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)

Chronic obstructive pulmonary disease (COPD) patients undergo infectious exacerbations whose frequency identifies a clinically meaningful phenotype. Mouse models have been mostly used to separately study both COPD and the infectious processes, but a reliable model of the COPD frequent exacerbator phenotype is still lacking. Accordingly, we first established a model of single bacterial exacerbation by nontypeable Haemophilus influenzae (NTHi) infection on mice with emphysema-like lesions. We characterized this single exacerbation model combining both noninvasive in vivo imaging and ex vivo techniques, obtaining longitudinal information about bacterial load and the extent of the developing lesions and host responses. Bacterial load disappeared 48 hours post-infection (hpi). However, lung recovery, measured using tests of pulmonary function and the disappearance of lung inflammation as revealed by micro-computed X-ray tomography, was delayed until 3 weeks post-infection (wpi). Then, to emulate the frequent exacerbator phenotype, we performed two recurrent episodes of NTHi infection on the emphysematous murine lung. Consistent with the amplified infectious insult, bacterial load reduction was now observed 96 hpi, and lung function recovery and disappearance of lesions on anatomical lung images did not happen until 12 wpi. Finally, as a proof of principle of the use of the model, we showed that azithromycin successfully cleared the recurrent infection, confirming this macrolide utility to ameliorate infectious exacerbation. In conclusion, we present a mouse model of recurrent bacterial infection of the emphysematous lung, aimed to facilitate investigating the COPD frequent exacerbator phenotype by providing complementary, dynamic information of both infectious and inflammatory processes., This work was supported by the Departamento de Universidad, Innovación y Transformación Digital, Gobierno de Navarra [PC150-151-152]; Ministerio de
Ciencia, Innovación y Universidades (MICIU), Gobierno de España [RTI2018-096369-B-I00]; MICIU, Gobierno de España [RED2018-102469-T]; MICIU, Gobierno de España [SAF2015-66520-R]; MICIU, Gobierno de España [RTI2018-094494-B-C222]; Departamento de Salud, Gobierno de Navarra [03/2016]; Sociedad Española de Neumología y Cirugía Torácica [31/2015].

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.

DNA regulatory elements that dictate how the bacterial pathobiont Haemophilus influenzae infects and adapts to the airways of immunocompromised patients suffering from chronic obstructive pulmonary disease (COPD) are poorly understood. This is in part due to the scarcity of research integrating genetic and epigenetic perspectives to shed light on the role of distinct bacterial adaptive strategies within the human airways. In this work, global fitness profiling of H. influenzae mutants by high-throughput transposon mutant sequencing within the mouse lung identified Dam methyltransferase as an in vivo requirement for respiratory infection. Equally, single-molecule real-time sequencing methylome analyses found undermethylation of GATC motifs within putative regulatory elements and revealed the first case of phenotypic variation controlled by variable Dam methylation in H. influenzae. Moreover, RNA sequencing differential gene expression disclosed a novel regulatory network where Dam methyltransferase positively regulates the expression of the ferric uptake regulator (Fur), which in turn represses the expression of the fumarate nitrate reductase (FNR) regulator and, subsequently, of a repertoire of genes that belong to the FNR regulon and encode bacterial anaerobic defenses against, among others, reactive nitrogen species produced within the diseased airways. Our results present a multifactorial regulatory network where the interplay between the Fur and FNR master transcriptional regulators is controlled epigenetically by Dam methylation. We put forward the notion that this network regulates H. influenzae survival in diseased airway niches with high nitrosative stress where damage reduces the amount of oxygen in the lungs, as encountered in COPD., We thank Dr. Beatriz Rapún-Araiz for technical help.
N.L.-L. was funded by a PhD studentship from Regional Navarra Government, Spain (0011-1408-2017-000000). C.G.-C. was funded by a PhD studentship from AEI (PRE2019-088382). J.A.-L. was funded by a PhD studentship from Regional Navarra Government, Spain (reference 0011-1408-2020-000007). This work has been funded by grants from MICIU RTI2018-096369-B-I00 and PID2021-125947OB-I00; 875/2019 from SEPAR; PI003 Micro-EPOC, PC150, and PC136 from Regional Navarra Government to J.G. CIBER is an initiative from Instituto de Salud Carlos III, Madrid, Spain., Peer reviewed

Simple sequence repeats (SSRs) are insertion-deletion mutational hotspots causing phase variation in bacterial genomes. When located in intergenic regions, SSR variation impacts transcription and may change post-transcriptional regulatory element targeting. Here, we show that transcription, 5'-UTR structure, and messenger RNA (mRNA) processing are key to bacterial SSR phase variation. Expression of the Haemophilus influenzae HMW1A adhesin is inversely proportional to the (5'-ATCTTTC)n SSR length upstream of the hmw1A gene. This repeat region is transcribed as part of the hmw 5'-UTR, which acts as a cis-acting regulatory element via a long hairpin structure involving 10 repeats on its left arm and a G-rich region on its right arm. The double strand-specific endoribonuclease RNase III processes hmw1 mRNA at this 5'-UTR hairpin increasing HMW1A expression. Deep-sequencing-based SSR quantification in bacterial populations showed that RNase III is also required for changes in SSR count. Moreover, mutations that disrupt the 5'-UTR hairpin in the repeat sequence or in the G-rich region impair HMW1A expression and repeat length changes. This supports hmw 5'-UTR and RNase III contribution to the mechanism of SSR number expansion and contraction. Overall, we present novel regulatory roles for the hmw 5'-UTR in bacterial adaptation, where its transcription, folding, and RNase III processing are essential to HMW1A expression and phase-variable changes., This work has been funded by grants from Agencia Estatal de Investigación (AEI), Spain, references RTI2018-096369-B-I00, PID2021-125947OB-I00, and PID2024-155918OB-I00 to J.G.; PC150 and PC136 from Regional Navarra Govern to J.G.; and AEI PID2022-136696NB-I00 to A.T.A. Centro de Investigación Biomédica en Red (CIBER) is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain. Funding to pay the Open Access publication charges for this article was provided by grants from AEI PID2021-125947OB-I00 to J.G., and PID2022-136696NB-I00 to A.T.A., Peer reviewed

Trabajo presentado para lograr el título de Doctor por la Universidad Pública de Navarra, Programa de Doctorado en Biotecnología.--2022-03-08.--Calificación: Sobresaliente Cum Laude, 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., 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., Peer reviewed

Trabajo presentado para lograr el título de Doctor por la Universidad Pública de Navarra, Programa de Doctorado en Biotecnología.--2020-12-14.--Calificación: Sobresaliente Cum Laude, 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., 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)., Peer reviewed

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammation and impaired airway immunity, providing an opportunistic platform for nontypeable <i>Haemophilus influenzae</i> (NTHi) infection. In this context, therapies targeting not only overactive inflammation without significant adverse effects, but also infection are of interest. Increasing evidence suggests that polyphenols, plant secondary metabolites with anti-inflammatory and antimicrobial properties, may be protective. Here, a <i>Cistus salviifolius</i> plant extract containing quercetin, myricetin, and punicalagin was shown to reduce NTHi viability. Analysis of these polyphenols revealed that quercetin has a bactericidal effect on NTHi, does not display synergies, and that bacteria do not seem to develop resistance. Moreover, quercetin lowered NTHi airway epithelial invasion through a mechanism likely involving inhibition of Akt phosphorylation, and reduced the expression of bacterially-induced proinflammatory markers <i>il-8</i>, <i>cxcl-1</i>, <i>il-6</i>, <i>pde4b</i>, and <i>tnfα</i>. We further tested quercetin’s effect on NTHi murine pulmonary infection, showing a moderate reduction in bacterial counts and significantly reduced expression of proinflammatory genes, compared to untreated mice. Quercetin administration during NTHi infection on a zebrafish septicemia infection model system showed a bacterial clearing effect without signs of host toxicity. In conclusion, this study highlights the therapeutic potential of the xenohormetic molecule quercetin against NTHi infection., This work has been funded by grants from MINECO SAF2015-66520-R and RTI2018-096369-B-I00,
PI011 from Economical Development Department, Regional Navarra Govern, Spain, and 31/2015 from SEPAR
to J.G., by grant from MINECO DRTI2018-094494-B-C22 (MCIU/AEI/FEDER, UE) to C.O.S., by grants from
MINECO AGL2015-67995-C3-1-R and RTI2018-096724-B-C21, PROMETEO/2016/006 from Generalitat Valenciana,
and CIBERobn (CB12/03/30038) to V.M, by grant 03/2016 from Health Department, Regional Navarra Govern,
Spain to J.G., C.O.S. and J.L, and by grant CIBERES (CB06/06/1102) to M.M. CIBER is an initiative from Instituto
de Salud Carlos III (ISCIII), Madrid, Spain.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and impaired airway immunity, which provides an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. Clinical evidence supports that the COPD airways present increased concentrations of glucose, which may facilitate proliferation of pathogenic bacteria able to use glucose as a carbon source. NTHi metabolizes glucose through respiration-assisted fermentation, leading to the excretion of acetate, formate, and succinate. We hypothesized that such specialized glucose catabolism may be a pathoadaptive trait playing a pivotal role in the NTHi airway infection. To find out whether this is true, we engineered and characterized bacterial mutant strains impaired to produce acetate, formate, or succinate by inactivating the ackA, pflA, and frdA genes, respectively. While the inactivation of the pflA and frdA genes only had minimal physiological effects, the inactivation of the ackA gene affected acetate production and led to reduced bacterial growth, production of lactate under low oxygen tension, and bacterial attenuation in vivo. Moreover, bacterially produced acetate was able to stimulate the expression of inflammatory genes by cultured airway epithelial cells. These results back the notion that the COPD lung supports NTHi growth on glucose, enabling production of fermentative end products acting as immunometabolites at the site of infection. Thus, glucose catabolism may contribute not only to NTHi growth but also to bacterially driven airway inflammation. This information has important implications for developing nonantibiotic antimicrobials, given that airway glucose homeostasis modifying drugs could help prevent microbial infections associated with chronic lung disease., We thank Dr. I. Rodriguez-Arce for technical support. N.L.-L. is funded by a PhD studentship from Regional Navarra Govern, Spain, reference 0011-1408-2017-000000. J.H. is the recipient of an Australian Commonwealth Government Research Training Program Award. This work has been funded by grants from MINECO SAF2015-66520-R and RTI2018-096369-B-I00, from the Health Department, Regional Navarra Govern, Spain, reference 03/2016, from SEPAR 31/2015 to J.G., and from the National Health and Medical Research Council (NHMRC, GNT1043532) to U.K. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

13 pags., 5 figs. -- This article belongs to the Special Issue Galectins: Their Network and Roles in Infection/Immunity/Tumor Growth Control 2021, Galectins bind various pathogens through recognition of distinct carbohydrate structures. In this work, we examined the binding of four human galectins to the Gram-negative bacteria Klebsiella pneumoniae (Kpn) and non-typeable Haemophilus influenzae (NTHi), which display different surface glycans. In particular, Kpn cells are covered by a polysaccharide capsule and display an O-chain-containing lipopolysaccharide (LPS), whereas NTHi is not capsulated and its LPS, termed lipooligosacccharide (LOS), does not contain O-chain. Binding assays to microarray-printed bacteria revealed that galectins-3, -4, and -8, but not galectin-1, bind to Kpn and NTHi cells, and confocal microscopy attested binding to bacterial cells in suspension. The three galectins bound to array-printed Kpn LPS. Moreover, analysis of galectin binding to mutant Kpn cells evidenced that the O-chain is the docking point for galectins on wild type Kpn. Galectins-3, -4, and -8 also bound the NTHi LOS. Microarray-assisted comparison of the binding to full-length and truncated LOSs, as well as to wild type and mutant cells, supported LOS involvement in galectin binding to NTHi. However, deletion of the entire LOS oligosaccharide chain actually increased binding to NTHi cells, indicating the availability of other ligands on the bacterial surface, as similarly inferred for Kpn cells devoid of both O-chain and capsule. Altogether, the results illustrate galectins’ versatility for recognizing different bacterial structures, and point out the occurrence of so far overlooked galectin ligands on bacterial surfaces., This research was funded by the Spanish Ministry of Science, Innovation, and Universities, the Spanish State Research Agency, and the European Regional Development Fund (Grants RTI2018-099985-B-I00, RTI2018-096369-B-I00, and RTI2018-094751-B-C21, MCIU/AEI/FEDER, UE), and by the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health
Carlos III (ISCIII)., Peer reviewed

20 pags., 7 figs., 2 tabs., 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 switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth., This work has been funded by grants SAF2015-66520-R and RTI2018-096369-B-I00
from MINECO, 875/2019 from SEPAR, and PC150-151-152 from the Gobierno de Navarra
to J.G. and RTI2018-099985-B-I00 to M.M. CIBER is an initiative from the Instituto de
Salud Carlos III (ISCIII), Madrid, Spain. A.T.-A. is funded by a Ministry of Science and
Innovation grant (PID2019-105216GB-I00)

Chronic obstructive pulmonary disease (COPD) patients undergo infectious exacerbations whose frequency identifies a clinically meaningful phenotype. Mouse models have been mostly used to separately study both COPD and the infectious processes, but a reliable model of the COPD frequent exacerbator phenotype is still lacking. Accordingly, we first established a model of single bacterial exacerbation by nontypeable Haemophilus influenzae (NTHi) infection on mice with emphysema-like lesions. We characterized this single exacerbation model combining both noninvasive in vivo imaging and ex vivo techniques, obtaining longitudinal information about bacterial load and the extent of the developing lesions and host responses. Bacterial load disappeared 48 hours post-infection (hpi). However, lung recovery, measured using tests of pulmonary function and the disappearance of lung inflammation as revealed by micro-computed X-ray tomography, was delayed until 3 weeks post-infection (wpi). Then, to emulate the frequent exacerbator phenotype, we performed two recurrent episodes of NTHi infection on the emphysematous murine lung. Consistent with the amplified infectious insult, bacterial load reduction was now observed 96 hpi, and lung function recovery and disappearance of lesions on anatomical lung images did not happen until 12 wpi. Finally, as a proof of principle of the use of the model, we showed that azithromycin successfully cleared the recurrent infection, confirming this macrolide utility to ameliorate infectious exacerbation. In conclusion, we present a mouse model of recurrent bacterial infection of the emphysematous lung, aimed to facilitate investigating the COPD frequent exacerbator phenotype by providing complementary, dynamic information of both infectious and inflammatory processes., This work was supported by the Departamento de
Universidad, Innovación y Transformación Digital,
Gobierno de Navarra [PC150-151-152]; Ministerio de
Ciencia, Innovación y Universidades (MICIU), Gobierno de
España [RTI2018-096369-B-I00]; MICIU, Gobierno de
España [RED2018-102469-T]; MICIU , Gobierno de España
[SAF2015-66520-R]; MICIU , Gobierno de España [RTI2018-
094494-B-C222]; Departamento de Salud, Gobierno de
Navarra [03/2016]; Sociedad Española de Neumología
y Cirugía Torácica [31/2015].

Objectives: To characterize the mechanisms of antimicrobial resistance and the prevalence of the polysaccharide capsule among urogenital and respiratory Haemophilus parainfluenzae isolates. Methods: Antimicrobial susceptibility was tested by microdilution. Fifty-five MDR strains were subjected to WGS and were phylogenetically compared with all the available H. parainfluenzae genomes from the NCBI database. The identification of the capsular bexA gene was performed by PCR in 266 non-MDR strains. Results: In 31 of the 42 ampicillin-resistant strains, blaTEM-1 located within Tn3 was identified. β-Lactamase-negative cefuroxime-resistant strains (n=12) presented PBP3 substitutions. The catS gene (n=14), the tet(M)-MEGA element (n=18) and FolA substitutions (I95L and F154V/S) (n=41) were associated with resistance to chloramphenicol, tetracycline plus macrolides, and co-trimoxazole, respectively. Thirty-seven isolates had a Tn10 harbouring tet(B)/(C)/(D)/(R) genes with (n=15) or without (n=22) catA2. Putative transposons (Tn7076-Tn7079), including aminoglycoside and co-trimoxazole resistance genes, were identified in 10 strains (18.2%). These transposons were integrated into three new integrative and conjugative elements (ICEs), which also included the resistance-associated transposons Tn3 and Tn10. The capsular operon was found only in the urogenital isolates (18/154, 11.7%), but no phylogenetic clustering was observed. The capsular operons identified were similar to those of Haemophilus influenzae serotype c and Haemophilus sputorum type 2. Conclusions: The identification of ICEs with up to three resistance-associated transposons suggests that these transferable elements play an important role in the acquisition of multidrug resistance in H. parainfluenzae. Moreover, the presence of polysaccharide capsules in some of these urogenital isolates is a cause for concern., This study was funded by the Fundación Española del Pulmón SEPAR (418/2017); the Instituto de Salud Carlos III through the Projects from the Fondo de Investigaciones Sanitarias (PI16/00977); CIBER de Enfermedades Respiratorias (CIBERES—CB06/06/0037; CB06/06/1102), co-funded by the European Regional Development Fund/European Social Fund (ERDF/ESF, ‘Investing in your future’) and CERCA Programme/Generalitat de Catalunya for institutional support; and the RTI2018-096369-B-100 grant. Bioinformatic analysis was supported by an Amazon Web Services (AWS) research grant to S.M.A.C. was supported by an FPU grant (Formación de Profesorado Universitario, FPU16/02202) from the Ministerio de Educación and S.M. was supported by a ‘Miguel Servet’ contract (CP19/00096) from the Instituto de Salud Carlos III.

Haemophilus influenzae has contributed to key bacterial genome sequencing hallmarks, as being not only the first bacterium to be genome-sequenced, but also starring the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, and pioneering Tn-seq methodologies. Over the years, the phenomenal and constantly evolving development of –omic technologies applied to a whole range of biological questions of clinical relevance in the H. influenzae-host interplay, has greatly moved forward our understanding of this human-adapted pathogen, responsible for multiple acute and chronic infections of the respiratory tract. In this way, essential genes, virulence factors, pathoadaptive traits, and multi-layer gene expression regulatory networks with both genomic and epigenomic complexity levels are being elucidated. Likewise, the unstoppable increasing whole genome sequencing information underpinning H. influenzae great genomic plasticity, mainly when referring to non-capsulated strains, poses major challenges to understand the genomic basis of clinically relevant phenotypes and even more, to clearly highlight potential targets of clinical interest for diagnostic, therapeutic or vaccine development. We review here how genomic, transcriptomic, proteomic and metabolomic-based approaches are great contributors to our current understanding of the interactions between H. influenzae and the human airways, and point possible strategies to maximize their usefulness in the context of biomedical research and clinical needs on this human-adapted bacterial pathogen., N.L.-L. is funded by a PhD studentship from Regional Navarra Govern, Spain, reference 0011-1408-2017-000000. C.G.-C. is funded by a PhD studentship from Agencia Española de Investigación (AEI), Spain, reference PRE2019-088382. This work has been funded by grants from MINECO RTI2018-096369-B-I00, 875/2019 from SEPAR, and PC150-151–152 from Gobierno de Navarra to J.G. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

Once relegated to the supply of energy and biosynthetic precursors, it is now indubitable that metabolism mediates most of physiological processes. In the context of bacterial–host interactions where virulence is the outcome (commonly termed bacterial pathogenesis) metabolism expands far beyond its canonical role in bacterial proliferation. In addition to all sorts of recognized molecular determinants or virulence factors (toxins, flagella, translocated effectors, adhesins, invasins, etc.), bacterial pathogens are equipped with specific metabolic traits to circumvent immune defenses and antimicrobial killing, thus facilitating colonization and proliferation within their hosts. As the implementation of high-throughput technologies elevates the pathogenesis field to the era of big data, it concurrently creates considerable challenges for our ability to interpret large data sets and identify factors that impact infectious processes. Metabolic modelling is emerging as a powerful tool allowing the integration and coherent organization of large data sets into the context of biological networks providing non-intuitive insights on biological systems that experimental analysis alone cannot provide. Here, we take a snapshot of the recent understanding of bacterial metabolism and the bacterial–host metabolic interplay during infection, and highlight key outcomes and challenges for the practical implementation of bacterial metabolic modelling computational tools in the pathogenesis field (summarized in Fig. 1)., This work has been funded by grants from MINECORTI2018-096369-B-I00, 875/2019 from SEPAR, PC150-151-152 from Gobierno de Navarra to J.G and fromMICIU through RobExplode PID2019-108458RB-I00 toJ.N. CIBER is an initiative from Instituto de Salud CarlosIII (ISCIII), Madrid, Spain.

24 pags., 6 figs., 3 tabs., Expediting drug discovery to fight antibacterial resistance requires holistic approaches at system levels. In this study, we focused on the human-adapted pathogen Haemophilus influenzae, and by constructing a high-quality genome-scale metabolic model, we rationally identified new metabolic drug targets in this organism. Contextualization of available gene essentiality data within in silico predictions identified most genes involved in lipid metabolism as promising targets. We focused on the b-ketoacyl-acyl carrier protein synthase III FabH, responsible for catalyzing the first step in the FASII fatty acid synthesis pathway and feedback inhibition. Docking studies provided a plausible three-dimensional model of FabH in complex with the synthetic inhibitor 1-(5-(2-fluoro-5-(hydroxymethyl)phenyl) pyridin-2-yl)piperidine-4-acetic acid (FabHi). Validating our in silico predictions, FabHi reduced H. influenzae viability in a dose- and strain-dependent manner, and this inhibitory effect was independent of fabH gene expression levels. fabH allelic variation was observed among H. influenzae clinical isolates. Many of these polymorphisms, relevant for stabilization of the dimeric active form of FabH and/or activity, may modulate the inhibitory effect as part of a complex multifactorial process with the overall metabolic context emerging as a key factor tuning FabHi activity. Synergies with antibiotics were not observed and bacteria were not prone to develop resistance. Inhibitor administration during H. influenzae infection on a zebrafish septicemia infection model cleared bacteria without signs of host toxicity. Overall, we highlight the potential of H. influenzae metabolism as a source of drug targets, metabolic models as target-screening tools, and FASII targeting suitability to counteract this bacterial infection., This work has been funded by grants from MICIU/
AEI RTI2018-096369-B-I00, 875/2019 from SEPAR, PC150-151-152 from Gobierno de
Navarra to J.G., by PID2019-108458RB-I00 MCIU/AEI /10.13039/501100011033 to J.N.,
and by RTI2018-099985-B-I00 to M.M. CIBER is an initiative from Instituto de Salud
Carlos III (ISCIII), Madrid, Spain

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. The elderly, and immunocompromised respiratory patients suffering from cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), bronchiectasis, neutrophilic asthma, or silicosis are also highly targeted by respiratory pathogens, which often accelerates the fatal progression of the underlying chronic disease. Accordingly, understanding microbial pathogenicity and host immunity against respiratory infections is essential for the rational development of new and more effective therapeutics., This work was supported by grants from MINECO
SAF2015-66520-R and RTI2018-096369-B-I00, from
Health Department, Regional Govern from Navarra,
Spain, reference 03/2016, and from SEPAR 31/2015 to JG,
and also from MINECO BFU2015-72190-EXP to JG-A.
CIBER is an initiative from Instituto de Salud Carlos
III (ISCIII), Madrid.

Resumen del trabajo presentado en la XIII Reunión del Grupo de Microbiología Molecular de la SEM, celebrada en Granada (España), del 7 al 9 de septiembre de 2022

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 quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as Haemophilus influenzae, 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 H. influenzae in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of Haemophilus parainfluenzae 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., This study was funded by the Fundación Española del Pulmón SEPAR (88/2016 to
D.H. and 1116/2020 to S.M.); Fondo de Investigaciones Sanitarias (PI16/00977 to S.M.);
Fundació Catalana de Pneumologia, FUCAP (Beca Albert Agustí 2017 to D.H.); Ministerio
de Ciencia, Innovación y Universidades (MICIU; RTI2018-096369-B-I00 to J.G.); and Centro de
Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES; CB06/06/0037 and
CB06/06/1102), an initiative of the Instituto de Salud Carlos III (ISCIII). The European Regional
Development Fund/European Social Fund (ERDF/ESF; “Investing in your future”) also provided
financial support, and CERCA Program/Generalitat de Catalunya provided institutional support.
Bioinformatic analysis was supported by an Amazon Web Services (AWS) research grant (to
S.M.). S.S. received financial support by Menarini. A.C.-S. was supported by Formación de
Profesorado Universitario from the Ministerio de Educación of Spain (FPU16/02202), and S.M.
was supported byMiguel Servet contract (CP19/00096) (ISCIII).
We declare that there is no conflict of interest regarding the publication of this article.

Haemophilus influenzae is a human-adapted bacterial pathogen that causes airway infections. Bacterial and host elements associated with the fitness of H. influenzae within the host lung are not well understood. Here, we exploited the strength of in vivo-omic analyses to study host-microbe interactions during infection. We used in vivo transcriptome sequencing (RNA-seq) for genome-wide profiling of both host and bacterial gene expression during mouse lung infection. Profiling of murine lung gene expression upon infection showed upregulation of lung inflammatory response and ribosomal organization genes, and downregulation of cell adhesion and cytoskeleton genes. Transcriptomic analysis of bacteria recovered from bronchoalveolar lavage fluid samples from infected mice showed a significant metabolic rewiring during infection, which was highly different from that obtained upon bacterial in vitro growth in an artificial sputum medium suitable for H. influenzae. In vivo RNA-seq revealed upregulation of bacterial de novo purine biosynthesis, genes involved in non-aromatic amino acid biosynthesis, and part of the natural competence machinery. In contrast, the expression of genes involved in fatty acid and cell wall synthesis and lipooligosaccharide decoration was downregulated. Correlations between upregulated gene expression and mutant attenuation in vivo were established, as observed upon purH gene inactivation leading to purine auxotrophy. Likewise, the purine analogs 6-thioguanine and 6-mercaptopurine reduced H. influenzae viability in a dose-dependent manner. These data expand our understanding of H. influenzae requirements during infection. In particular, H. influenzae exploits purine nucleotide synthesis as a fitness determinant, raising the possibility of purine synthesis as an anti-H. influenzae target. IMPORTANCE In vivo-omic strategies offer great opportunities for increased understanding of host-pathogen interplay and for identification of therapeutic targets. Here, using transcriptome sequencing, we profiled host and pathogen gene expression during H. influenzae infection within the murine airways. Lung pro-inflammatory gene expression reprogramming was observed. Moreover, we uncovered bacterial metabolic requirements during infection. In particular, we identified purine synthesis as a key player, highlighting that H. influenzae may face restrictions in purine nucleotide availability within the host airways. Therefore, blocking this biosynthetic process may have therapeutic potential, as supported by the observed inhibitory effect of 6-thioguanine and 6-mercaptopurine on H. influenzae growth. Together, we present key outcomes and challenges for implementing in vivo-omics in bacterial airway pathogenesis. Our findings provide metabolic insights into H. influenzae infection biology, raising the possibility of purine synthesis as an anti-H. influenzae target and of purine analog repurposing as an antimicrobial strategy against this pathogen., We are grateful to Lucía Caballero and Sergio Cuesta for technical support. We are grateful to the generosity of APEPOC members (Asociación de pacientes con EPOC).
N.L.-L. was funded by a PhD studentship from Regional NavarraGovern, Spain, no. 0011-1408-2017-000000. C.G.-C. is funded by a PhD studentship from AEI, PRE2019-088382. J.A.-L. is funded by a PhD studentship from Regional Navarra Govern, Spain, no. 0011-1408-2020-000007. This work has been funded by grants MICIU RTI2018-096369-B-I00 PID2021-125947OB-I00, no. 875/2019 from SEPAR, PI003 Micro-EPOC, PC150 from Gobierno de Navarra to J.G. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.
We have no competing interests to declare.

Genomic diversity of nontypeable H. influenzae strains confers phenotypic heterogeneity. Multiple strains of H. influenzae can be simultaneously isolated from clinical specimens, but we lack detailed information about polyclonal infection dynamics by this pathogen. A long-term barrier to our understanding of this host-pathogen interplay is the lack of genetic tools for strain engineering and differential labeling. Here, we present a novel plasmid toolkit named pTBH (toolbox for Haemophilus), with standardized modules for fluorescent or bioluminescent labeling, adapted to H. influenzae requirements but designed to be versatile so it can be utilized in other bacterial species. We present detailed experimental and quantitative image analysis methods, together with proof-of-principle examples, and show the ample possibilities of 3D microscopy, combined with quantitative image analysis, to model H. influenzae polyclonal infection lifestyles and unravel the co-habitation and co-infection dynamics of this respiratory pathogen., 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-B-C22, 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., Peer reviewed

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., The author(s) declare financial support was received for the research, authorship, and/or publication of this article. 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., Peer reviewed

Trabajo presentado en la Jornada CIBERES - CIBERINFEC, celebradas en Madrid (España), los días 15 y 16 de junio de 2023, Introduction
Haemophilus influenzae is a human-adapted pathogen causing chronic lower airway infections and recurrent exacerbations in chronic obstructive pulmonary disease (COPD) patients. Unraveling H. influenzae virulence mechanisms will result in identifying targets for drug development. By using transposon insertion sequencing (Tnseq), we screened bacterial genes required for infection in a murine model of airway infection, identified and validated the methyltransferase Dam.
Objectives
To study the role of Dam GATC methylation in the regulation of H. influenzae gene expression, and the contribution of such epigenetic regulation to this host-pathogen interplay.
Methods
We followed two complementary approaches:
(i) RNA sequencing (RNA-seq) to profile differential gene expression when comparing WT and dam mutant strains.
(ii) methylome analysis in a panel of H. influenzae PacBio clinical strain genomes to screen hypo/hemi-methylated GATC sites in non-coding regions, as potential elements of epigenetic regulation of gene expression.
Results
The oxygen sensitive fumarate and nitrate reductase (FNR) encoding gene, together with the FNR regulon genes ytfE, dmsA and cydD, were overexpressed upon dam inactivation. Further analysis identified GATC motifs in the fnr, dmsA and cydD promoter regions, and Dam methylation of these sites was confirmed. Conversely, methylome analyses recurrently showed GATC hypo/hemi-methylation in a region containing two GATC motifs upstream of the high temperature protein G (htpG) encoding gene, the proximal one overlapping with a putative FNR binding site. Analysis of such GATC sites revealed possible phenotypic heterogeneity in the above mentioned proximal motif, further tested by ad hoc generation of fluorescent reporter strains for single-cell analyses.
Conclusions
Together, our results shed light on Dam methyltransferase contribution to H. influenzae pulmonary infection, highlight epigenetic regulation of the H. influenzae FNR regulon and its likely involvement in airway infection and bacterial response to environment stress conditions., C.G.-C. is funded by PhD studentship PRE2019-088382. This work has been funded by grant MICIU RTI2018-096369-B-I00 and PID2021-125947OB-I00 to J.G. CIBER is an initiative from ISCIII.

Trabajo presentado en el Cell Symposia: Infection Biology in the Age of the Microbiome, celebrado en París (Francia) del 7 al 9 de junio de 2023, Haemophilus influenzae is a human-adapted bacterial pathogen causing airway infections. We used RNAseq and Tnseq to profile bacterial genome-wide gene expression and mutant fitness during lung infection. Integration of these data sets drawed a comprehensive landscape in terms of bacterial metabolic requirements during infection. Genes involved in tryptophan and serine biosynthesis were identified, and auxotrophy of these aminoacids dampened in vivo bacterial fitness. Moreover, transcriptome analyses showed a likely long 3¿UTR overlapping event in the mtr-sdaCA region, where mtr encodes a tryptophan transporter, sdaC a L-serine transporter, and sdaA a L-serine desaminase. Biological significance and contribution of this tryptophan-serine metabolic axis to H. influenzae infection is being elucidated by combining transport/metabolism mutant phenotyping, analysis of regulatory elements, and evaluation of natural antibiotics as potential inhibitors of tryptophan synthesis. Our results shed light on these aminoacids contribution to H. influenzae infection, and their potential as a source of therapeutic targets., J.A.-L. is funded by a PhD studentship from Gobierno Navarra,
reference 0011-1408-2020-000007. This work has been funded by
grants MICIU RTI2018-096369-B-I00 and PID2021-1259470B-100 to
J.G. CIBER is an initiative from Instituto de Salud Carlos III;

Biofilm formation by the pathobiont Haemophilus influenzae is associated with human nasopharynx colonization, otitis media in children, and chronic respiratory infections in adults suffering from chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD). β-lactam and quinolone antibiotics are commonly used to treat these infections. However, considering the resistance of biofilm-resident bacteria to antibiotic-mediated killing, the use of antibiotics may be insufficient and require being replaced or complemented with novel strategies. Moreover, unlike the standard minimal inhibitory concentration assay used to assess antibacterial activity against planktonic cells, standardization of methods to evaluate anti-biofilm drug activity is limited. In this work, we detail a panel of protocols for systematic analysis of drug antimicrobial effect on bacterial biofilms, customized to evaluate drug effects against H. influenzae biofilms. Testing of two cinnamaldehyde analogs, (E)-trans-2-nonenal and (E)-3-decen-2-one, demonstrated their effectiveness in both H. influenzae inhibition of biofilm formation and eradication or preformed biofilms. Assay complementarity allowed quantifying the dynamics and extent of the inhibitory effects, also observed for ampicillin resistant clinical strains forming biofilms refractory to this antibiotic. Moreover, cinnamaldehyde analog encapsulation into poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles allowed drug vehiculization while maintaining efficacy. Overall, we demonstrate the usefulness of cinnamaldehyde analogs against H. influenzae biofilms, present a test panel that can be easily adapted to a wide range of pathogens and drugs, and highlight the benefits of drug nanoencapsulation towards safe controlled release., We are grateful to Alex Mira (FISABIO Foundation, Valencia, Spain)
for providing access to the xCELLigence RTCA instrument. Funding
Agencies and grant numbers: PhD studentship from Regional Navarra
Govern, Spain, reference 0011-1408-2020-000007. Margarita Salas
grant from Ministerio de Universidades, Spain, funded by the EU-Next
Generation EU. This work has been funded by grants from AEI MICIU
RTI2018-096369-B-I00, PID2021-125801OB-I00, MICIU PDI2021-
122409OB-C22; from SEPAR 875/2019; from Gobierno de Navarra
PC150-151-152 and PC136-137-138; Atracción de Talento (Modalidad
1) program from Comunidad de Madrid, Spain, 2019-T1/IND-12906;
Nanomedicine CSIC Hub, Spain, PIE202180E048. CIBER is an initiative
from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

Trabajo presentado para lograr el título de Doctor por la Universidad Pública de Navarra, Departamento de Agronomía, Biotecnología y Alimentación, Programa de Doctorado en Biotecnología.--Calificación: Sobresaliente Cum Laude, [ES] 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. [...], [EN] 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. [...], AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/RTI2018-096369-B-I00

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.

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.