New therapies for COPD-related pulmonary infections unveiled by inspecting bacterial interactions

Universidade do Minho Escola de Engenharia Beatriz Maciel Ferreira New therapies for COPD-related pulmonary infections unveiled by inspecting bacterial interactions janeiro de 2023 New therapies for COPD-related pulmonary infections unveiled by inspecting bacterial interactions Título Título Título Título Título Título Título Beatriz Maciel Ferreira UMinho | 2023 Universidade do Minho Escola de Engenharia Beatriz Maciel Ferreira New therapies for COPD-related pulmonary infections unveiled by inspecting bacterial interactions Dissertação de Mestrado Mestrado em Biotecnologia Trabalho efetuado sob a orientação da Doutora Paula Alexandra da Silva Jorge janeiro de 2023 I DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-CompartilhaIgual CC BY-NC-SA https://creativecommons.org/licenses/by-nc-sa/4.0/ II Acknowledgements The help and support of several people were crucial for the accomplishment of the dissertation, and therefore, I am grateful to all the people who contributed directly or indirectly in some way to the completion of my dissertation. To my supervisor, Dr. Paula, for sympathy, availability, dedication, patience, comprehension, and coordination. For all the knowledge transmitted at a theoretical level as well as in terms of laboratory techniques. For all the support, critical thinking, suggestions, and clarification of all my questions during this work. Grateful for everything! To the MOP laboratory group, for the good atmosphere, sympathy, availability, and clarification of all procedures and questions throughout the development of the laboratory work. To my family and friends in general, for the moments of sharing and happiness, companionship, dedication, care, support, and exchange of experience that made me evolve personally and academically. To my parents, words will never be enough to thank them. Thank you for helping me in everything, for giving me confidence, for believing in me and in my capacity to continue to accomplish my goals. Finally, to Nelson, my great support, for making my days happier, thank you for your care, comprehension, attention, patience, knowledge sharing, and encouragement to overcome the difficulties that arose throughout this dissertation. I'm grateful to everyone. III STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. IV Novas terapias para infecções pulmonares relacionadas com a DPOC por inspeção de interações bacterianas Resumo Anualmente, milhões de pessoas morrem devido à doença pulmonar obstrutiva crónica (DPOC). Esta doença incurável é marcada por exacerbações que são principalmente provocadas por bactérias formadoras de biofilmes, onde NTHi e a sua interação com outras bactérias aumentam a resistência aos agentes antimicrobianos. Portanto, entender o papel das bactérias e das suas interações na DPOC é crucial para o desenvolvimento de novos fármacos capazes de combater a resistência destas aos antibióticos. O principal objetivo desta dissertação consistiu no estudo das condições ótimas de crescimento de biofilmes de H. influenzae e de S. aureus e das interações bacterianas que se estabelecem nesses biofilmes, bem como a avaliação da suscetibilidade das duas espécies a uma seleção de péptidos antimicrobianos (AMPs). O impacto da renovação do meio de cultura em biofilmes de H. influenzae revelou inexistência de células cultiváveis quando este não foi renovado às 24 h, possivelmente devido a metabolitos tóxicos, ao esgotamento de nutrientes, ou à existência de células viáveis mas não cultiváveis. Os resultados demonstraram que as diferenças no crescimento de H. influenzae em diferentes marcas do meio de crescimento (Oxoid, Liofilchem e VWR) podem dever-se à qualidade do meio usado, dado que o meio mais caro (Oxoid) foi o que resultou em menos variabilidade, tanto em biofilmes de uma ou duas espécies. Portanto, foi decidido usar esta marca para os ensaios subsequentes. Os resultados da dinâmica populacional de biofilmes mistos de H. influenzae com diferentes concentrações iniciais de S. aureus sugerem que ambas as espécies podem estar a beneficiar o crescimento uma da outra. Do estudo da influência da colonização sequencial nos biofilmes, não se verificou a influência das espécies colonizadoras na formação dos biofilmes de outra espécie. Na avaliação do efeito dos exoprodutos produzidos por uma espécie na formação de biofilmes da outra espécie, não se observaram quaisquer efeitos, pelo que a concentração dos exoprodutos pode ter sido insuficiente para causar um impacto ou as células do biofilme podem não ser responsivas aos exoprodutos produzidos. Por fim, o AMP tachyplesin I demonstrou melhor ação bacteriostática e bactericida contra ambas as estirpes em estudo, revelando resultados promissores para trabalhos futuros. V Os resultados deste trabalho permitiram extrair informações úteis, nomeadamente condições ótimas de crescimento e tipo de interações estabelecidas in vitro , que ajudarão o grupo de pesquisa na busca de potenciais agentes antimicrobianos para combater os biofilmes formados por estas bactérias. Palavras-chave: DPOC; Exacerbações; Haemophilus influenzae ; Interações bacterianas; Staphylococcus aureus . VI New therapies for COPD-related pulmonary infections unveiled by inspecting bacterial interactions Abstract Every year, millions of people die from chronic obstructive pulmonary disease (COPD). This incurable disease is marked by exacerbations that are mainly caused by biofilm-forming bacteria, where NTHi and its interaction with other bacteria increase resistance to antimicrobial agents. Therefore, understanding the role of bacteria and their interactions in COPD is crucial for the development of new drugs capable of combating their resistance to antibiotics. The main objective of this dissertation was to study the optimal conditions for the growth of H. influenzae and S. aureus biofilms and the bacterial interactions that are established in these biofilms, as well as the evaluation of the susceptibility of the two species to a selection of antimicrobial peptides (AMPs). The impact of renewing the culture medium on H. influenzae biofilms revealed the absence of culturable cells when the medium was not renewed after 24 h, possibly due to toxic metabolites, nutrient depletion, or the existence of viable but non-culturable cells. The results showed that the differences in H. influenzae growth in different brands of growth medium (Oxoid, Liofilchem, and VWR) could be due to the quality of the medium used, given that the most expensive medium (Oxoid) was the one that resulted in less variability, both in biofilms of one or two species. Therefore, it was decided to use this brand for subsequent assays. The population dynamics results of mixed H. influenzae biofilms with different initial concentrations of S. aureus suggest that both species could be benefiting each other's growth. From the study of the influence of sequential colonization on biofilms, the influence of colonizers species on the formation of biofilms of another species was not verified. In assessing the effect of exoproducts produced by one species on biofilm formation by another species, no effects were observed, so the concentration of exoproducts may have been insufficient to cause an impact, or the biofilm cells may not be responsive to the exoproducts produced. Finally, AMP tachyplesin I showed better bacteriostatic and bactericidal action against both strains under study, revealing promising results for future work. The results of this work allowed extracting useful information, namely optimal growth conditions and type of interactions established in vitro , which will help the research group in the search for potential antimicrobial agents to combat the biofilms formed by these bacteria. Keywords: Bacterial interactions; COPD; Exacerbations; Haemophilus influenzae ; Staphylococcus aureus . XIII Abbreviations: HI 2 = H. influenzae from the second cryovial (in use), HI 6 = H. influenzae from the sixth cryovial (newly opened). .................................................................................................................... 35 Figure 7 – Quantification of culturable H. influenzae (A) and total biomass (B) of 48 h H. influenzae biofilms grown in sBHI from Oxoid and Liofilchem with media replacement at 24 h. Standard deviations are indicated by the error bars. Statistically significancy: *** p<0.001 and **** p<0.0001. Abbreviations: HI 2 = H. influenzae from the second cryovial (in use), HI 6 = H. influenzae from the sixth cryovial (newly opened). ........................................................................................................................................... 36 Figure 8 - Quantification of culturable H. influenzae (A) and total biofilm biomass (B) from 24 h biofilms of H. influenzae (HI) and of H. influenzae with 1% of S. aureus (1% SA) grown in sBHI from Liofilchem and Oxoid. Statistically significant differences: **** p < 0.0001. Standard deviations are indicated by error bars. ................................................................................................................................................ 38 Figure 9 - Quantification of culturable H. influenzae (A) and total biofilm biomass (B) from 48 h biofilms of H. influenzae (HI) and of H. influenzae with 1% of S. aureus (1% SA) grown in sBHI from Liofilchem and Oxoid without media replacement at 24 h. Statistically significant differences are represented by *** p < 0.001 and **** p < 0.0001. Standard deviations are indicated by error bars. .............................. 38 Figure 10 - Quantification of culturable H. influenzae (A) and total biofilm biomass (B) from 48 h biofilms of H. influenzae (HI) and of H. influenzae with 1% of S. aureus (1% SA) grown in sBHI from Liofilchem and Oxoid with media replacement at 24 h. Statistically significant differences are represented by ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Standard deviations are indicated by error bars. .............. 40 Figure 11 - Quantification of culturable H. influenzae and S. aureus (A) and total biofilm biomass (B) from 24 h double-species biofilms with different initial concentrations of S. aureus (1%, 10%, 25%, and 50% V/V). Statistically significant differences are represented by * p <0.05, ** p < 0.01, *** p < 0.001, and #### p < 0.0001. Standard deviations are indicated by error bars. The symbols * and # represent comparisons with H. influenzae and S. aureus control biofilms, respectively (single-species control). .. 41 Figure 12 - Quantification of culturable H. influenzae and S. aureus (A) and total biofilm biomass (B) from 48 h double-species biofilms with different initial concentrations of S. aureus (1%, 10%, 25%, and 50% V/V) without media replacement at 24h. Statistically significant are represented by **** p < 0.0001, and # p < 0.05. The symbols * and # represent comparisons with H. influenzae and S. aureus control biofilms, respectively. ....................................................................................................................... 42 Figure 13 – Quantification of culturable H. influenzae and S. aureus (A) and total biofilm biomass (B) from 48 h double-species biofilms of H. influenzae with different initial concentrations of S. aureus (1%, 10%, 25%, and 50% V/V) with media replacement at 24 h. Statistically significant differences are XIV represented by * p < 0.05. Standard deviations are indicated by error bars. Note: Due to unforeseen technical reasons, there is no data for the culturability of H. influenzae in H. influenzae biofilms with 50% of S. aureus. ..................................................................................................................................... 43 Figure 14 - Quantification of culturable S. aureus and H. influenzae cells (A) and quantification of biomass (B) from double-species biofilms in which H. influenzae was the second colonizer. Controls correspond to 48 h S. aureus and 24 h H. influenzae single-species biofilms. The statistical differences are represented by: * p < 0.05 and ** p < 0.01. Standard deviations are indicated by error bars. ...... 45 Figure 15 - Quantification of culturable H. influenzae and S. aureus cells (A) and quantification of biomass (B) from double-species biofilms in which S. aureus was the second colonizer for 24 h. Controls correspond to 48 h H. influenzae and 24 h S. aureus single-species biofilms. The statistical differences are depicted as: * p < 0.05 and **** p < 0.0001. Standard deviations are indicated by error bars. .... 46 Figure 16 – Effect of SA CFS on HI culturable biofilm cells (A) and of HI CFS on SA culturable biofilm cells (B). The statistically significant differences are represented as: **** p < 0.0001. Standard deviations are indicated by error bars. ............................................................................................................... 48 XV List of Tables Table 1 - Functional categories controlled by QS (Grandclément et al., 2016). ................................. 14 Table 2 - Summary of NTHi biofilm resistance and tolerance mechanisms and their respective model and experimental effects (adapted from Weeks et al., 2021). ............................................................. 23 Table 3 - Calibration curve equations for the two bacterial strains provided by the research group. ... 27 Table 4 – Antimicrobial activity of temporin A, tachyplesin I, palm-KGKPEG, citropin 1.1, and ciprofloxacin against H. influenzae DSM 4690 (ATCC 33391, NCTC 8143) and S. aureus (ATCC 25923). MIC and MBC are expressed in mg/L. .............................................................................................. 49 1 Chapter 1. Introduction This chapter presents the contextualization and motivation of this dissertation, namely explaining the severity of COPD, characterizing the pathology, revealing the main bacteria responsible for the critical periods of the disease, and not least, the problem in the treatment of these infections due to antibiotic resistance by these microorganisms. Furthermore, this chapter conveys the main objectives of this dissertation given this scenery and the structure of the document. 1.1 Context and Motivation Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide that mainly affects the respiratory system (GOLD, 2022). According to the World Health Organization (WHO), in 2019, 3.23 million people died due to COPD, with almost 90% of these deaths reported in LMICs (WHO, 2021). COPD is a common, preventable, and progressive debilitating disease that although treatable, it has no cure and its appearance is mainly related to smoking habits, genetic factors, and pollution (Awokola et al., 2022; Jiang et al., 2016; Weinberger et al., 2019; WHO, 2021). This disease has a high socio-economic burden and includes two pathologies, namely emphysema, demarcated by the destruction of the lung parenchyma, and chronic bronchitis, characterized by mucus hypersecretion, resulting in chronic productive cough (GOLD, 2022; Leung et al., 2017; Vogelmeier et al., 2020). COPD is characterized by frequent exacerbations that are denoted by a sudden decline in lung function and the consequent worsening of symptoms (GOLD, 2022). These exacerbations are caused by pathogenic bronchial colonization, of which 50% are related to bacterial infections, often resulting in poor quality of life, increased hospitalizations, and higher mortality rates (Saxena et al., 2016; Vogelmeier et al., 2020). These bacterial infections are the result of bacterial adhesion to the pulmonary epithelium and the consequent formation of biofilms (Short et al., 2021). Biofilms are aggregates of bacteria in a selfproduced polymeric matrix that normally include different microbial species, which interact with each other benefiting or harming one another (Melton & Anderson, 2019; Welp & Bomberger, 2020). Specifically, in COPD exacerbations, non-typable Haemophilus influenzae (NTHi) is the most prevalent isolated species, followed by Moraxella catarrhalis , Streptococcus pneumoniae , Staphylococcus aureus , Pseudomonas aeruginosa , and Klebsiella pneumoniae (Leung et al., 2017; Short et al., 2021; Su et al., 2018). In recent times, there has been a global increasing development of antibiotic resistance on the part of bacteria (Jorge et al., 2019). This resistance compromises the effectiveness of infection treatments 2 and leads to the selection of resistant subpopulations that can make COPD exacerbations even more persistent and severe (Welp & Bomberger, 2020). It is, therefore, crucial to fully understand the interactions between the bacteria present in COPD-related infections in order to try to find new therapies adapted to this polymicrobial reality. Thus, the focus of this work was the study of H. influenzae and S. aureus biofilm interactions. H. influenzae was selected given its high relevance in COPD related infections, and S. aureus was chosen because it is the second least studied of the four prevalent species ( M. catarrhalis , S. pneumoniae , and P. aeruginosa ). In fact, up until February 2020, only nine articles were published relating S. aureus with H. influenzae in COPD, most of which only analyzed the co-occurrence of the two species in patients and none of studied them in the context of a biofilm (Amaro, 2022). 1.2 Main Objectives The main objective of this dissertation is to understand and characterize the biogeography of the polymicrobial consortia that often establishes in the lungs of patients with COPD, always focusing on NTHi. In this sense, this work was focused on the investigation of interactions of two species, namely H. influenzae and S. aureus , in biofilms, to try and identify the social relationships that they establish with each other. The rationale was to obtain information on the influence of growth conditions, specifically the brand of media used on H. influenzae and S. aureus biofilm formation, as well as the population dynamics between the two species with different initial concentrations. Sequential colonization was also investigated to understand whether the introduction of a species at a later stage induces changes to the biofilm and, moreover, if there is any benefit or damage to the total community. Finally, the effect of extracellular products that may eventually bring some benefit or harm to the biofilm community was also investigated, as well as the susceptibility of the bacteria to potential therapeutic agents, more specifically antimicrobial peptides (AMPs). This thesis was performed within the scope of an ESCMID Research Project, hence the information here acquired will later assist in the selection and testing of antimicrobial products for the eradication of these multispecies aggregates, with the ultimate goal of developing and evaluating new therapeutic strategies to treat COPD-related infections. 1.3 General Outline of the Thesis The structure of the thesis is composed by five chapters. Chapter 1 addresses the context, scope, main objectives, and organization of this thesis. Chapter 2 presents the state of the art encompassing theoretical knowledge about COPD, the relevance of NTHi in COPD, and the role of biofilms in the development of the disease. In addition, it addresses the increasing resistance to antibiotics by bacteria 3 and the role of antimicrobial peptides to treat COPD infections. Chapter 3 describes the procedures, the strains, the growth media, and the main material used to out the experiments. Chapter 4 reveals the results obtained during the experiments performed and their discussion. Finally, Chapter 5 contains the main conclusions and suggested future work. 4 Chapter 2. State of the Art Chapter 2 presents the definition of COPD, describes the two pathologies associated with this disease, its origin, and the acute periods of the disease. Furthermore, it gives theorical knowledge about the bacteria involved in the COPD related infections and the strategical mechanisms that allow them to survive in the hosts. This section also addresses the impact of antimicrobial resistance in COPD exacerbation treatment as well as the promise laying in antimicrobial peptides to treat them. 2.1 Chronic Obstructive Pulmonary Disease (COPD) 2.1.1 Definition Chronic Obstructive Pulmonary Disease (COPD) is a common progressive disease that is currently considered the most prevalent chronic respiratory disease worldwide (Frazer, 2020; Hatipoglu, 2018). It is one of the leading causes of death in the world, presenting high rates of mortality and morbidity, being an economically expensive public health problem (Brody et al., 2020; Frazer, 2020; GOLD, 2021). In this disease, structural (central airways, peripheral airways, lung parenchyma, and pulmonary vasculature) and functional (gas exchange, inflation, and airflow) changes occur in the lungs, being mainly characterized by airway obstruction and persistent airflow limitation (Davidson & Bai, 2005; Papandrinopoulou et al., 2012). Generally, these limitations are caused by excessive mucus production, thickening of airway walls resulting from edema or muscle hypertrophy, and structural changes in lung tissue, such as loss of lung elasticity and tissue destruction (Frazer, 2020; Papandrinopoulou et al., 2012; Vogelmeier et al., 2017). COPD includes two disorders, namely chronic bronchitis and emphysema. Chronic bronchitis is caused by changes in the mucus-secreting system (Weinberger et al., 2019). In this case, there is an increase in mucus-secreting glands and goblet cells responsible for bronchial secretions. This increase induces thickening of the airway walls and excessive production of mucus causing blockage of the lumen (Papandrinopoulou et al., 2012). In addition, the bronchial walls show cell infiltration and fibrosis resulting from the inflammatory process (Weinberger et al., 2019). In turn, emphysema is a pathology characterized by destruction of the parenchyma (alveolar walls), enlargement of air spaces distal to the terminal bronchiole, loss of elastin, and abnormalities in the formation of the lung elastic fiber (Mecham, 2018; Weinberger et al., 2019). In this condition, as there is gradual destruction of the alveoli, gas exchange is compromised, which can lead to aggravated airflow obstruction (Weinberger et al., 2019). 5 Although airflow obstruction has different origins in these two pathologies, patients often have the characteristics of both. Beyond the damage caused to the lungs, COPD may be associated with extrapulmonary dysfunctions (Jaitovich & Barreiro, 2018). For example, it has been shown that modifications in brain function may be caused by COPD due to airway obstruction, hypoxia, and inflammatory mediators (M. Yin et al., 2019). Furthermore, changes and destruction of lung tissue increase the vulnerability of patients with COPD to develop heart diseases (André et al., 2019). COPD also contributes to skeletal muscle dysfunction that causes high rates of hospitalizations. This dysfunction arises from muscle atrophy and changes in fibers, metabolism, and anatomy (Jaitovich & Barreiro, 2018). 2.1.2 Causes and Symptoms COPD is a complex and heterogeneous disease that can be caused by multiple factors such as smoking, environmental pollution, and genetic factors (Weinberger et al., 2019). Currently, smoking is a common cause of death by COPD (Kim et al., 2019). In 2030, 27% of deaths from COPD will be caused by smoking (Anzueto et al., 2015). Smoking causes serious damage to the bronchi, bronchioles, and pulmonary parenchyma. Besides that, there is overproduction of mucus and development of inflammatory processes mediated by inflammatory cells (leukotriene B4, interleukin-8, and tumor necrosis factor-α) that contribute to tissue degradation (Weinberger et al., 2019). These factors and the release of reactive oxygen species (ROS) contribute to the progression of the disease. Due to smoking habits, alterations in the structure of the bronchioles (small airways) and the appearance of fibrosis occur. Consequently, it severely hinders the passage of air, which in COPD patients is worrisome (Weinberger et al., 2019). Environmental pollution has a major impact on public health. It is considered an important risk factor that can cause or aggravate COPD (Jiang et al., 2016). The main risk factors associated with COPD are prolonged exposure to industrial pollution, traffic, and the combustion of fuels (Manisalidis et al., 2020). Exposure to air pollutants may also cause exacerbations of the disease and increased COPD morbidity and mortality (Manisalidis et al., 2020). In fact, air pollutants, such as carbon monoxide, nitrogen oxide, sulfur dioxide, ground-level ozone, and particulate matter pollution, have adverse effects on this disease (Jiang et al., 2016). Moreover, volatile organic compounds, dioxins, and polycyclic aromatic hydrocarbons also contribute to the worsening of the disease (Manisalidis et al., 2020). According to the literature, genetic factors also contribute to the development of COPD (Weinberger et al., 2019). Deficiency in the α1-antitrypsin glycoprotein is the most well-known genetic factor that causes COPD (Silverman, 2020). As stated by Weinberg and collaborators, α1-antitrypsin is produced in the liver and circulates in the blood. In normal conditions, α1-antitrypsin is an enzyme that inhibits the action of 6 serine proteases, protecting the lungs from their harmful effects (for example, emphysema with shortness of breath, coughing, and wheezing) (Wise & Hopkins, 2020). This protein is encoded by the SERPINA1 gene. When this gene is altered, there are modifications in the protein's structure, in its production, and its liberation. People with this genetic inheritance have reduced levels of α1-antitrypsin in the blood, and the protein may have an abnormal structure, which induces dysfunctions. The ZZ genotype is considered the most important form of α1-antitrypsin deficiency and is highly associated with premature development of emphysema (Weinberger et al., 2019). In addition to the causes mentioned, COPD may also be associated with advancing age. Despite not being a fully clarified subject, successive damage throughout life due to exposure to agents and the aging of the airways and lung parenchyma can contribute to COPD. Furthermore, there is a propensity for the development of COPD when lung infections occur in childhood, and the factors that affect lung growth from birth to adolescence also contribute to increase the probability of developing this disease (GOLD, 2021). Symptoms caused by COPD have a major impact on patients’ life quality and well-being (Vogelmeier et al., 2020). According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), dyspnea is the symptom that is most evident in COPD (GOLD, 2021). This disabling symptom is characterized by difficulty in breathing and is associated with anxiety (Anzueto & Miravitlles, 2017; Vogelmeier et al., 2020). Usually, the first symptom that appears in COPD is chronic cough (GOLD, 2021). Other symptoms, such as prolonged sputum production, are associated with inflammatory mediators that are indicative of bacterial exacerbation. In situations of aggravated illness, it may be related to fatigue, anorexia, and loss of weight. Depression also appears to be associated with COPD, which contributes to worsening health status and greater susceptibility to exacerbations and hospitalizations (GOLD, 2021; Vogelmeier et al., 2020). 2.1.3 Epidemiology in the World, Europe, and Portugal Nowadays, it is estimated that 328 million people suffer from COPD worldwide. This illness is considered a "silent killer” in lowand middle-income countries (LMICs), accounting for over 90% of deaths (Quaderi & Hurst, 2018). A rise in COPD is expected over the next 40 years due to the continuous exposure to risk factors, increase in smoking in developing countries, and the aging of the world population. Even more serious is that, in 2060, there may be more than 5.4 million annual deaths from COPD (GOLD, 2021). In the past, COPD was predominantly a disease that affected men (Aryal et al., 2014). However, nowadays the prevalence of COPD is equal in men and women. This change may be due to the increase 7 in tobacco consumption by women (GOLD, 2021). In addition, women's emancipation and cultural changes have led to women being more exposed to risk factors (Aryal et al., 2014). In terms of susceptibility, some studies report that women are more susceptible than men (GOLD, 2021). However, the relationship between genre, COPD, and susceptibility is complex, since biological and hormonal mechanisms can influence the way the disease manifests itself (Aryal et al., 2014). In Europe, studies on COPD are scarce and there is a lack of knowledge about the disease (Gibson et al., 2013). A study carried out in 19 European countries on people over 40 years of age estimates that there is a prevalence of COPD of 12.4%. However, this percentage may not be correct due to the absence of information and irregularities in the distribution of data across Europe (Blanco et al., 2018). Another study estimates that, in Europe, there is a predominance of COPD between 4% and 10% (Miravitlles et al., 2016). According to the European Lung Foundation (ELF) and the European Respiratory Society (ERS), prolonged exposure to risk factors in the workplace is responsible for 15% to 20% of cases. Recent studies reveal that, in the European Union, the prevalence of COPD in men and women was not significantly different, which demonstrates an increase in the prevalence of COPD in the female population. In addition, there is evidence that COPD is a critical health problem for women, with more and more cases of COPD among women (Ntritsos et al., 2018). According to the Portuguese Lung Society (Sociedade Portuguesa do Pulmão - SPP), in Portugal, COPD is one of the main causes of death, affecting approximately 800 thousand people. In addition, it has a major impact on public health and is considered the chronic respiratory disease with the highest mortality rates. It is estimated that one in seven Portuguese over 40 years of age has COPD. In 2016, 20.7% of deaths from respiratory disease were caused by COPD (SPP, 2019). In 2017, it was responsible for 2 627 deaths, representing 2.4 % of total deaths in the country. The disease affected more men than women, and about 95% of deaths occurred in people over 65 years (INE, 2019). In 2018, there were 2 834 deaths, and the mortality increased by 7.9 % compared to the previous year (INE, 2020). In Portugal, COPD cases have been on the rise and, as this disease is underdiagnosed, the number of people who have COPD may be much higher (Munhá, 2020). Besides, the diagnosis is often made late and, consequently, when patients turn to the doctor, the disease is already at a very advanced stage. Thus, it is extremely important to detect the disease in the early stages, which will allow to control and slow the disease progression. For this, it is necessary to raise awareness and inform individuals about the existence of COPD, its associated risk factors, and associated symptoms (Simão & Carvalho, 2018). The truth is that this chronic respiratory disease is not given due importance despite its severity. It is urgent that 14 Table 1 - Functional categories controlled by QS (Grandclément et al., 2016). Functional categories Examples Cell maintenance and proliferation Exoenzymes production, siderophores synthesis, sporulation, acid resistance Cell behaviours Biofilm formation and dispersal, motility, adhesion Horizontal gene transfer Plasmid conjugation, competence Interactions with host and other microbes Virulence factors, exopolysaccharide production, bioluminescence, antibiotics, host colonization factors The properties of autoinducers and the response they induce in coordinating population behaviors ensure bacterial survival and propagation in natural environments where a variety of bacterial species coexist (Federle & Bassler, 2003; P. Smith & Schuster, 2019). Intraspecies and interspecies communication is carried out by autoinducers (Sheela et al., 2019). In intraspecies communication, only the species of bacteria that produces the autoinducer can detect and respond to it. Intraspecies QS has differences between Gram-positive and Gram-negative bacteria. In Gram-negative bacteria, the QS system specifically uses the acyl-homoserine lactone (AHL) autoinducer. AHL is produced and freely diffused into and out of the cell and only members of the same species recognize and respond to the peptide. In Grampositive bacteria, QS is mediated by oligopeptides or autoinducing peptides (AIPs) that are transported to the extracellular medium via oligopeptide transporters because the bacterial membrane is not permeable to AIPs. This type of communication with bacteria of the same species is relevant when they live together with several species of bacteria and in an environment with similar chemical structures, as it allows them to distinguish themselves from other species, assess their numbers, and coordinate particular behaviors of the species. In addition to the species-specific QS communication system, all bacteria have the interspecies QS communication system (Federle & Bassler, 2003). Autoinducer-2 (AI-2) is one of the main signaling molecules in the bacterial QS process with the ability to control many processes, such as the production of virulence factors, biofilm formation and motility. These molecules are produced by Grampositive and Gram-negative bacteria and participate in the regulation of gene expression and physiological behaviors of bacteria in interspecies communication (Armbruster et al., 2011; Sheela et al., 2019). This type of communication is evident when some species cannot produce their own autoinducers (AI-2), however they have receptors for the autoinducers of other species (Windsor, 2020). Bacteria can also produce AI-2 through protein synthesis, but it is not detected by the receptor and can be used to regulate physiological behaviors of other bacteria (J. Zhao et al., 2018). QS communication plays a facilitating role in several functions, such as protection from toxins, nutrient starvation response, competition with other bacteria for resources and limited space, and 15 survival, as well as establishing symbiosis (Nadell et al., 2016; Sheela et al., 2019). In bacterial cooperation, production by bacteria of extracellular products, costly to produce and shareable for the entire population (public goods) coordinated by QS, can benefit neighboring cells in biofilm communities (K. Zhao et al., 2019). Examples of public goods include extracellular enzymes important for nutrient digestion, biosurfactants that promote bacterial motility to the substrate, and toxins that damage host tissue to obtain nutrients. Other examples of public goods are exopolysaccharides that provide structure and protection to biofilms, siderophores that capture iron from the environment, as well as AIs described above (P. Smith & Schuster, 2019). The production of molecules by bacteria are not always considered public goods and may establish competitive relationships. For example, bacterial QS can be blocked by inhibiting the production, delivery, or detection of AIs. Blockade of QS is performed by enzymes, such as lactonases or acylases, which inactivate the AI and consequently interrupt cell-to-cell communication. Other competitive relationships relate to competition for nutrients mainly for iron, production of antibiotics by bacteria (bacteriocins), such as piocin, and production of molecules to kill or interfere with microbial growth, as well as transfer of toxins by physical contact (Welp & Bomberger, 2020). 2.4 Biofilms in COPD Chronic respiratory diseases, including COPD, are often associated with the formation of polymicrobial biofilms (Scoffield & Wu, 2019; Welp & Bomberger, 2020). Polymicrobial biofilms are complex and dynamic mixed species communities that are constantly evolving (F. Harrison et al., 2020; Welp & Bomberger, 2020). Patients with COPD have the ideal conditions for biofilm formation due to impaired ciliary clearance system and inflammatory hyperplasia of mucus secretion. NTHi is the bacterium that initiates the process of colonization and attachment to respiratory epithelial cells. In human lungs, this microorganism can survive in the mucus-rich environment and has available hemin and NAD essential for its survival. It has an arsenal of adhesins, such as type TfP and OMP P1, that bind to the ICAM-1 protein and the CEACAM1 glycoprotein located on the host cell surface (Figure 1) that allow the adhesion and invasion of lung cells (Figure 3). The production and development of EPS by aggregates of NTHi allows the entry of new bacterial species. The polymicrobial consortium develops microcolonies with water and oxygen channels due to the development of complex intraspecific and interspecific interactions, changes in metabolism and gene expression that drive the water and oxygen gradient. The quorum sensing system allows the dispersion of the biofilm with consequent dissemination and colonization of new sites. This cycle causes 16 the infection to persist and become chronic, leading to tolerance and resistance to antibiotics and to the host's immune system (Weeks et al., 2021). Figure 3 - Life cycle of biofilm in COPD. (A) Biofilm formation begins with the reversible attachment of NTHi to a suitable surface of the airliquid interface of the tracheobronchial respiratory mucosa composed of columnar epithelium predominated by ciliated epithelial cells interspersed with secretory cells and basal cells. (B) Irreversible fixation occurs due to the presence of NTHi TfP and OMP P1 adhesins that bind to specific proteins (ICAM-1, CEACAM-1, CLEC7A) present in respiratory epithelial cells. NTHi also has adhesins that bind to mucus proteins such as mucin and lactoferrin. (C) The production of EPS by NTHi favors the acquisition of secondary colonizers. (D) Biofilm develops with the entry of secondary colonizers. The species present initiate complex intraspecific and interspecific interactions, and changes occur in gene expression and metabolism. As a result, nutrient and oxygen gradients are formed, and bacterial differentiation takes place. The biofilm matures and nutrient and water channels are formed. (E) Bacteria disperse in planktonic form or in biofilm due to the quorum sensing system. (F) The process ends with the colonization of suitable substrates (Weeks et al., 2021). In a polymicrobial biofilm, ecological interactions, for example, synergism, commensalism, mutualism, competition, etc., between bacterial species or strains are extremely important for the functioning of the biofilm ecosystem, as well as for the expression of virulent or persistent phenotypes. The diversity of the polymicrobial lung microbiome may favor interspecific interactions between bacteria. These interspecific interactions can be indirect due to biotic and abiotic changes that favor the proliferation of secondary colonizers in airway diseases. For example, NTHi infection increases inflammation that can result in microbial changes and COPD exacerbations. Furthermore, is also increases airway obstruction and the production of a viscous substrate by upregulating MUC2, which favors the secondary colonization of bacteria over time (Weeks et al., 2021). Interspecific interactions can also be more direct when there is the establishment of multi-species biofilms that cooperate in substrate adhesion and stabilize the biofilm 17 structure, such as the multi-species biofilms of NTHi and S. pneumoniae that together produce and share an EPS. NTHi and S. pneumoniae often co-colonize the respiratory tract of patients with COPD, interacting synergistically, promoting initial adhesion to the substrate, biofilm formation, and survival. Thus, together, these bacteria develop an EPS matrix made up of TfP, LOS, eDNA, QS signals, proteins, and carbohydrates essential in cooperative adhesion and stability of the biofilm structure (Kyd et al., 2016; Weeks et al., 2021). In addition, H. influenzae produces -lactamases that protect S. pneumoniae from treatment with -lactams. However, they also establish a competitive relationship because, as S. pneumoniae develops, the pH of the biofilm decreases and there is production of a biocide, hydrogen peroxide, which kills NTHi (Kyd et al., 2016). Despite killing NTHi, the production of this biocide in the long term stimulates the production of neutrophils that together make a selective pressure of persistent and ROS-tolerant strains of NTHi. Furthermore, hydrogen peroxide is a source of nutrients and DNA due to bacterial death and senescent host cells that maintain the biofilm (Weeks et al., 2021). M. catarrhalis , responsible for 10% of exacerbations in COPD, also favors NTHi colonization after infection due to biotic changes. Specifically, its binding to epithelial cilia reduces the frequency of their beats, compromising mucociliary clearance with consequent formation of mucus plugs that are colonized by NTHi. In otitis media (OM) models, the highly active catalase production by M. catarrhalis protects NTHi from the bactericidal effect of S. pneumoniae hydrogen peroxide (Bair & Campagnari, 2020). The presence of M. catarrhalis in the COPD lung could have the same effect. As interactions play an important role in biofilm stability, treatment of polymicrobial biofilm infections should be focused on these interactions (F. Harrison et al., 2020). COPD prevention and treatment may require understanding microbial interactions that modify diversity and the microbial community (Welp & Bomberger, 2020). However, many of the microbial interactions in the respiratory tract are still unknown because the presence of mixed species in chronic infections is often confirmed through PCR and sequencing. These techniques do not consider the spatial organization of the bacterial community, so crucial information about the composition of the biofilm aggregate, the spatial organization, and the possible interactions between different species may be lost (Kvich et al., 2020). Recently, two innovative methods for the rapid diagnosis of H. influenzae biofilms were created. One of the methods is the identification of H. influenzae through molecular imaging in real time due to the use of an environmentally sensitive fluorophore 7-nitrobenz-2-oxa-1,3-diazole conjugated with polymyxin which fluoresces in contact with the lipid A component of Gram-negative bacteria. The other method involves similar smart probes capable of identifying cellular metabolites. Combined, these two methods may provide a selective labeling of molecules associated with H. influenzae biofilms, enabling their rapid 18 diagnosis in COPD lung and even in other conditions. Although there are studies of NTHi biofilms and their persistence during infection, they come from OM models and little information exists on these biofilms in COPD (Short et al., 2021). More research on NTHi biofilms is needed in order to characterize the biofilm formation capacity and the bacteria-host interactions that will allow selecting anti-biofilm therapeutic targets helping in the diagnosis and treatment of COPD patients (Weeks et al., 2021). 2.5 Antimicrobial Resistance (AMR) AMR is an emerging problem worldwide, contributing strongly to deadly bacterial infections (Jorge et al., 2019). In 2019, it is estimated that AMR was responsible for at least 1.27 million deaths worldwide and by 2050 the number is predicted to increase dramatically to 10 million annual deaths (O’Neill, 2016). AMR is a natural process in which microorganisms (such as bacteria, fungi, viruses and parasites) develop defense mechanisms to counteract the lethal effects of antimicrobials (e.g. antibiotics, antifungals, antivirals, antimalarials) giving rise to multidrug-resistant organisms (MDR) or “superbugs” (Aslam et al., 2018; CDC, 2021; WHO, 2017). However, the excessive or inappropriate use of antimicrobials accelerates the AMR process, which leads to the dissemination of these microorganisms and their resistance mechanisms (WHO, 2017). According to the WHO, antibiotic resistance is, today, one of the greatest threats to public health, development, and food security (WHO, 2020). Although antibiotics play a crucial role in the treatment of infections, due to their overuse and inadequate use, it is becoming a problem worldwide (Jorge et al., 2019). Worryingly, if antibiotics lose their effectiveness altogether, the ability to treat infections and public health will be compromised (CDC, 2020). The main causes of the emergence of antimicrobial resistance are: (1) the excessive and unnecessary use of antibiotics in livestock, fish farming, and agriculture; (2) overconsumption of antibiotics due to medical overprescription, self-medication, incorrect antibiotic use, and over-the-counter accessible antibiotics; (3) lack of standard guidelines for antibiotic use; (4) poor infection control in hospitals and clinics; (5) lack of rapid laboratory tests; (6) lack of development of new antibiotics; (7) lack of hygienization and sanitation practices; (8) access to counterfeit drugs; (9) release of unmetabolized antibiotics or their residues into the environment (Aslam et al., 2018; CDC, 2019b; Jorge et al., 2019). There are four general mechanisms of bacterial resistance, which are (1) limited uptake of a drug, for example in gram-negative bacteria, the limitation of drug permeability due to the presence of an outer membrane, or by mutations in the number, type and size of porins present in the outer membrane, which may be restricted the entry of hydrophobic antibiotics such as β-lactams, fluoroquinolones, tetracyclines, 19 and chloramphenicol. In gram-positive bacteria, such as S. aureus , there is greater peptidoglycan synthesis with more D-Ala-D-Ala residues that bind to the antibiotic vancomycin, preventing its binding to the target site (C Reygaert, 2018; Christaki et al., 2019; Lowy, 2003); (2) drug target modification. Changes occur in the antibiotic binding targets present in bacterial cells, due to mutations, such as changes in penicillin binding proteins (PBP) that confer resistance to β-lactam antibiotics or by enzymatic action, such as 23SrRNA methylation that confers cross-resistance to macrolides, and lincosamides (C Reygaert, 2018; Christaki et al., 2019); (3) drug inactivation by degradation. For example, the destruction of the antibiotic by enzymes , such as β-lactamases , or by the transfer of a chemical group, called transferases (e . g ., acetyl, phosphoryl, and adenyl groups) (C Reygaert, 2018); and (4) drug efflux systems with the function of pumping toxic molecules out of the cell to regulate the internal bacterial environment (Christaki et al., 2019). In addition, antibiotic resistance can also be performed through mechanisms of horizontal gene transfer through plasmids, transposons or integrons or being an integral part of bacterial chromosomal DNA (M. Kyd et al., 2011). Recently, there has been an increasing development of bacterial resistance to the main antibiotics used to treat infections (Aslam et al., 2018; Short et al., 2021). This resistance compromises the effectiveness of the treatment and leads to the selection of resistant subpopulations that can make COPD exacerbations even more persistent and severe. For example, in exacerbations, the overuse and widespread use of antibiotics for the treatment of patients in primary and secondary health care contributes largely to the resistance of bacteria (Beasley et al., 2012; CDC, 2019a; Short et al., 2021). Antibiotic treatment is not necessary in all patients with COPD and avoiding its unnecessary use is important to limit the development of antimicrobial resistance (MacLeod et al., 2021). Antibiotics should be prescribed in case of moderate or severe exacerbation and, according to the GOLD parameters, patients should have three symptoms: increased dyspnea, sputum volume, and sputum purulence; having two symptoms, one of which is increased sputum purulence; or needing ventilation (invasive and non-invasive) (GOLD, 2021; Nissly & Prasad, 2014; Siddiqi & Sethi, 2008). The main antibiotics used in these situations are the advanced macrolides (azithromycin, clarithromycin), ketolide (telithromycin), cephalosporin (cefuroxime, cefpodoxime or cefdinir), doxycycline, trimethoprim/sulfamethoxazole, fluoroquinolone (moxifloxacin, gemifloxacin, levofloxacin), and amoxicillin/clavulanate (Siddiqi & Sethi, 2008). A systematic review to determine the prevalence, patterns, risk factors, and consequences of AMR in COPD revealed that at least one of the three bacteria H. influenzae , S. pneumoniae , and M. catarrhalis present in COPD patients exhibits high levels of resistance to at least one of the antibiotics 20 amoxicillin, doxycycline, and clarithromycin, which may contribute to treatment failure and emergence of resistance (D. Smith et al., 2022). According to GOLD, 2022, treatment of azithromycin and clarithromycin for one year in patients prone to exacerbations reduces the risk of exacerbations compared with usual care (GOLD, 2022). However, long-term treatment with macrolides, namely azithromycin and clarithromycin, can increase bacterial resistance to this type of antibiotics (Y. Cui et al., 2018). Macrolide resistance can be caused by modification of macrolide target sequences, as well as macrolide efflux pump systems (Djamin et al., 2020). In addition, ketolides can also be inhibited by the AcrAB efflux pump present in H. influenzae , and the overproduction of efflux pumps by P. aeruginosa in COPD contributes to resistance mechanisms acting on the inhibition of fluoroquinolones, -lactams, aminoglycosides, and polymyxin B (M. Kyd et al., 2011). Acquisition or expression of β-lactamases is a common resistance mechanism that causes βlactamic ring destruction of β-lactam antibiotics. H. influenzae , M. catarrhalis , and P. aeruginosa , produce this enzyme and, therefore, the use of these antibiotics, such as amoxicillin, to treat COPD related infections is not recommended. In H. influenzae , inhibition of β-lactams occurs either through the production of β-lactamases or via the alteration of PBPs. Most strains only have one mechanism, but many strains have both and are known to be β-lactamase-positive amoxicillin clavulanate-resistant. Also, the alterations in porins, mentioned above, may be involved in the resistance mechanisms in COPD, where, for example, the alteration in porin 2 present in H. influenzae partially contributes to the resistance to ampicillin, penicillin, cephalothin, and chloramphenicol (M. Kyd et al., 2011). In view of the resistance mechanisms expressed by bacteria, namely the production of βlactamases, alteration of membrane permeability, efflux pump systems, and mutations in microbial targets, the selection of antimicrobial therapy for exacerbations is a challenge since it is necessary to obtain microbial clearance and avoidance of microbial stimulation (M. Kyd et al., 2011; Short et al., 2021). To circumvent this adversity, mucolytic agents are currently being prescribed on a large scale by clinicians as they decrease mucus viscosity and increase its fluidity, helping with microbial clearance (Papi et al., 2020; Weeks et al., 2021). Specifically, erdosteine can be used as an adjunct to therapy in COPD as it has a significant potentiating effect of antibiotics against chronic respiratory infections (Papi et al., 2020). 2.6 The Role of H. influenzae Biofilms in AMR The formation of biofilms is a form of AMR and is considered a recurring problem due to their persistence in infections (Jorge et al., 2019; Welp & Bomberger, 2020). Biofilms protect bacteria and 21 make them tolerant to antibiotics, thus hampering the clinical treatment of a biofilm related infection (Melton & Anderson, 2019). In biofilms, due to poor growth conditions, it is common to find tolerance in resident bacteria. Tolerance can be defined as the ability of the microorganism to survive a brief exposure to high antibacterial concentrations. Therefore, the antibiotic takes longer to kill the bacteria. This happens because biofilms have a gradient of oxygen and nutrients, which makes the bacteria that are in the lower layers have less access to them. This phenomenon induces these bacteria to reduce their metabolic activity and enter a slow growth or state of dormancy, giving rise to the persister cell phenotype. When a bacterial subpopulation has a persistent phenotype, although the antimicrobial can eliminate the other cells, the subpopulation survives the treatment and can grow again, causing the infection to relapse. This persistence phenotype is one of the main causes of the inefficiency of some antimicrobials (Jorge et al., 2019). In fact, a proteomics study indicated that H. influenzae biofilms in a semi-dormant state have reduced cellular metabolism and protein synthesis (Post et al., 2014). This altered cellular activity enhances the limited diffusion of drugs through the biofilm ECM components, and the decreased protein synthesis minimizes the activity of protein synthesis inhibitor antibiotics, such as macrolides (Short et al., 2021). In biofilms, the matrix that surrounds them also contributes to their high resistance/tolerance to external stresses, namely the action of antimicrobials and the immune system (Jorge et al., 2019). The production of a thick ECM by H. influenzae contributes to resistance to neutrophils and NETs, blocks macrophage access to biofilm cells, thus preventing phagocytosis, and aids in cleavage of immunoglobulin (IgA), which is considered a major component of the immune system and a first-line defense mechanism of the mucosal surface, via IgA proteases. The eDNA belonging to the ECM is also an important component of antibiotic resistance, because it stimulates biofilm formation and binds to human β-defensins present in greater amounts in COPD compared to healthy individuals, reducing their antimicrobial properties (Short et al., 2021). H. influenzae is tolerant to imipenem and, in a biofilm community, this tolerance can be strengthened due to the differential behavior of biofilm cells, such as fluctuating gene expression and cell metabolism that can lead to the development of heteroresistant cell phenotypes and persistence (Short et al., 2021). Interestingly, β-lactam antibiotics, probably due to a stress response, induce the formation of H. influenzae biofilms. Furthermore, in polymicrobial biofilms, M. catarrhalis has the ability to secrete β-lactamases, which confer protection to H. influenzae from ampicillin treatment, and the signaling molecule AI-2 from H. influenzae induces resistance of M. catarrhalis to clarithromycin and trimethoprimsulfamethoxazole (Jorge et al., 2019). The synergetic interaction of the in vitro polymicrobial biofilm of 22 both H. influenzae and S. pneumoniae can also promote protection from the effects of antibiotics, since the production of β-lactamases by H. influenzae and the production of ECM by both bacteria were able to protect S. pneumoniae biofilms from amoxicillin treatment in chinchillas’ middle ear in OM models (Short et al., 2021). NTHi biofilms in COPD lung are currently being studied, but most available information on the biofilm life of these bacteria is given through OM studies or in vitro bacterial monoculture models (Weeks et al., 2021). NTHi biofilms exhibit a series of mechanisms of resistance and tolerance to several commonly used antibiotics (Table 2). For example, in OM, the antibiotics ciprofloxacin, azithromycin, and amoxicillin, which are clinically effective for planktonic states of NTHi, are ineffective for NTHi biofilms. In addition, the effect of the antibiotics chlorhexidine, glucuronate, ampicillin, and ciprofloxacin is also ineffective due to the rich eDNA matrix (Weeks et al., 2021). A recent study with a COPD model of ferret lung exposed to cigarette smoke showed that NTHi bacteria have the ability to aggregate and express genes related to growth, QS, and environmental and oxidative stress tolerance, which induces formation and persistence of NTHi biofilms (Hunt et al., 2020). Thus, with the increase in AMR, it is necessary to find new ways to combat these versatile and adaptable microorganisms. Therefore, it is essential to study and explore the mechanisms of bacterial resistance, characterize the biofilm formation capacity of NTHi, identify the host-pathogen interactions in COPD, as well as anti-biofilm therapeutic targets. These strategies can have a positive impact on diagnosis and treatment, not only in COPD, but also in terms of other diseases, with the ultimate goal of minimizing this greatest source of mortality and morbidity (C Reygaert, 2018; Weeks et al., 2021). 23 Table 2 - Summary of NTHi biofilm resistance and tolerance mechanisms and their respective model and experimental effects (adapted from Weeks et al., 2021). Mechanism and Drug Model/Experiment Experimental effects Citation Gentamycin tolerance Clinical CF1 NTHi isolates adhered to human airway epithelia Biofilms survived treatment with high gentamycin concentration. (Starner et al., 2006) β-lactam antibiotics & carbohydrate metabolism NTHi biofilms grown on airway epithelia Increase in carbohydrate metabolism gene expression in response to sub-MIC ampicillin and amoxicillin. (Wu et al., 2014) eDNA-rich EPS matrix protects against antimicrobials 8 clinical NTHi isolates static biofilms in vitro Biocide resistance mediated by the cohesive and protective properties of the biofilm matrix. (Izano et al., 2009) Low metabolic activity protects biofilms against βlactam antibiotics Metabolomic and proteomic analysis of 814 proteins across biofilm and planktonic strains. 127 differentially expressed products suggested that NTHi biofilms survive βlactam antibiotics in a dormant state with decreased energy metabolism and protein synthesis. (Post et al., 2014) 2.7 AMPs against bacteria Bacterial resistance to antibiotics, which are one of the main ways of treating infections, is a major concern in modern medicine. Currently, the slow pace of discovery and development of new antibiotics in the face of the rapid spread of resistance by these microorganisms to these drugs causes serious impacts on the economy and public health (Aslam et al., 2018; Hernández-Aristizábal & Ocampo-Ibáñez, 2021). Therefore, it is urgent to find new antimicrobial drugs against bacteria. AMPs are multifunctional molecules of the innate immune system of prokaryotic and eukaryotic organisms that act against infections caused by bacteria, fungi, viruses, and some protozoa (HernándezAristizábal & Ocampo-Ibáñez, 2021; Wu et al., 2018). Generally, in nature, the different types of AMPs are constituted by four, six, or eight cysteines interconnected by disulfide bridges that confer stability, have between 12 and 50 amino acids, α-helical structure, are mostly cationic, and show amphiphilicity (Denardi et al., 2022; Hernández-Aristizábal & Ocampo-Ibáñez, 2021; H. Wang et al., 2022). The α1 CF – Cystic Fibrosis 30 ANOVA and two-way ANOVA using Bonferroni's multicomparison test. The significant differences between the groups are represented in the graphs by the asterisk (*) or the cardinal (#) symbols, where */# equals p < 0.05, **/## equals p < 0.001, ***/### equals p < 0.001, and ****/#### equals p < 0.0001. 31 Chapter 4. Results and Discussion Chapter 4 is composed of the description and depiction of the results obtained, their critical analysis as well as their discussion. 4.1 Impact of media renewal on the growth of H. influenzae biofilm To study the impact of growth media renewal in the biofilm formation of H. influenzae , 48 h biofilms were formed, with and without media replacement at 24 h. Based on the analysis of Figure 4, it is possible to verify that, when there is no replacement of media at 24 h, there are no culturable bacteria at 48 h. On the other hand, when the medium is replenished at 24 h, there are culturable bacteria at 48 h. The results suggest that sBHI medium replacement affects the amount of culturable bacteria present in the biofilm, since there are statistically significant differences between H. influenzae counts without sBHI medium replacement compared to H. influenzae counts with sBHI medium replacement (p < 0.0001) (Figure 4). These results are in agreement with those previously obtained by our research group (data not published). Figure 4 - Quantification of culturable H. influenzae in 48 h biofilms grown in sBHI (Liofilchem), with and without media replacement at 24 h. On the graph, standard deviations are indicated by error bars. Statistically significant differences: **** p<0.0001. The development of biofilms and their bacterial and final chemical composition are affected by the amount, availability, and type of nutrients and by their perfusion to the cells inside of the biofilm (Bowden & Li, 1997; Dunne, 2002). Hence, the absence of culturable cells observed could be explained by bacterial unviability caused by consumption and consequent depletion of nutrients by the bacteria over time, such as the growth factors hemin and NAD. H. influenzae cells are not well adapted to survival under non-optimal conditions, requiring an enriched culture medium, namely BHI, with adequate supplementation of hemin and NAD (Poje & Redfield, 2003). 32 The toxic metabolites produced by the bacteria themselves are also another factor that could explain the absence of culturable bacteria. Dunne describes that, in bacterial biofilms, bacterial growth is limited by the expression of quorum-sensing molecules released in response to nutrient limitation and the accumulation of toxic by-products (Dunne, 2002). H. influenzae is subject to oxidative stress resulting from its own metabolism during aerobic growth (Harrison et al., 2012). Oxidative stress can cause damage to all types of bacterial cell components including proteins, lipids, and DNA, leading to bacterial death (Ezraty et al., 2017; Fasnacht & Polacek, 2021). A third hypothesis to explain the absence of culturable cells is that H. influenzae could be viable but not culturable (VBNC). In the VBNC state, the biofilm bacteria are alive but do not grow or divide, and their presence is not detectable by conventional methods (they do not form colonies on solid media and do not change the appearance of the broth) (Năşcuţiu, 2010). The VBNC state is a unique survival strategy in the face of adverse environmental conditions such as antibiotic pressure, high or low temperature, starvation, chlorination, pH change, and oxygen stress (Ramamurthy et al., 2014). It is known that VBNC has been associated with longer periods of biofilm formation and, furthermore, the central areas of the same tend to be hypoxic, with limited resources, and acidic due to the deposits of metabolic residues that consequently induce the VBNC state (Ayrapetyan et al., 2018; Castro et al., 2022). The VBNC state can be reversible under favorable growth conditions with an ideal energy source and stoichiometric ratio of carbon to inorganic elements (Ramamurthy et al., 2014). 4.2 Influence of Growth Media Brand in H. influenzae Biofilm Formation Previous assays within the research group found behavioral variations in the growth of H. influenzae . The assays were highly consistent for about 5 months, but variability started to be more worrisome afterwards (data not published). Based on Chapter 3 of the book " Haemophilus influenzae protocols", growing H. influenzae on cheaper growth media can cause these growth problems as well as plating problems (Poje & Redfield, 2003). As such, the influence of media brand on H. influenzae biofilm growth was tested. 4.2.1 H. influenzae Cryovial Variability in Different Brands of Growth Media Surprisingly, the variability detected in the growth of H. influenzae by the research group was suspected to correlate with different cryovials of the same strain. Although all the vials were prepared and stocked at the same time, repeated use of some could be compromising the cells. For this reason, the differential growth between two cryovials was tested, one of which was already in use and the other was opened for the first time. In addition, it was also investigated whether the variability in the growth of these 33 cryovials was dependent or not on the brand of growth media used, namely, Oxoid (most expensive), VWR (average price), and Liofilchem (cheaper and used in all previous studies within the group). For this study, 24 h and 48 h biofilms of H. influenzae (with and without media replacement) were formed in sBHI of different brands. The sBHI from VWR was discarded at the beginning of the assay because HI 2 ( H. influenzae from cryovial 2, in use) did not grow in it, hence no comparison was performed for this brand. However, this was in itself an interesting result, since it was possible to see that there is in fact differences in bacterial growth dependent on the media brand and the cryovial used. 4.2.1.1 24 h Biofilms Figure 5 illustrates the results of culturable cells and total biomass obtained in the cultivation of 24 h H. influenzae biofilms. It is possible to observe that, in sBHI from Oxoid, the number of culturable cells (Figure 5-A) as well as the amount of total biomass of the biofilm (Figure 5-B) is similar for the two cryovials. In contrast, with the sBHI from Liofilchem, HI 2 has fewer culturable cells (1 log) (p < 0.01) (Figure 5-A) and drastically lower biomass (p < 0.0001) (Figure 5-B) compared to HI 6 ( H. influenzae from cryovial 6, newly opened). In addition, it is observed that the Oxoid brand reached a higher number of culturable cells compared to the Liofilchem brand (Figure 5-A) and, in terms of biomass (Figure 5-B), sBHI from Oxoid performed equal for HI 6 and better for HI 2. Figure 5 - Quantification of culturable H. influenzae cells (A) and total biomass (B) of 24 h H. influenzae biofilms grown in sBHI from Oxoid and Liofilchem. Standard deviations are indicated by error bars. Statistically significancy: ** p < 0.001 and **** p < 0.0001. Abbreviations: HI 2 = H. influenzae from the second cryovial (in use), HI 6 = H. influenzae from the sixth cryovial (newly opened). The growth differences observed suggest that the proliferation of H. influenzae may be dependent on the brand of sBHI used, which could be related to its higher or lower quality. The results also showed that the amount of biomass (Figure 5-B) does not directly correlate with the amount of culturable cells, since HI 6 produced a similar amount of biomass in both brands but had a lower amount of culturable 34 cells when grown in sBHI from Liofilchem (Figure 5-A). This similarity in the amount of biomass in both brands can be explained by the CV method which, in addition to quantifying viable cells, also quantifies dead cells and extracellular matrix (Xu et al., 2016). Interestingly, the amount of biomass differed significantly for the Liofilchem brand. This phenomenon can be explained by the greater production of extracellular matrix of HI 6 compared to HI 2, even though they are of the same strain. This could indicate that HI 2 suffered modifications derived from its use that caused cells to lower their ability to produce matrix. Additionally, it was verified that biofilms with 24 h have culturable cells of H. influenzae . Thus, the problems mentioned in the previous section (4.1), such as lack of nutrients and production of toxic metabolites by H. influenzae that can affect the culturability of the biofilm, are not yet present in biofilms cultured for 24 h. 4.2.1.2 48 h Biofilms without Media Renewal Figure 6 shows the same comparison as before but for 48 h biofilms without media renewal at 24 h. Compared to 24 h H. influenzae biofilms, the amount of culturable cells (Figure 5-A) reduced for all conditions, except for HI 2 with the sBHI from Liofilchem (Figure 6-A). This could indicate that the cell growth of vial HI 2 was slower than that of vial HI 6 in Liofilchem and, therefore, they managed to grow a little more from 24 h to 48 h, as they could still be at the end of the exponential phase or in the stationary phase. In the other three conditions, as the cells could have grown faster, they could have already reached the dead phase or in the biofilm detachment stage, resulting in lower cell culturability at 48 h. For sBHI from Oxoid, the amount of culturable HI 2 cells was 2 logs lower compared to HI 6 (p < 0.05) (Figure 6-A) but the amount of biomass was identical for both cryovials (Figure 6-B). Furthermore, Figure 6-A shows that there is a significant difference (p < 0.01) in the number of culturable cells between HI 2 and HI 6 for sBHI from Liofilchem. This can be explained by the same reason described above. In terms of biomass, there is a significant reduction (p < 0.0001) between the two cryovials (Figure 6-B) that was also observed in the 24 h H. influenzae biofilms (Figure 5-B) and discussed above. It is noteworthy that the amount of culturable cells and biomass differs between brands and cryovials, except for the amount of biomass in cryovials of sBHI from Oxoid (Figure 6-B). 35 Figure 6 - Quantification of culturable H. influenzae (A) and total biomass (B) of 48 h H. influenzae biofilms grown in sBHI from Oxoid and Liofilchem without media replacement at 24h. Standard deviations are indicated by error bars. Statistically significancy: * p < 0.05 ** p < 0.01, and **** p < 0.0001. Abbreviations: HI 2 = H. influenzae from the second cryovial (in use), HI 6 = H. influenzae from the sixth cryovial (newly opened). Interestingly, culturable cells of these 48 h biofilms without media renewal were observed for both brands (Figure 6-A), contrary to the initial experience (Figure 4), which was performed with sBHI from Liofilchem. This confirmed the variability previously observed by the research group and eliminated the operator as the possible causing factor. This event can be related to the universal stress protein (UspA). Although lack of sBHI replacement is associated with loss of culturability probably due to deficiency of nutrients and production of toxic metabolites, as discussed previously, it is known that H. influenzae can survive due to increased expression of UspA. According to the literature, when bacteria are exposed to stressful environmental conditions that compromise cell viability, UspA expression is increased, enhancing the bacterial survival rate (Sousa & McKay, 2001). In this case, the probable stress caused by nutrient depletion and production of reactive oxygen species resulting from its own metabolism, which could compromise cell viability, may have induced an increase in UspA expression. 4.2.1.3 48 h Biofilms with Media Renewal Finally, Figure 7 shows the same comparisons as before but for 48 h biofilms with media renewal at 24 h. It is possible to observe that the amount of culturable H. influenzae cells for sBHI from Oxoid was significantly higher (p < 0.001) (Figure 7-A) in vial HI 2 than in vial HI 6. Regarding total biomass, there was similar production for both vials (Figure 7-B). The amount of culturable H. influenzae cells for sBHI from Liofilchem was also higher (p < 0.001) (Figure 7-A) in HI 2 than in HI 6. In contrast, the amount of biomass produced was significantly lower (p < 0.0001) (Figure 7-B) in HI 2 compared to HI 6. This difference in the quantification of biomass between cryovials for sBHI from Liofilchem (p < 0.0001) (Figure 7-B) has also been observed previously (Figure 5-B and 6-B). 36 Figure 7 – Quantification of culturable H. influenzae (A) and total biomass (B) of 48 h H. influenzae biofilms grown in sBHI from Oxoid and Liofilchem with media replacement at 24 h. Standard deviations are indicated by the error bars. Statistically significancy: *** p<0.001 and **** p<0.0001. Abbreviations: HI 2 = H. influenzae from the second cryovial (in use), HI 6 = H. influenzae from the sixth cryovial (newly opened). Comparing the results of 48 h biofilms without (Figure 6) and with media renewal (Figure 7), it is possible to verify an increase in the number of culturable cells for both cryovials (Figure 7-A) and total biomass (Figure 7-B) for sBHI from Oxoid. As verified in the initial experiment (Figure 4), the replacement of sBHI at 24 h showed again that the restitution of nutrients and the possible removal of toxic metabolites could be what is here influencing cell culturability. In conclusion, it was found that there were behavioral differences between HI 2 and HI 6 cryovials, even though they were from the same strain and established at the same time. Thus, it was seen that vial HI 2, which had already been opened and consequently subjected to more temperature variations, due to its freezing and thawing, had a slower bacterial growth compared to the vial HI 6, which was opened for the first time to be used in this experiment. Furthermore, H. influenzae growth differences were dependent on the brand of growth medium used, and variability and poorer/slower growth and biomass formation were most noticeable for sBHI from Liofilchem. The results of sBHI from Oxoid were more consistent among the cryovials tested. Thus, the Oxoid brand was chosen to carry on the remainder assays. 4.3 Population Dynamics in Dual-species Biofilms In biofilms, the interaction of bacteria with each other is inevitable. These interactions can be cooperative or competitive, and can influence the growth and survival of species, having a spatial and temporal impact on the formation of a highly organized community (Giaouris et al., 2015). Throughout the development of the biofilm, microorganisms are distributed in a non-random manner depending on 37 social interactions and nutrient availability (W. Liu et al., 2018; Paula et al., 2020). In a multispecies biofilm, there are interactions that benefit the whole group, such as the availability of synthesized compounds and the escape of host defenses, but there are also harmful interactions, such as species elimination due to competition from different coexisting species (Reigada et al., 2021). Investigation of population dynamics is essential for understanding interactions between species, making possible the selection of drugs that can inhibit interactions between species and their ability to form multispecies biofilms (Tikhomirova & Kidd, 2013a). 4.3.1 Population Dynamics of Dual-Species Biofilms in Growth Media of Different Brands Given the variability observed previously regarding the use of different media brands in the growth of H. influenzae biofilms, a preliminary assay was performed to check if the media brand had the same effects when S. aureus was mixed with H. influenzae . In this way, 24 h and 48 h H. influenzae biofilms (single-species control) and H. influenzae biofilms with 1% of S. aureus (starting inoculum %) were formed in sBHI from Liofilchem and from Oxoid. 4.3.1.1 24 h Biofilms The results of 24 h H. influenzae biofilms grown in sBHI from Liofilchem show statistically significantly lower culturable cells when compared to Oxoid (p < 0.0001) (Figure 8-A), in contrast to the amount of biomass, which is similar) (Figure 8-B). This fact occurs for the same reasons explained in the previous study, namely the greater probability of sBHI from Liofilchem to cause growth problems. In terms of total biomass, although equal values were obtained, biofilm composition may be different, as it may contain a greater amount of dead cells for sBHI from Liofilchem due to the lower culturable cells counts. The results of H. influenzae biofilms grown with 1% S. aureus showed no significant difference in the number of culturable cells (Figure 8-A) or biomass (Figure 8-B) between the two brands. Interestingly, the amount of culturable H. influenzae cells was higher in the double-species biofilm when compared with the single-species control for sBHI from Liofilchem (Figure 8-A). Thus, S. aureus may have a positive influence on H. influenzae growth, possibly by providing the necessary nutrients to maintain the culturability of H. influenzae cells. In fact, H. influenzae and S. aureus were already reported to establish a cooperative relationship in polymicrobial infections, in which H. influenzae reached a greater number of colonies when S. aureus was the resident colonizer due to nutrients that the latter provided, such as hemin and NAD (Nair et al., 2014). In this case, the mechanism for obtaining nutrients by H. influenzae was different since sBHI does not have erythrocytes for the hemolysins produced by S. aureus to cause 38 their lysis and release hemin and NAD. In terms of total biomass, this was maintained for all biofilms in the different brands. However, its composition may vary because, for the reasons explained previously. Figure 8 - Quantification of culturable H. influenzae (A) and total biofilm biomass (B) from 24 h biofilms of H. influenzae (HI) and of H. influenzae with 1% of S. aureus (1% SA) grown in sBHI from Liofilchem and Oxoid. Statistically significant differences: **** p < 0.0001. Standard deviations are indicated by error bars. 4.3.1.2 48 h Biofilms without Media Renewal In the 48 h H. influenzae biofilms without sBHI replacement, a greater number of culturable cells were obtained for sBHI from Oxoid (p < 0.0001) (Figure 9-A). As for the quantification of the total biomass, the H. influenzae biofilms cultivated in sBHI from Liofilchem reached the highest amount of biomass (p < 0.0001) (Figure 9-B), but they were the ones that obtained the lowest number of culturable cells (Figure 9-A), therefore it can be inferred that the amount of biomass comes mainly from dead cells and/or matrix. In biofilms with two species, the number of culturable H. influenzae cells was lower for sBHI from Liofilchem (p < 0.001) (Figure 9-A), as well as the total biomass (p < 0.0001) (Figure 9-B). Figure 9 - Quantification of culturable H. influenzae (A) and total biofilm biomass (B) from 48 h biofilms of H. influenzae (HI) and of H. influenzae with 1% of S. aureus (1% SA) grown in sBHI from Liofilchem and Oxoid without media replacement at 24 h. Statistically significant differences are represented by *** p < 0.001 and **** p < 0.0001. Standard deviations are indicated by error bars. 39 In this case, it is again notorious that the presence of S. aureus resulted in a greater amount of culturable H. influenzae cells. As described above, S. aureus can promote the culturability of H. influenzae due to nutrient supply. It is also important to note that when S. aureus is present, the amount of biomass is lower in sBHI from Liofilchem compared to the single-species control. This may be due to S. aureus making H. influenzae produce less matrix, for example. Comparing H. influenzae biofilms at 24 h (Figure 8-A) and at 48 h without media renewal, it was observed that there was a significant decrease in cell culturability, namely for sBHI from Liofilchem (approximately 3 logs) as well as from Oxoid (approximately 1.5 logs) (Figure 9-A). Furthermore, while in the 24 h double-species biofilms (Figure 8-A), the number of culturable cells was similar for the different brands, in this case, the number of culturable cells was lower (approximately 1 log) for Liofilchem (Figure 9-A). Thus, it appears that the lack of sBHI renewal impaired the culturability of H. influenzae . In the quantification of biofilm biomass (Figure 9-B), it was shown that, over the 48 h without sBHI replacement, the amount of biomass produced was lower for both biofilms in comparison to the 24 h biofilms (Figure 8-B). Furthermore, the amount of biomass showed more variability between the Liofilchem and Oxoid brands in the 48 h biofilms (Figure 9-B), which did not happen in the 24 h biofilms (Figure 8-B). Results once again show that the quality of the growth media coupled with the lack of sBHI replacement can influence H. influenzae growth. This is mainly shown when the 24 h and 48 h biofilms are compared. For example, when H. influenzae biofilms are cultivated in sBHI from Liofilchem, they often reach a lower number of culturable cells in relation to sBHI from Oxoid and, when they are cultivated for a period of 48 h without media replacement, they tend to decrease cell culturability. Interestingly, S. aureus positively influenced the amount of culturable H. influenzae cells, which leads to the inference that S. aureus may play an important role in providing nutrients necessary for the development of H. influenzae . 4.3.1.3 48 h Biofilms with Media Renewal In the 48 h H. influenzae biofilms with media renewal at 24 h, the number of culturable cells was higher for sBHI from Oxoid (p < 0.01) (Figure 10-A). Regarding the amount of biomass, there was a higher production for sBHI from Liofilchem (p < 0.0001) (Figure 10-B) and, as previously explained, it may come from dead cells and/or matrix, since there was less cells cultured. For double-species biofilms, the number of culturable H. influenzae cells (p < 0.01) (Figure 10-A) and the amount of biomass (p < 0.001) (Figure 10-B) was greater for sBHI from Oxoid. Furthermore, it was observed that the H. influenzae biofilm had a higher amount of biomass than the double-species biofilm (Figure 10-B). 46 the double-species biofilm had a higher biomass production compared to the 24 h single-species control (p < 0.0001) (Figure 15-B). Figure 15 - Quantification of culturable H. influenzae and S. aureus cells (A) and quantification of biomass (B) from double-species biofilms in which S. aureus was the second colonizer for 24 h. Controls correspond to 48 h H. influenzae and 24 h S. aureus single-species biofilms. The statistical differences are depicted as: * p < 0.05 and **** p < 0.0001. Standard deviations are indicated by error bars. The insertion of S. aureus into the pre-established H. influenzae biofilm after 24 hours had no influence on adhesion or biofilm formation because the number of culturable cells obtained was similar to the controls and the biomass was similar to the first colonizer. Given that S. aureus is known to be able to co-colonize with other bacteria, namely with H. influenzae , when it was introduced into the preestablished biofilm, it may have developed a cooperative relationship with H. influenzae (Nair et al., 2014). Although colonization of the secondary species had no significant effects on the existing biofilm of the other species, Esin and colleagues found that pre-colonization by H. influenzae promoted adhesion and biofilm formation of S. aureus in tympanostomy tubes (TTs), suggesting that there is a process active and synergistic in which H. influenzae promotes the attachment of S. aureus , such as cell-to-cell signaling, which is a critical early step in biofilm formation (Esin et al., 2015). In conclusion, although the literature describes that the pre-establishment of the biofilm by one species can promote the adhesion, biofilm formation, and growth of other bacteria, in this study, it was seen that there was no influence of the colonizer species on the adhesion and biofilm formation of another species. 47 4.5 Interspecies Influence of Exoproducts on Biofilm Formation Biofilm-forming bacteria can release EPS such as polysaccharides, small amounts of proteins, DNA, and other molecules into the surrounding environment that can help or harm other bacteria present in the biofilm, affecting their evolutionary dynamics (Nadell et al., 2008). In addition, through the QS cellcell communication system, there is the production and release of AIs that provide a platform for intraand interspecies crosstalk and the control of diverse bacterial activities, such as the formation and development of the biofilm, secretion of virulence factors, and survival in a constantly changing environment (Sahreen et al., 2022). CFS is the liquid resulting from the removal of bacterial cells and contains metabolites from bacterial growth (some listed above) and residual nutrients from the medium used and has been studied mainly due to its antimicrobial potential against bacteria. Even though there is no literature about the effect of S. aureus and H. influenzae CFS on H. influenzae and S. aureus biofilms, respectively, some experiments with CFS against these strains have been done. For example, in a study by Coleman and colleagues, on the in vitro ability of alpha haemolytic streptococcus (AHS) and lactobacilli (LBs) from indigenous Australian children to inhibit the growth of respiratory pathogens, it was shown that LBs-free cells were effective in inhibiting H. influenzae , reducing the initial concentration by 3 logs (Coleman et al., 2022). Another study on the use of extracellular extracts of lactic acid bacteria (LAB) and bifidobacteria for the inhibition of S. aureus demonstrated that CFS, mainly produced by LAB, drastically inhibited the growth of the pathogen S. aureus (Hor & Liong, 2014). On the other hand, exoproducts can also benefit the biofilm. A study by Hou et al. found that the biofilm of an EPS-producing S. aureus ATCC 12600 exhibits a higher resistance to mechanical pressure than that of a non-EPS-producing S. aureus 5298, due to an immediate increase in polysaccharide content (Hou et al., 2018). Another example includes the reduction of penetration by some antibiotics into the S. aureus biofilm due to exoproducts (Yin et al., 2019). Additionally, the EPS matrix formed by H. influenzae provides a protective barrier that allows them to survive the stress caused by antimicrobial agents and the host's immune response (Tikhomirova & Kidd, 2013b). In order to study the influence of exoproducts from one species on biofilms of another species, CFS from H. influenzae was added to 48 h S. aureus biofilms at 24 h and vice versa. The results show that the amount of culturable cells from HI biofilms with SA CFS and from SA biofilms with HI CFS was slightly higher (p < 0.0001) (Figure 16) when compared to the respective controls. However, these differences are not biologically significant, given the value proximity. 48 Figure 16 – Effect of SA CFS on HI culturable biofilm cells (A) and of HI CFS on SA culturable biofilm cells (B). The statistically significant differences are represented as: **** p < 0.0001. Standard deviations are indicated by error bars. The bacterial viability of the 48 h biofilms may be related to the addition of CFS.This could be due to the reduction in the number of cells competing for nutrients, as a result of removing planktonic cells and adding CFS from 24 h biofilms that still have nutrients and EPS of the species (such as polysaccharides, proteins, eDNA, and lipids) that enhance the overall structure of the biofilm (Di Martino, 2018; Mani-López et al., 2022). Compared to the controls, the exoproducts of S. aureus and H. influenzae did not have a negative or positive influence on H. influenzae and S. aureus biofilms, respectively (Figure 16). This may have occurred because exoproducts produced by biofilms during 24 h may not be at a high enough concentration to have an effect on the amount of culturable cells. Another factor is that S. aureus and H. influenzae biofilms may simply not be susceptible to the exoproducts produced by the other species. 4.6 Susceptibility H. influenzae and S. aureus to AMPs The susceptibility of H. influenzae and S. aureus to four synthetic AMPs, namely temporin A, tachyplesin I, palm-KGK-PEG, and citropin 1.1, was assessed by the MIC and MBC (Table 4). Results show that the two species have different patterns of susceptibility to the chosen AMPs. Tachyplesin I showed the best bacteriostatic and bactericidal activity against both species. In H. influenzae , the second most active AMP was palm-KGK-PEG, followed by citropin 1.1 and, lastly, temporin A. In S. aureus , after tachyplesin I, temporin A was the second best, followed by palm-KGK-PEG, and finally citropin 1.1. To ensure that the assays were well performed, ciprofloxacin, which acts against both species, was used as a control. 49 Table 4 – Antimicrobial activity of temporin A, tachyplesin I, palm-KGKPEG, citropin 1.1, and ciprofloxacin against H. influenzae DSM 4690 (ATCC 33391, NCTC 8143) and S. aureus (ATCC 25923). MIC and MBC are expressed in mg/L. H. influenzae S. aureus AMPs/Antibiotic MIC MBC MIC MBC Temporin A 64 1024 16 128 - 256 Tachyplesin I 32 128 16 - 32 16 - 32 Palm-KGK-PEG 64 128 - 256 32 - 64 64 - 128 Citropin 1.1 64 - 256 256 - 512 64 - 128 128 Ciprofloxacin < 0.015 0.06 - 0.125 0.001 > 0.002 According to EUCAST tabulated breakpoint values, the MIC values of ciprofloxacin indicate that H. influenzae and S. aureus are not resistant to this antibiotic (EUCAST, 2022b). This is in accordance to what is described for these strains , hence the assays were considered well executed (Chan et al., 2017; Kashef et al., 2020). All AMPs under study have their mechanism of action targeting the bacterial membrane (Alves et al., 2016; Boland & Separovic, 2006; C. Liu et al., 2018; Mangoni & Shai, 2009). Tachyplesin I was the AMP that showed the greatest efficacy against H. influenzae and S. aureus , which might be explained by the fact that, in addition to targeting the bacterial membrane, it also acts at the intracellular level, targeting DNA (C. Liu et al., 2018; Yonezawa et al., 1992; Yu et al., 2020). For tachyplesin I, the MIC values were high (32 mg/L and 16 – 32 mg/L for H. influenzae and S. aureus , respectively) (Table 4) compared to MIC values found in the literature (0.8 - 12.5 mg/L) for Gram-negative ( H. influenzae ) and Gram-positive ( S. aureus ) bacteria (Xue et al., 2018). Repeated AMP freezing and defrosting cycles to make aliquots and its previous use can have made the AMP susceptible to degradation, contributing to lower antimicrobial activity and consequently higher MIC values (GenScript, 2022). On the other hand, it is possible that these strains may be resistant to tachyplesin I. However, this hypothesis would have to be confirmed through other assays. Temporin A showed greater antimicrobial activity against S. aureus compared to H. influenzae . This result is consistent with the literature, since temporins are mainly effective and act quickly against Gram-positive bacteria. The activity against S. aureus presents MIC values (16 mg/L) within those mentioned in the literature, since generally the MIC of temporins ranges from 2.5 to 20 mg/L for Gram- 50 positive bacteria (Mangoni & Shai, 2009). For H. influenzae , the MIC value was substantially lower than what is published in the literature for Gram-negative bacteria (100-400 mg/L) (Rosenfeld et al., 2006). Citropin 1.1 is considered to have a broad spectrum of action against both Gram-positive and Gramnegative bacteria (Boland & Separovic, 2006). In this study, citropin 1.1 demonstrated greater antibacterial activity against the Gram-positive bacteria S. aureus . In susceptibility researches with S. aureus (ATCC 25923), citropin 1.1 showed greater antimicrobial activity with lower MIC and MBC values (16 mg/L) (Jorge et al., 2017; Neubauer et al., 2017) than the values obtained here (MIC of 64 - 128 mg/L and MBC of 128 mg/L). The problems that may be associated with the higher MIC and MBC values obtained are the same as those previously reported for the AMP tachyplesin I. Palm-KGK-PEG is a pegylated lipopeptide that demonstrated better antibacterial activity against S. aureus compared to H. influenzae . According to the literature, Palm-KGK-NH2 (without PEG) has a potent antibacterial activity and is an excellent alternative to antibiotics since it has a low propensity for inducing microbial resistance (Alves et al., 2016; Avrahami & Shai, 2004). In addition, the association of AMP with PEG has the advantage of increasing water solubility, protecting the peptide constituents from protease degradation, producing larger conjugates that avoid rapid renal filtration to prolong circulation in the bloodstream, and further decreasing the potential effects of cytotoxicity against mammalian cells (Z. Cui et al., 2021). Similar activity of this lipopeptide without PEG against S. aureus was found in previous studies where, moreover, the activity against Gram-positive bacteria was also stronger compared to Gramnegative bacteria (Alves et al., 2016; Paduszynska et al., 2019). Given that tachyplesin I demonstrated the best bactericidal and bacteriostatic activity against H. influenzae and S. aureus , it was considered the best AMP of the four AMPs tested. 51 Chapter 5. Conclusions and Future Work COPD, a chronic, progressive, and incurable disease, is the third leading cause of mortality worldwide. The main causes of this disease are smoking, pollution and genetic deficiency of alpha-1 antitrypsin. Exacerbations, which are characterized by a worsening of COPD symptoms, occur recurrently and are mainly caused by bacterial infections. These infections are dangerous because they encompass the formation of polymicrobial biofilms, usually composed of NTHi with other bacteria, that often resist the administered antibiotics. The ineffectiveness of these drugs leads to a reduction in the quality of life of patients, comorbidities, and high mortality rates. As the presence of several species can aggravate bacterial resistance and infection resolution, knowledge on the bacterial interactions taking place is urgent to design better treatments. This study examined some of the growth conditions of H. influenzae and S. aureus biofilms, both isolated and combined, including the effect of renewal the growth media and the influence of the different brands of growth media on the formation of biofilms. It also investigated the dynamics of bacterial interactions that occur within the double-species biofilms namely the impact of sequential colonization and of exoproducts, and the susceptibility of both species to AMPs. The results showed that the lower number of culturable H. influenzae cells in 48 h biofilms without sBHI renewal at 24 h was probably due to nutrient depletion, given that when media is replaced at 24 h, the number of culturable cells is higher, but toxic metabolites produced by the bacteria itself or the existence of VBNC could also be involved. The results also revealed differences in the growth of H. influenzae when different media brands were used, which may be due to the quality of the medium, and that the repeated use of HI cryovials may impact bacterial quality and cause lower growth rates. It was also found that the growth of H. influenzae , both in single and in double-species biofilms, had less variability when it was grown in sBHI from Oxoid, making this the chosen brand for the following assays. Regarding the dynamics of double-species biofilms with different initial concentrations of S. aureus , it was highlighted that H. influenzae and S. aureus appear to improve each other’s culturability, but most differences encountered were small. Although it is reported in the literature that the density of H. influenzae increases when S. aureus is the previous colonizer, in this case, the colonizer species had no influence on the formation of the biofilm of the other species. Additionally, the results also showed that the exoproducts of one species had no influence on the formation of the biofilms of the other species, which could be due to the concentration of the exoproduct not being enough to trigger an effect, or the cells of the biofilms not be susceptible to the exoproducts produced by the other species. Finally, of the four AMPs tested, tachyplesin I showed the best bacteriostatic and bactericidal action against H. 52 influenzae and S. aureus , thus being more promising for further development of treatments against the double-species consortia. Hopefully, the investigation of the growth conditions and of the interactions between these two species conducted in this work will provide the research team with valuable insights for the development of antimicrobial agents to treat infections caused by these bacteria. To enhance the research conducted in this thesis, future work should include the discrimination of the microbial population of biofilms, namely viable, non-viable, and VBNC cells, for example through flow cytometry. Further investigations may be carried out, such as the identification of extracellular metabolites produced by biofilms when cultured with and without sBHI renewal, by mass spectrometry (MS). Another fundamental point would be the analysis of the spatial arrangement of the different species in the biofilm, where the fluorescence in situ hybridization (FISH) technique could be used. For a more realistic study, biofilms could be grown in artificial mucus that mimics the mucus in the airways. It would be interesting to compare all the characterizations indicated above between the different growth media (sBHI and artificial mucus). Since, in chronic infections, most biofilms form small aggregates of cells not attached to a surface but embedded in host material, the biofilms, in addition to being formed on microtiter plates, could also be formed on alginate beads (Sønderholm et al., 2017). Alginate beads represent a simple and flexible model of in vivo biofilms. This would allow us to understand biofilm formation in different structures: microtiter plates (adhered biofilm) vs alginate granules (aggregated biofilm). Finally, for the development of a possible drug, it would be essential to predict resistance for generations of H. influenzae and S. aureus to tachyplesin I. Resistance is tested for bacterial generations because a bacterial cell that is exposed to an antimicrobial agent can develop resistance and transmit this resistance to her descendants through her genetic material. By testing resistance in several generations of bacteria, it will be possible to determine how easily resistance is transmitted and how quickly it spreads within a population. In this way, the susceptibility test for bacterial generations could be performed. Given the significance of biofilms in this context, it would also be important to study the susceptibility of these bacteria to tachyplesin I when they are in single and double biofilms in order to gain a comprehensive understanding of how they are affected by this promising AMP. 53 References Ahearn, C. P., Gallo, M. C., & Murphy, T. F. (2017). Insights on persistent airway infection by non-typeable Haemophilus influenzae in chronic obstructive pulmonary disease. In Pathogens and disease (Vol. 75, Issue 4, p. 42). Oxford Academic. https://doi.org/10.1093/femspd/ftx042 Alves, D., Magalhães, A., Grzywacz, D., Neubauer, D., Kamysz, W., & Pereira, M. O. (2016). Coimmobilization of Palm and DNase I for the development of an effective anti-infective coating for catheter surfaces. Acta Biomaterialia , 44 , 313–322. https://doi.org/10.1016/j.actbio.2016.08.010 Amaro, B. (2022). COPD associated pneumonia: polymicrobial biofilms and new antimicrobial combinations . (master´s thesis). University of Minho, Braga. Retrieved from https://www.ceb.uminho.pt/Publications/Details/55563 André, S., Conde, B., Fragoso, E., Boléo-Tomé, J. P., Areias, V., & Cardoso, J. (2019). COPD and Cardiovascular Disease. Pulmonology , 25 (3), 168–176. https://doi.org/10.1016/j.pulmoe.2018.09.006 Anzueto, A., Heijdra, Y., Hurst, J. R., Anzueto, A., Heijdra, Y., & Hurst, J. R. (2015). Controversies in COPD . European Respiratory Society. https://doi.org/10.1183/2312508X.10018914 Anzueto, A., & Miravitlles, M. (2017). Pathophysiology of dyspnea in COPD. In Postgraduate Medicine (Vol. 129, Issue 3, pp. 366–374). Taylor and Francis Inc. https://doi.org/10.1080/00325481.2017.1301190 Armbruster, C. E., Pang, B., Murrah, K., Juneau, R. A., Perez, A. C., Weimer, K. E. D., & Swords, W. E. (2011). RbsB (NTHI_0632) mediates quorum signal uptake in nontypeable Haemophilus influenzae strain 86-028NP. Molecular Microbiology , 82 (4), 836. https://doi.org/10.1111/J.13652958.2011.07831.X Aryal, S., Diaz-Guzman, E., & Mannino, D. M. (2014). Influence of sex on chronic obstructive pulmonary disease risk and treatment outcomes. In International Journal of COPD (Vol. 9, pp. 1145–1154). Dove Medical Press Ltd. https://doi.org/10.2147/COPD.S54476 Aslam, B., Wang, W., Arshad, M. I., Khurshid, M., Muzammil, S., Rasool, M. H., Nisar, M. A., Alvi, R. F., Aslam, M. A., Qamar, M. U., Salamat, M. K. F., & Baloch, Z. (2018). Antibiotic resistance: a rundown of a global crisis. In Infection and Drug Resistance (Vol. 11, pp. 1645–1658). Dove Medical Press Ltd. https://doi.org/10.2147/IDR.S173867 Avrahami, D., & Shai, Y. (2004). A New Group of Antifungal and Antibacterial Lipopeptides Derived from Non-membrane Active Peptides Conjugated to Palmitic Acid. Journal of Biological Chemistry , 54 279 (13), 12277–12285. https://doi.org/10.1074/JBC.M312260200 Awokola, B. I., Amusa, G. A., Jewell, C. P., Okello, G., Stobrink, M., Finney, L. J., Mohammed, N., Erhart, A., & Mortimer, K. J. (2022). Chronic obstructive pulmonary disease in sub-Saharan Africa. The International Journal of Tuberculosis and Lung Disease , 26 (3), 232. https://doi.org/10.5588/IJTLD.21.0394 Ayrapetyan, M., Williams, T., & Oliver, J. D. (2018). Relationship between the Viable but Nonculturable State and Antibiotic Persister Cells. Journal of Bacteriology , 200 (20). https://doi.org/10.1128/JB.00249-18 Bahar, A. A., & Ren, D. (2013). Antimicrobial Peptides. Pharmaceuticals , 6 (12), 1543. https://doi.org/10.3390/PH6121543 Bair, K. L., & Campagnari, A. A. (2020). Moraxella catarrhalis Promotes Stable Polymicrobial Biofilms With the Major Otopathogens. Frontiers in Microbiology , 10 . https://doi.org/10.3389/FMICB.2019.03006/FULL Baltzer, S. A., & Brown, M. H. (2011). Antimicrobial Peptides – Promising Alternatives to Conventional Antibiotics. Microbial Physiology , 20 (4), 228–235. https://doi.org/10.1159/000331009 Batoni, G., Maisetta, G., & Esin, S. (2016). Antimicrobial peptides and their interaction with biofilms of medically relevant bacteria. Biochimica et Biophysica Acta (BBA) - Biomembranes , 1858 (5), 1044– 1060. https://doi.org/10.1016/J.BBAMEM.2015.10.013 Beasley, V., Joshi, P. V, Singanayagam, A., Molyneaux, P. L., Johnston, S. L., & Mallia, P. (2012). COPD28286-lung-microbiology-and-exacerbations-in-copd. International Journal of COPD , 2012 , 555– 569. https://doi.org/10.2147/COPD.S28286 Blanco, I., Diego, I., Bueno, P., Fernández, E., Casas-Maldonado, F., Esquinas, C., Soriano, J. B., & Miravitlles, M. (2018). Geographical distribution of COPD prevalence in Europe, estimated by an inverse distance weighting interpolation technique. International Journal of COPD , 13 , 57–67. https://doi.org/10.2147/COPD.S150853 Blasi, F., Page, C., Rossolini, G. M., Pallecchi, L., Matera, M. G., Rogliani, P., & Cazzola, M. (2016). The effect of N-acetylcysteine on biofilms: Implications for the treatment of respiratory tract infections . https://doi.org/10.1016/j.rmed.2016.06.015 Boland, M. P., & Separovic, F. (2006). Membrane interactions of antimicrobial peptides from Australian tree frogs. Biochimica et Biophysica Acta (BBA) - Biomembranes , 1758 (9), 1178–1183. https://doi.org/10.1016/J.BBAMEM.2006.02.010 Bouquet, J., Tabor, D. E., Silver, J. S., Nair, V., Tovchigrechko, A., Griffin, M. P., Esser, M. T., Sellman, 55 B. R., & Jin, H. (2020). Microbial burden and viral exacerbations in a longitudinal multicenter COPD cohort. Respiratory Research , 21 (1), 1–13. https://doi.org/10.1186/s12931-020-01340-0 Bowden, G. H., & Li, Y. H. (1997). Nutritional influences on biofilm development. Advances in Dental Research , 11 (1), 81–99. https://doi.org/10.1177/08959374970110012101 Brody, H., Hodson, R., Rooke, J., Haggart, A., Duncan, K., & Hodson, R. (2020). Chronic obstructive pulmonary disease. Nature Outlook , 581 (1). https://doi.org/https://doi.org/10.1038/d41586020-01371-z Bustamante-Marin, X. M., & Ostrowski, L. E. (2017). Cilia and mucociliary clearance. Cold Spring Harbor Perspectives in Biology , 9 (4). https://doi.org/10.1101/cshperspect.a028241 C Reygaert, W. (2018). An overview of the antimicrobial resistance mechanisms of bacteria. AIMS Microbiology , 4 (3), 482–501. https://doi.org/10.3934/microbiol.2018.3.482 Castro, J., Lima, Â., Sousa, L. G. V., Rosca, A. S., Muzny, C. A., & Cerca, N. (2022). Crystal Violet Staining Alone Is Not Adequate to Assess Synergism or Antagonism in Multi-Species Biofilms of Bacteria Associated With Bacterial Vaginosis. Frontiers in Cellular and Infection Microbiology , 11 . https://doi.org/10.3389/FCIMB.2021.795797/FULL CDC. (2019a). Antibiotic Resistance and NARMS Surveillance | NARMS | CDC . https://www.cdc.gov/narms/faq.html CDC. (2019b). CDC Global Health - Infographics - Antibiotic Resistance The Global Threat . https://www.cdc.gov/globalhealth/infographics/antibioticresistance/antibiotic_resistance_global_threat.htm CDC. (2020). About Antibiotic Resistance | Antibiotic/Antimicrobial Resistance | CDC . https://www.cdc.gov/drugresistance/about.html CDC. (2021). About Antibiotic Resistance | CDC . https://www.cdc.gov/drugresistance/about.html Chan, C. L., Richter, K., Wormald, P. J., Psaltis, A. J., & Vreugde, S. (2017). Alloiococcus otitidis forms multispecies biofilm with Haemophilus influenzae: Effects on antibiotic susceptibility and Growth in Adverse Conditions. Frontiers in Cellular and Infection Microbiology , 7 (AUG), 344. https://doi.org/10.3389/FCIMB.2017.00344/FULL Christaki, E., Marcou, M., & Tofarides, A. (2019). Antimicrobial Resistance in Bacteria: Mechanisms, Evolution, and Persistence. Journal of Molecular Evolution 2019 88:1 , 88 (1), 26–40. https://doi.org/10.1007/S00239-019-09914-3 Coleman, A., Håkansson, A., Grahn Håkansson, E., Cottrell, K., Bialasiewicz, S., Zaugg, J., & Cervin, A. (2022). In vitro Inhibition of respiratory pathogens by lactobacillus and alpha haemolytic 62 Polymicrobial Infections. Infection and Immunity , 82 (6), 2162. https://doi.org/10.1128/IAI.00059-14 Năşcuţiu, A.-M. (2010). Bacterii viabile necultivabile [Viable non-culturable bacteria]. Bacteriologia, virusologia, parazitologia, epidemiologia (Bucharest, Romania : 1990), 55(1), 11–18 . https://pubmed.ncbi.nlm.nih.gov/21038700/ Neubauer, D., Jaśkiewicz, M., Migoń, D., Bauer, M., Sikora, K., Sikorska, E., Kamysz, E., & Kamysz, W. (2017). Retro analog concept: comparative study on physico-chemical and biological properties of selected antimicrobial peptides. Amino Acids , 49 (10), 1755–1771. https://doi.org/10.1007/S00726-017-2473-7/TABLES/6 Nissly, T., & Prasad, S. (2014). PURLs: Should you consider antibiotics for exacerbations of mild COPD? The Journal of Family Practice , 63 (4), E11. /pmc/articles/PMC4042897/ Ntritsos, G., Franek, J., Belbasis, L., Christou, M. A., Markozannes, G., Altman, P., Fogel, R., Sayre, T., Ntzani, E. E., & Evangelou, E. (2018). Gender-specific estimates of COPD prevalence: A systematic review and meta-analysis. In International Journal of COPD (Vol. 13, pp. 1507–1514). Dove Medical Press Ltd. https://doi.org/10.2147/COPD.S146390 O’Neill, J. (2016). TACKLING DRUG-RESISTANT INFECTIONS GLOBALLY: FINAL REPORT AND RECOMMENDATIONS THE REVIEW ON ANTIMICROBIAL RESISTANCE CHAIRED BY JIM O’NEILL . Oliveira, A. S., Munhá, J., Bugalho, A., Guimarães, M., Reis, G., & Marques, A. (2018). Identification and assessment of COPD exacerbations. In Pulmonology (Vol. 24, Issue 1, pp. 42–47). Elsevier Espana S.L.U. https://doi.org/10.1016/j.rppnen.2017.10.006 Paduszynska, M. A., Maciejewska, M., Neubauer, D., Golacki, K., Szymukowicz, M., Bauer, M., & Kamysz, W. (2019). Influence of short cationic lipopeptides with fatty acids of different chain lengths on bacterial biofilms formed on polystyrene and hydrogel surfaces. Pharmaceutics , 11 (10). https://doi.org/10.3390/pharmaceutics11100506 Papandrinopoulou, D., Tzouda, V., & Tsoukalas, G. (2012). Lung compliance and chronic obstructive pulmonary disease. Pulmonary Medicine , 2012 . https://doi.org/10.1155/2012/542769 Papi, A., Avdeev, S., Calverley, P. M. A., Cordeiro, C. R., Jesenak, M., Koblížek, V., Petkova, D., Rogliani, P., Tarraf, H., Tzanakis, N., Ulmeanu, R., Uzaslan, E., & Yochai, A. (2020). Use of mucolytics in COPD: A Delphi consensus study. Respiratory Medicine , 175 , 106190. https://doi.org/10.1016/J.RMED.2020.106190 Paula, A. J., Hwang, G., & Koo, H. (2020). Dynamics of bacterial population growth in biofilms resemble spatial and structural aspects of urbanization. Nature Communications 2020 11:1 , 11 (1), 1–14. 63 https://doi.org/10.1038/s41467-020-15165-4 Pavord, I. D., Jones, P. W., Burgel, P. R., & Rabe, K. F. (2016). Exacerbations of COPD. In International journal of chronic obstructive pulmonary disease: Vol. 11 Spec (Issue Spec Iss, pp. 21–30). Dove Press. https://doi.org/10.2147/COPD.S85978 Pinto, A. M., Cerqueira, M. A., Bañobre-Lópes, M., Pastrana, L. M., & Sillankorva, S. (2020). Bacteriophages for chronic wound treatment: From traditional to novel delivery systems. Viruses , 12 (2), 1–29. https://doi.org/10.3390/v12020235 Pirrone, M., Pinciroli, R., & Berra, L. (2016). Microbiome, biofilms, and pneumonia in the ICU. Current Opinion in Infectious Diseases , 29 (2), 160–166. https://doi.org/10.1097/QCO.0000000000000255 Poje, G., & Redfield, R. J. (2003). General methods for culturing Haemophilus influenzae. Methods in Molecular Medicine , 71 , 51–56. https://doi.org/10.1385/1-59259-321-6:51 Post, D. M. B., Held, J. M., Ketterer, M. R., Phillips, N. J., Sahu, A., Apicella, M. A., & Gibson, B. W. (2014). Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry. BMC Microbiology , 14 (1), 1–16. https://doi.org/10.1186/S12866-014-0329-9/TABLES/2 Quaderi, S. A., & Hurst, J. R. (2018). The unmet global burden of COPD. In Global Health, Epidemiology and Genomics (Vol. 3, pp. 1–3). Cambridge University Press. https://doi.org/10.1017/gheg.2018.1 Ramamurthy, T., Ghosh, A., Pazhani, G. P., & Shinoda, S. (2014). Current perspectives on viable but non-culturable (VBNC) pathogenic bacteria. Frontiers in Public Health , 2 (JUL), 103. https://doi.org/10.3389/FPUBH.2014.00103/BIBTEX Reigada, I., San-Martin-Galindo, P., Gilbert-Girard, S., Chiaro, J., Cerullo, V., Savijoki, K., Nyman, T. A., Fallarero, A., & Miettinen, I. (2021). Surfaceome and Exoproteome Dynamics in Dual-Species Pseudomonas aeruginosa and Staphylococcus aureus Biofilms. Frontiers in Microbiology , 12 . https://doi.org/10.3389/FMICB.2021.672975/FULL Ritchie, A. I., & Wedzicha, J. A. (2020). Definition, Causes, Pathogenesis, and Consequences of Chronic Obstructive Pulmonary Disease Exacerbations. In Clinics in Chest Medicine (Vol. 41, Issue 3, pp. 421–438). W.B. Saunders. https://doi.org/10.1016/j.ccm.2020.06.007 Rosenfeld, Y., Barra, D., Simmaco, M., Shai, Y., & Mangoni, M. L. (2006). A Synergism between Temporins toward Gram-negative Bacteria Overcomes Resistance Imposed by the Lipopolysaccharide Protective Layer. Journal of Biological Chemistry , 281 (39), 28565–28574. 64 https://doi.org/10.1074/JBC.M606031200 Sahreen, S., Mukhtar, H., Imre, K., Morar, A., Herman, V., & Sharif, S. (2022). Exploring the Function of Quorum Sensing Regulated Biofilms in Biological Wastewater Treatment: A Review. International Journal of Molecular Sciences , 23 (17), 9751. https://doi.org/10.3390/IJMS23179751 Saxena, S., Ramnani, V. K., Nema, S., Tripathi, K., Dave, L., & Srivastava, N. (2016). Bacteriological Profile in Acute Exacerbation of Chronic Obstructive Lung Disease (AECOPD). Annals of International Medical and Dental Research , 2 (5). https://doi.org/10.21276/aimdr.2016.2.5.mb1 Scoffield, J., & Wu, H. (2019). Microbial biofilms. In Encyclopedia of Microbiology (Issue September 2016). Elsevier Inc. https://doi.org/10.1016/B978-0-12-801238-3.99205-5 Sheela, G. M., Prathyusha, A. M. V. N., Neelapu, N. R. R., & Bramhachari, P. V. (2019). Intra and interspecies communication in microbes: Living with complex and sociable neighbors. Implication of Quorum Sensing System in Biofilm Formation and Virulence , 7–16. https://doi.org/10.1007/978981-13-2429-1_2 Shimizu, K., Yoshii, Y., Morozumi, M., Chiba, N., Ubukata, K., Uruga, H., Hanada, S., Saito, N., Kadota, T., Ito, S., Wakui, H., Takasaka, N., Minagawa, S., Kojima, J., Hara, H., Numata, T., Kawaishi, M., Saito, K., Araya, J., … Kuwano, K. (2015). Pathogens in COPD exacerbations identified by comprehensive real-time PCR plus older methods. International Journal of COPD , 10 (1), 2009– 2016. https://doi.org/10.2147/COPD.S82752 Short, B., Carson, S., Devlin, A. C., Reihill, J. A., Crilly, A., MacKay, W., Ramage, G., Williams, C., Lundy, F. T., McGarvey, L. P., Thornbury, K. D., & Martin, S. L. (2021). Non-typeable Haemophilus influenzae chronic colonization in chronic obstructive pulmonary disease (COPD). Critical Reviews in Microbiology , 47 (2), 192–205. https://doi.org/10.1080/1040841X.2020.1863330 Siddiqi, A., & Sethi, S. (2008). Optimizing antibiotic selection in treating COPD exacerbations. International Journal of Chronic Obstructive Pulmonary Disease , 3 (1), 31. https://doi.org/10.2147/COPD.S1089 Silverman, E. K. (2020). Genetics of COPD. In Annual Review of Physiology (Vol. 82, pp. 413–431). Annual Reviews Inc. https://doi.org/10.1146/annurev-physiol-021317-121224 Simão, P., & Carvalho, J. (2018). DPOC – Doença Pulmonar Obstrutiva Crónica - Unidade Local de Saúde de Matosinhos . http://www.ulsm.min-saude.pt/mais-saude/dpoc-doenca-pulmonar-obstrutivacronica/ Smith, D., Gill, A., Hall, L., & Turner, A. M. (2022). Prevalence, Pattern, Risks Factors and Consequences of Antibiotic Resistance in COPD: A Systematic Review. 65 Https://Doi.Org/10.1080/15412555.2021.2000957 , 18 (6), 672–682. https://doi.org/10.1080/15412555.2021.2000957 Smith, P., & Schuster, M. (2019). Public goods and cheating in microbes. Current Biology , 29 (11), R442– R447. https://doi.org/10.1016/J.CUB.2019.03.001 Sønderholm, M., Kragh, K. N., Koren, K., Jakobsen, T. H., Darch, S. E., Alhede, M., Jensen, P. Ø., Whiteley, M., Kühl, M., & Bjarnsholt, T. (2017). Pseudomonas aeruginosa aggregate formation in an alginate bead model system exhibits in vivo-like characteristics. Applied and Environmental Microbiology , 83 (9), 113–130. https://doi.org/10.1128/AEM.00113-17 Sousa, M. C., & McKay, D. B. (2001). Structure of the Universal Stress Protein of Haemophilus influenzae. Structure , 9 (12), 1135–1141. https://doi.org/10.1016/S0969-2126(01)00680-3 SPP. (2019). Sociedade Portuguesa de Pneumonologia - SPP . https://www.sppneumologia.pt/noticias/dpo-que-portugueses-desconhecem-doenca-que-e-umadas-principais-causas-de-morte-no-nosso-pais Sriram, K. B., Cox, A. J., Clancy, R. L., Slack, M. P. E., & Cripps, A. W. (2018). Nontypeable Haemophilus influenzae and chronic obstructive pulmonary disease: a review for clinicians. In Critical Reviews in Microbiology (Vol. 44, Issue 2, pp. 125–142). Taylor and Francis Ltd. https://doi.org/10.1080/1040841X.2017.1329274 Stepanović, S., Vuković, D., Dakić, I., Savić, B., & Švabić-Vlahović, M. (2000). A modified microtiter-plate test for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods , 40 (2), 175–179. https://doi.org/10.1016/S0167-7012(00)00122-6 Su, Y. C., Jalalvand, F., Thegerström, J., & Riesbeck, K. (2018). The Interplay Between Immune Response and Bacterial Infection in COPD: Focus Upon Non-typeable Haemophilus influenzae. Frontiers in Immunology , 9 (NOV), 2530. https://doi.org/10.3389/FIMMU.2018.02530 Tikhomirova, A., & Kidd, S. P. (2013a). Haemophilus influenzae and Streptococcus pneumoniae : living together in a biofilm. Pathogens and Disease , 69 (2), 114–126. https://doi.org/10.1111/2049632X.12073 Tikhomirova, A., & Kidd, S. P. (2013b). Haemophilus influenzae and Streptococcus pneumoniae : living together in a biofilm. Pathogens and Disease , 69 (2), 114–126. https://doi.org/10.1111/2049632X.12073 van Bragt, J. J. M. H., Brinkman, P., de Vries, R., Vijverberg, S. J. H., Weersink, E. J. M., Haarman, E. G., de Jongh, F. H. C., Kester, S., Lucas, A., in ’t Veen, J. C. C. M., Sterk, P. J., Bel, E. H. D., & Maitlandvan der Zee, A. H. (2020). Identification of recent exacerbations in COPD patients by electronic 66 nose. ERJ Open Research , 6 (4), 00307–02020. https://doi.org/10.1183/23120541.00307-2020 Vogelmeier, C. F., Criner, G. J., Martinez, F. J., Anzueto, A., Barnes, P. J., Bourbeau, J., Celli, B. R., Chen, R., Decramer, M., Fabbri, L. M., Frith, P., Halpin, D. M. G., López Varela, M. V., Nishimura, M., Roche, N., Rodriguez-Roisin, R., Sin, D. D., Singh, D., Stockley, R., … Agusti, A. (2017). Global Strategy for the Diagnosis, Management and Prevention of Chronic Obstructive Lung Disease 2017 Report: GOLD Executive Summary. In Respirology (Vol. 22, Issue 3). https://doi.org/10.1111/resp.13012 Vogelmeier, C. F., Román-Rodríguez, M., Singh, D., Han, M. L. K., Rodríguez-Roisin, R., & Ferguson, G. T. (2020). Goals of COPD treatment: Focus on symptoms and exacerbations. In Respiratory Medicine (Vol. 166, p. 105938). W.B. Saunders Ltd. https://doi.org/10.1016/j.rmed.2020.105938 Wang, H., Niu, M., Xue, T., Ma, L., Gu, X., Wei, G., Li, F., & Wang, C. (2022). Development of antibacterial peptides with efficient antibacterial activity, low toxicity, high membrane disruptive activity and a synergistic antibacterial effect. Journal of Materials Chemistry B , 10 (11), 1858–1874. https://doi.org/10.1039/D1TB02852A Wang, J., Dou, X., Song, J., Lyu, Y., Zhu, X., Xu, L., Li, W., & Shan, A. (2019). Antimicrobial peptides: Promising alternatives in the post feeding antibiotic era. Medicinal Research Reviews , 39 (3), 831– 859. https://doi.org/10.1002/MED.21542 Weeks, J. R., Staples, K. J., Spalluto, C. M., Watson, A., & Wilkinson, T. M. A. (2021). The Role of NonTypeable Haemophilus influenzae Biofilms in Chronic Obstructive Pulmonary Disease. Frontiers in Cellular and Infection Microbiology , 11 . https://doi.org/10.3389/FCIMB.2021.720742 Weinberger, S. E., Cockrill, B. A., & Mandel, J. (2019). Chronic Obstructive Pulmonary Disease. In Principles of Pulmonary Medicine (pp. 93–112). Elsevier. https://doi.org/10.1016/B978-0-32352371-4.00009-X Welp, A. L., & Bomberger, J. M. (2020). Bacterial Community Interactions During Chronic Respiratory Disease. In Frontiers in Cellular and Infection Microbiology (Vol. 10, p. 213). Frontiers Media S.A. https://doi.org/10.3389/fcimb.2020.00213 WHO. (2017). Antimicrobial Resistance | WHO | Regional Office for Africa . https://www.afro.who.int/health-topics/antimicrobial-resistance WHO. (2020). Antibiotic resistance . Antibiotic Resistance. https://www.who.int/news-room/factsheets/detail/antibiotic-resistance WHO. (2021). Chronic obstructive pulmonary disease (COPD) . https://www.who.int/news-room/fact- 67 sheets/detail/chronic-obstructive-pulmonary-disease-(copd) Windsor, J. (2020). How Quorum Sensing Works . https://asm.org/Articles/2020/June/How-QuorumSensing-Works Wise, R. A., & Hopkins, J. (2020). Alpha-1 Antitrypsin Deficiency - Lung and Airway Disorders - MSD Manual Consumer Version . https://www.msdmanuals.com/en-pt/home/lung-and-airwaydisorders/chronic-obstructive-pulmonary-disease-copd/alpha-1-antitrypsin-deficiency Wu, Q., Patočka, J., & Kuča, K. (2018). Insect antimicrobial peptides, a mini review. Toxins , 10 (11), 1– 17. https://doi.org/10.3390/toxins10110461 Xu, Z., Liang, Y., Lin, S., Chen, D., Li, B., Li, L., & Deng, Y. (2016). Crystal Violet and XTT Assays on Staphylococcus aureus Biofilm Quantification. Current Microbiology , 73 (4), 474–482. https://doi.org/10.1007/s00284-016-1081-1 Xue, Q., Liu, X. Bin, Lao, Y. H., Wu, L. P., Wang, D., Zuo, Z. Q., Chen, J. Y., Hou, J., Bei, Y. Y., Wu, X. F., Leong, K. W., Xiang, H., & Han, J. (2018). Anti-infective biomaterials with surface-decorated tachyplesin I. Biomaterials , 178 , 351–362. https://doi.org/10.1016/J.BIOMATERIALS.2018.05.008 Yin, M., Wang, H., Hu, X., Li, X., Fei, G., & Yu, Y. (2019). Patterns of brain structural alteration in COPD with different levels of pulmonary function impairment and its association with cognitive deficits. BMC Pulmonary Medicine , 19 (1), 203. https://doi.org/10.1186/s12890-019-0955-y Yin, W., Wang, Y., Liu, L., & He, J. (2019). Biofilms: The Microbial “Protective Clothing” in Extreme Environments. International Journal of Molecular Sciences , 20 (14). https://doi.org/10.3390/IJMS20143423 Yonezawa, A., Kuwahara, J., Fujii, N., & Sugiura, Y. (1992). Binding of Tachyplesin I to DNA Revealed by Footprinting Analysis: Significant Contribution of Secondary Structure to DNA Binding and Implication for Biological Action. Biochemistry , 31 (11), 2998–3004. https://doi.org/10.1021/BI00126A022/SUPPL_FILE/BI00126A022_SI_001.PDF Yu, R., Wang, J., So, L. Y., Harvey, P. J., Shi, J., Liang, J., Dou, Q., Li, X., Yan, X., Huang, Y. H., Xu, Q., Kaas, Q., Chow, H. Y., Wong, K. Y., Craik, D. J., Zhang, X. H., Jiang, T., & Wang, Y. (2020). Enhanced Activity against Multidrug-Resistant Bacteria through Coapplication of an Analogue of Tachyplesin i and an Inhibitor of the QseC/B Signaling Pathway. Journal of Medicinal Chemistry , 63 (7), 3475– 3484. https://doi.org/10.1021/ACS.JMEDCHEM.9B01563/ASSET/IMAGES/LARGE/JM9B01563_000 8.JPEG 68 Zhang, Q. Y., Yan, Z. Bin, Meng, Y. M., Hong, X. Y., Shao, G., Ma, J. J., Cheng, X. R., Liu, J., Kang, J., & Fu, C. Y. (2021). Antimicrobial peptides: mechanism of action, activity and clinical potential. Military Medical Research , 8 (1). https://doi.org/10.1186/S40779-021-00343-2 Zhao, J., Quan, C., Jin, L., & Chen, M. (2018). Production, detection and application perspectives of quorum sensing autoinducer-2 in bacteria. Journal of Biotechnology , 268 , 53–60. https://doi.org/10.1016/J.JBIOTEC.2018.01.009 Zhao, K., Liu, L., Chen, X., Huang, T., Du, L., Lin, J., Yuan, Y., Zhou, Y., Yue, B., Wei, K., & Chu, Y. (2019). Behavioral heterogeneity in quorum sensing can stabilize social cooperation in microbial populations. BMC Biology , 17 (1), 1–15. https://doi.org/10.1186/S12915-019-06393/FIGURES/6