G.); from SEPAR, 875/2019 (J.G.); from Gobierno de Navarra, PC150 and PC136 (J.G.) and PC151 and PC137 (C.O.S.). CIBER is an initiative from Instituto de Salud Carlos III, Madrid, Spain. Experimental design: B.R.A., I.S.B., M.L.D., A.T.A., C.O.S., J.G.; experimental work: B.R.A., I.S.B., J.A.L., B.E., M.L.D., C.O.M., A.F.C.; data analysis: B.R.A., I.S.B., M.L.D., M.A.; writing of the manuscript: J.G., C.O.S.; correction of the manuscript: all authors; funding¿ I.C.D., S.B., C.O.S., J.G.; Funding text 2: J.A.-L. is funded by a PhD studentship from Regional Navarra Govern, Spain, reference 0011-1408-2020-000007. C.S.M. is funded by a Formación de Profesorado Universitario PhD studentship from the Spanish Ministry of Science and Innovation (MCINN), Spain, reference FPU20/06252. This work has been funded by grants from Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación (MCIU/AEI/10.13039/50110011033) and FEDER funds EU, RTI2018-094494-BC22, PDI2021-122409OB-C22 (C.O.S.), and RTI2018-096369-B-I00, PID2021-125947OB-I00 (J.G.); from SEPAR, 875/2019 (J.G.); from Gobierno de Navarra, PC150 and PC136 (J.G.) and PC151 and PC137 (C.O.S.). CIBER is an initiative from Instituto de Salud Carlos III, Madrid, Spain.

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 and 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 for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 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. 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 P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent 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 quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as , an opportunistic respiratory colonizing bacteria that play an important role in exacerbations. This study contributes to a better understanding of COPD progression by characterizing the clinical evolution of in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of as a reservoir and source of resistance dissemination, is a cause for concern that may lead to therapeutic failure. Furthermore, genetic variations in cell wall and inorganic ion metabolism coding genes likely favor bacterial adaptation to host selective pressures. Therefore, the bacterial pathoadaptive evolution in these severe COPD patients raise our awareness of the possible spread of macrolide resistance and selection of host-adapted clones.

Respiratory infections pose a continuous threat to humans due to their easy dissemination via aerial transmission. As a consequence, they are leading causes of mortality and morbidity worldwide. Lower respiratory tract infections (LRTI) remained the deadliest communicable diseases causing 3 million deaths worldwide in 2016 (1). Similarly, although the number of tuberculosis (TB) deaths tends to decrease, it is still among the top 10 causes of global mortality with a yearly death burden of about 1.6 million (2). The growing emergence of bacterial antibiotic resistance is a major global challenge for the coming years, and several major respiratory pathogens are included in the WHO priority list of bacteria for which new antibiotics are urgently needed (3). In terms of target population, children under the age of five are the most susceptible hosts to a plethora of respiratory pathogens.