BUSCADOR RECOLECTA

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.

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 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;

The global threat posed by antimicrobial resistance demands urgent action and the development of effective drugs. Lower respiratory tract infections remain the deadliest communicable disease worldwide, often challenging to treat due to the presence of bacteria that form recalcitrant biofilms. There is consensus that novel anti-infectives with reduced resistance compared with conventional antibiotics are needed, leading to extensive research on innovative antibacterial agents. This review explores the recent progress in lipid-based nanomedicines developed to counteract bacterial respiratory infections, especially those involving biofilm growth; focuses on improved drug bioavailability and targeting and highlights novel strategies to enhance treatment efficacy while emphasizing the importance of continued research in this dynamic field., This work was supported by Atracción de Talento program (Modalidad 1) from Comunidad de Madrid (Spain) (Reference 2019-T1/IND-12906), Nanomedicine CSIC Hub (Spain) funding (PIE 202180E048), Spanish Ministry of Science and Innovation (PID2021-123238OB-I00, PID2021-125947OB-I00, PDC2022-133493-I00, RED2022-134299-T), Spanish Society of Pulmonology and Thoracic Surgery (SEPAR) (875/2019), Gobierno de Navarra (Spain) (PC150, PC136) and Comunidad de Madrid (Spain) (P2022/BMD-7333). CIBER is an initiative from Instituto de Salud Carlos III (ISCIII) (Spain). EM Arroyo-Urea benefits from the FPU fellowship program of the Spanish Ministry of Universities (reference FPU21/04116). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

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.

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.