Metagenomics Applied to the Respiratory Mycobiome in Cystic Fibrosis.

Cystic fibrosis (CF) is a genetic disorder characterized by chronic microbial colonization and inflammation of the respiratory tract (RT), leading to pulmonary exacerbation (PEx) and lung damage. Although the lung bacterial microbiota has been extensively studied, the mycobiome remains understudied. However, its importance as a contributor to CF pathophysiology has been highlighted. The objective of this review is to provide an overview of the current state of knowledge regarding the mycobiome, as described through NGS-based studies, in patients with CF (pwCF).Several studies have demonstrated that the mycobiome in CF lungs is a dynamic entity, exhibiting a lower diversity and abundance than the bacterial microbiome. Nevertheless, the progression of lung damage is associated with a decrease in fungal and bacterial diversity. The core mycobiome of the RT in pwCFs is mainly composed of yeasts (Candida spp., Malassezia spp.) and molds with lower abundance. Some fungi (Aspergillus, Scedosporium/Pseudallescheria) have been demonstrated to play a role in PEx, while the involvement of others (Candida, Pneumocystis) remains uncertain. The "climax attack" ecological model has been proposed to explain the complexity and interplay of microbial populations in the RT, leading to PEx and lung damage. NGS-based studies also enable the detection of intra- and interkingdom correlations between fungi and bacteria. Further studies are required to ascertain the biological and pathophysiological relevance of these correlations. Finally, with the recent advent of CFTR modulators, our understanding of the pulmonary microbiome and mycobiome in pwCFs is about to change.

In Vitro Safety Study on the Use of Cold Atmospheric Plasma in the Upper Respiratory Tract.

Cold atmospheric plasma (CAP) devices generate reactive oxygen and nitrogen species, have antimicrobial and antiviral properties, but also affect the molecular and cellular mechanisms of eukaryotic cells. The aim of this study is to investigate CAP treatment in the upper respiratory tract (URT) to reduce the incidence of ventilator-associated bacterial pneumonia (especially superinfections with multi-resistant pathogens) or viral infections (e.g., COVID-19). For this purpose, the surface-microdischarge-based plasma intensive care (PIC) device was developed by terraplasma medical GmbH. This study analyzes the safety aspects using in vitro assays and molecular characterization of human oral keratinocytes (hOK), human bronchial-tracheal epithelial cells (hBTE), and human lung fibroblasts (hLF). A 5 min CAP treatment with the PIC device at the "throat" and "subglottis" positions in the URT model did not show any significant differences from the untreated control (ctrl.) and the corresponding pressurized air (PA) treatment in terms of cell morphology, viability, apoptosis, DNA damage, and migration. However, pro-inflammatory cytokines (MCP-1, IL-6, and TNFα) were induced in hBTE and hOK cells and profibrotic molecules (collagen-I, FKBP10, and αSMA) in hLF at the mRNA level. The use of CAP in the oropharynx may make an important contribution to the recovery of intensive care patients. The results indicate that a 5 min CAP treatment in the URT with the PIC device does not cause any cell damage. The extent to which immune cell activation is induced and whether it has long-term effects on the organism need to be carefully examined in follow-up studies in vivo.

Mucosal immunity in upper and lower respiratory tract to MERS-CoV.

Introduction: Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged as a deadly pathogen with a mortality rate of up to 36.2%. MERS-CoV can cause severe respiratory tract disease and multiorgan failure. Therefore, therapeutic vaccines are urgently needed. This intensive review explores the human immune responses and their immunological mechanisms during MERS-CoV infection in the mucosa of the upper and lower respiratory tracts (URT and LRT, respectively). Objective: The aim of this study is to provide a valuable, informative, and critical summary of the protective immune mechanisms against MERS-CoV infection in the URT/LRT for the purpose of preventing and controlling MERS-CoV disease and designing effective therapeutic vaccines. Methods: In this review, we focus on the immune potential of the respiratory tract following MERS-CoV infection. We searched PubMed, Embase, Web of Science, Cochrane, Scopus, and Google Scholar using the following terms: "MERS-CoV", "B cells", "T cells", "cytokines", "chemokines", "cytotoxic", and "upper and lower respiratory tracts". Results: We found and included 152 studies in this review. We report that the cellular innate immune response, including macrophages, dendritic cells, and natural killer cells, produces antiviral substances such as interferons and interleukins to prevent the virus from spreading. In the adaptive and humoral immune responses, CD4+ helper T cells, CD8+ cytotoxic T cells, B cells, and plasma cells protect against MERS-CoV infection in URT and LRT. Conclusion: The human nasopharynx-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) could successfully limit the spread of several respiratory pathogens. However, in the case of MERS-CoV infection, limited research has been conducted in humans with regard to immunopathogenesis and mucosal immune responses due to the lack of relevant tissues. A better understanding of the immune mechanisms of the URT and LRT is vital for the design and development of effective MERS-CoV vaccines.

The fate of airborne microfibers in the human respiratory tract in different microenvironments.

Αirborne microplastics (MPs) are considered an important exposure hazard to humans, especially in the indoor environment. Deposition and clearance of MPs in the human respiratory tract (HRT) was investigated using the ExDoM2 dosimetry model, modified to incorporate the deposition and clearance of MPs fibers. Fiber deposition was calculated via the fiber equivalent aerodynamic diameter determined using their properties such as size, density and dynamic shape factor. Scenario simulations were performed for elongated particles of cylindrical (base) diameters 1 µm and 10 µm and aspect ratios (ratio of fiber length to base diameter) 3, 10 and 100. Modelling results showed that the highest fiber deposition occurred in the extra-thoracic region due to large particles (fiber cylindrical diameter dp > 0.1 µm), whereas particle length (via the aspect ratio) had an influence mainly on smaller base-diameter fibers (dp < 0.1 µm) that deposited predominantly in the alveolar region. The ExDoM2 dosimetry model was also used to calculate fiber deposition in the HRT using experimental data for microplastic fiber and fragment concentrations in different microenvironments. The highest deposited number dose (220 fibers) after a 24-hour exposure was calculated in the microenvironment (bus) that had the highest fiber concentration (17.3 ± 2.4 fibers/m3). After clearance, the majority (66.4 %) of the average deposited fiber mass was transferred from the respiratory tract to the esophagus via mucociliary clearance, 32.6 % was retained in the respiratory tract, 1 % passed into the blood, and a very small amount (0.0004 %) was transferred to the lymph nodes.

Microbiome-Mucosal Immunity Nexus: Driving Forces in Respiratory Disease Progression.

The importance of the complex interplay between the microbiome and mucosal immunity, particularly within the respiratory tract, has gained significant attention due to its potential implications for the severity and progression of lung diseases. Therefore, this review summarizes the specific interactions through which the respiratory tract-specific microbiome influences mucosal immunity and ultimately impacts respiratory health. Furthermore, we discuss how the microbiome affects mucosal immunity, considering tissue-specific variations, and its capacity in respiratory diseases containing asthma, chronic obstructive pulmonary disease, and lung cancer. Additionally, we investigate the external factors which affect the relationship between respiratory microbiome and mucosal immune responses. By exploring these intricate interactions, this review provides valuable insights into the potential for microbiome-based interventions to modulate mucosal immunity and alleviate the severity of respiratory diseases.

Fine particulate matter manipulates immune response to exacerbate microbial pathogenesis in the respiratory tract.

Particulate matter with a diameter ≤2.5 µm (PM2.5) poses a substantial global challenge, with a growing recognition of pathogens contributing to diseases associated with exposure to PM2.5 Recent studies have focused on PM2.5, which impairs the immune cells in response to microbial infections and potentially contributes to the development of severe diseases in the respiratory tract. Accordingly, changes in the respiratory immune function and microecology mediated by PM2.5 are important factors that enhance the risk of microbial pathogenesis. These factors have garnered significant interest. In this review, we summarise recent studies on the potential mechanisms involved in PM2.5-mediated immune system disruption and exacerbation of microbial pathogenesis in the respiratory tract. We also discuss crucial areas for future research to address the gaps in our understanding and develop effective strategies to combat the adverse health effects of PM2.5.

Pathogen shape: Implication on pathogenicity via respiratory deposition.

The shape of environmental aerosols contributes to the discrepancy in their dynamic behavior compared to spherical particles, which have received inadequate consideration. We reported deposition patterns of aerosols and aerosol-transmissible pathogens in real human respiratory systems, taking into account their actual shape, using a validated computational-based model. We found that the shape of the aerosols significantly influenced its deposits and accessibility within the respiratory system, significantly in the tracheobronchial region. As an example, we estimated that over 180 % of differences in deposits in the trachea and bronchi were attributable to pathogens shape, inferring the underlying pathogenicity difference of these regions. These findings, capturing the spatial heterogeneity of pathogens and aerosols deposition in human respiratory system, have major implication for understanding the evolution of aerosol-related disease.

Practical Guidance for Clinical Microbiology Laboratories: Updated guidance for processing respiratory tract samples from people with cystic fibrosis.

SUMMARYThis guidance presents recommendations for clinical microbiology laboratories for processing respiratory samples from people with cystic fibrosis (pwCF). Appropriate processing of respiratory samples is crucial to detect bacterial and fungal pathogens, guide treatment, monitor the epidemiology of cystic fibrosis (CF) pathogens, and assess therapeutic interventions. Thanks to CF transmembrane conductance regulator modulator therapy, the health of pwCF has improved, but as a result, fewer pwCF spontaneously expectorate sputum. Thus, the collection of sputum samples has decreased, while the collection of other types of respiratory samples such as oropharyngeal and bronchoalveolar lavage samples has increased. To optimize the detection of microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia complex; other less common non-lactose fermenting Gram-negative bacilli, e.g., Stenotrophomonas maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species; and yeasts and filamentous fungi, non-selective and selective culture media are recommended for all types of respiratory samples, including samples obtained from pwCF after lung transplantation. There are no consensus recommendations for laboratory practices to detect, characterize, and report small colony variants (SCVs) of S. aureus, although studies are ongoing to address the potential clinical impact of SCVs. Accurate identification of less common Gram-negative bacilli, e.g., S. maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species, as well as yeasts and filamentous fungi, is recommended to understand their epidemiology and clinical importance in pwCF. However, conventional biochemical tests and automated platforms may not accurately identify CF pathogens. MALDI-TOF MS provides excellent genus-level identification, but databases may lack representation of CF pathogens to the species-level. Thus, DNA sequence analysis should be routinely available to laboratories for selected clinical circumstances. Antimicrobial susceptibility testing (AST) is not recommended for every routine surveillance culture obtained from pwCF, although selective AST may be helpful, e.g., for unusual pathogens or exacerbations unresponsive to initial therapy. While this guidance reflects current care paradigms for pwCF, recommendations will continue to evolve as CF research expands the evidence base for laboratory practices.

Spectrum detection and analysis of the epidemiological characteristics of infectious pathogens in the feline respiratory tract.

Our study was designed to investigate the original spectrum of feline respiratory tract infection and to provide a scientific basis for the clinical diagnosis and treatment of feline respiratory infections and for precise prevention and control measures. A total of 400 cats with upper respiratory tract infections from animal hospitals in 12 provinces in China were examined from November 2022 to October 2023 to investigate the epidemiology of feline calicivirus (FCV), feline herpes virus type 1 (FHV-1), influenza A virus (IAV), Mycoplasma felis, Chlamydia felis, and Bordetella bronchiseptica through loop-mediated isothermal amplification (LAMP) with microfluidic chip detection. The results showed that 396 of the 400 samples tested were positive for at least one of these pathogens, with an overall detection rate of 99.00%. The detection rates were as follows: FCV, 36.00% (144/400); M. felis, 34.00% (136/400); FHV-1, 21.50% (86/400); C. felis, 15.75% (63/400); B. b, 13.00% (52/400); IAV, 4.50% (18/400). There were no statistically significant differences in the detection rates of respiratory pathogens between different sexes, ages, seasons, breeds, or regions (P > 0.05). There were 88 mixed infections, giving a total mixed infection rate of 22.00% (88/400). It is worth noting that the detection rate of FCV at different ages and of FHV-1 in different sexes showed significant differences (P < 0.05). The highest rate of FCV infection was found in animals that were 1 to 2 years old, and the rate of FHV-1 infection in male cats was higher than that in female cats. The results showed that the spectrum of feline respiratory pathogens is complex, with diverse epidemiological characteristics and mixed infections, and some differences among different respiratory pathogens were found with regard to the sex, age, and breed of the cat. Studies should be continued to provide a scientific basis for precise prevention and control of feline respiratory diseases.

Changes in respiratory tract and gut microbiota in AR mice and their relationship with Th1/Th2/Treg.

BACKGROUND: The etiology of allergic rhinitis (AR) is not fully understood. Studies have shown that the maturation of children's immune systems is closely related to microecology. However, few studies have focused simultaneously on changes in respiratory and gut microbiota in AR and their correlation between microecological changes and Th1/Th2/Treg. OBJECTIVE: The aim is to investigate the pathogenesis of AR based on respiratory microecology, gut microecology, and Th1/Th2/Treg levels by applying microbiome techniques and correlation analysis. METHODS: Standardized OVA-induced AR mice were established. Serum OVA-sIgE, IL-4, IFN-γ, IL-10 were measured by ELISA, Tregs in lymph nodes were determined by flow cytometry, and the histological characteristics of nasal tissues were evaluated by Hematoxylin & Eosin (H&E). Nasal symptoms were observed to determine the reliability of the AR mouse model. Nasal lavage fluid (NALF) and fecal samples were collected after the last OVA challenge. The composition of respiratory microbiota in NALF and gut microbial in feces samples via 16S rRNA gene sequencing between the two groups, further explored the relationship between microbiota and Th1/Th2/Treg levels. RESULTS: In the AR group, the incidence of nose rubbing and sneezing in each mouse was significantly increased compared with the control group (all P < 0.001) and the inflammatory cell infiltration of NALF shows a significant increase in eosinophilic and neutrophilic infiltrates upon the AR group; H&E showed that the nasal mucosa of AR mice infiltration of massive eosinophils cells and neutrophils cells. OVA-sIgE and IL-4 in the AR group were increased (P < 0.01, P < 0.05) and IFN-γ, IL-10 were significantly decreased (P < 0.01, P < 0.05). Tregs showed a downward trend in the AR group, but there was no statistical difference. Compared with the control group, the respiratory microbiota of AR mice did not change significantly, while the gut microbiota changed significantly. In gut microbiota, compared to the control group, Shannon index in the AR group revealed a significant decrease at the genus level (P < 0.01), and Simpson index was significantly increased at all levels (all P < 0.05). PCoA also showed significant differences in beta diversity between the two groups (all P < 0.05). Compared to the control group, Deferribacteres at phylum level, Roseburia, Ruminiclostridium, Anaerotruncus at genus level were significantly decreased in the AR group (all P < 0.05). Spearman's rank correlation showed that OVA-sIgE was positively correlated with Bacteroidetes, Muribaculaceae and Erysipelotrichaceae (all P < 0.05); IL-4 was significantly negatively correlated with Epsilonbacteraeota and Deferribacteres (all P < 0.05). Treg was significantly positively correlated with Patescibacteria, Lachnospiraceae, and Saccharimonadaceae in gut microecology. CONCLUSION: Our results showed that the respiratory microbiota of AR mice was not significantly altered, but the gut microbiota varied significantly and there was a correlation between gut microbiota and Th1/Th2/Treg.

Detection of lumpy skin disease virus reads in the human upper respiratory tract microbiome requires further investigation.

Lumpy skin disease virus (LSDV), a double-stranded DNA virus from the Capripoxvirus genus, primarily affects Bos indicus, Bos taurus breeds, and water buffalo. Arthropod vectors, including mosquitoes and biting flies, are the main LSDV transmitters. Although LSDV is not zoonotic, this study unexpectedly detected LSDV reads in the upper respiratory tract microbiome of humans from rural and urban areas in Maharashtra, India. Nasopharyngeal and oropharyngeal swab samples collected for SARS-CoV-2 surveillance underwent whole-genome metagenomics sequencing, revealing LSDV reads in 25% of samples. Split kmer analysis provided insights into sample relatedness despite the low coverage of LSDV reads with the reference genome. Our findings, which include the detection of LSDV contigs aligning to specific locations on the reference genome, suggest a common source for LSDV reads, potentially shared water sources, or milk/milk products. Further investigation is needed to ascertain the mode of transmission and reason for the detection of LSDV reads in human upper respiratory tract.

Lower respiratory tract microbiome and lung cancer risk prediction in patients with diffuse lung parenchymal lesions.

Objective: In clinical practice, imaging manifestations of diffuse lung parenchymal lesions are common and indicative of various diseases, making differential diagnosis difficult. Some of these lesions are eventually diagnosed as lung cancer. Methods: Because respiratory microorganisms play an important role in lung cancer development, we searched for microbial markers that could predict the risk of lung cancer by retrospectively analyzing the lower respiratory tract (LRT) microbiome of 158 patients who were hospitalized in the First Affiliated Hospital of Guangzhou Medical University (March 2021-March 2023) with diffuse lung parenchymal lesions. The final diagnosis was lung cancer in 21 cases, lung infection in 93 cases, and other conditions (other than malignancy and infections) in 44 cases. The patient's clinical characteristics and the results of metagenomic next-generation sequencing of bronchoalveolar lavage fluid (BALF) were analyzed. Results: Body mass index (BMI) and LRT microbial diversity (Shannon, Simpson, species richness, and Choa1 index) were significantly lower (P< 0.001, respectively) and Lactobacillus acidophilus relative abundance in the LRT was significantly higher (P< 0.001) in patients with lung cancer. The relative abundance of L. acidophilus in BALF combined with BMI was a good predictor of lung cancer risk (area under the curve = 0.985, accuracy = 98.46%, sensitivity = 95.24%, and specificity = 100.00%; P< 0.001). Conclusion: Our study showed that an imbalance in the component ratio of the microbial community, diminished microbial diversity, and the presence of specific microbial markers in the LRT predicted lung cancer risk in patients with imaging manifestations of diffuse lung parenchymal lesions.

Histology Atlas of the Developing Mouse Respiratory System From Prenatal Day 9.0 Through Postnatal Day 30.

Respiratory diseases are one of the leading causes of death and disability around the world. Mice are commonly used as models of human respiratory disease. Phenotypic analysis of mice with spontaneous, congenital, inherited, or treatment-related respiratory tract abnormalities requires investigators to discriminate normal anatomic features of the respiratory system from those that have been altered by disease. Many publications describe individual aspects of normal respiratory tract development, primarily focusing on morphogenesis of the trachea and lung. However, a single reference providing detailed low- and high-magnification, high-resolution images of routine hematoxylin and eosin (H&E)-stained sections depicting all major structures of the entire developing murine respiratory system does not exist. The purpose of this atlas is to correct this deficiency by establishing one concise reference of high-resolution color photomicrographs from whole-slide scans of H&E-stained tissue sections. The atlas has detailed descriptions and well-annotated images of the developing mouse upper and lower respiratory tracts emphasizing embryonic days (E) 9.0 to 18.5 and major early postnatal events. The selected images illustrate the main structures and events at key developmental stages and thus should help investigators both confirm the chronological age of mouse embryos and distinguish normal morphology as well as structural (cellular and organ) abnormalities.

A model study on the effect of human's height variability in particle deposition and retained dose in the respiratory tract.

The objective of the current study was to investigate the impact of human's height variability to the deposition percentage, the deposited and the retained dose of particulate matter in the respiratory tract. In addition, the dose to the oesophagus, blood and lymph nodes was evaluated after particle clearance. A methodology which correlates anatomical and physiological parameters with height was adopted into an existing particle dosimetry model (Exposure Dose Model 2, ExDoM2). Model results showed that deposition of particles with aerodynamic diameter (dae) ranging from 0.001 to 10 µm depends on the competition between anatomical/physiological parameters, with the maximum effect induced from height variability to be observed for particles in the size range of 0.30 µm

Host and environmental factors shape upper airway microbiota and respiratory health across the human lifespan.

Our understanding of the normal variation in the upper respiratory tract (URT) microbiota across the human lifespan and how these relate to host, environment, and health is limited. We studied the microbiota of 3,104 saliva (<10 year-olds)/oropharynx (≥10 year-olds) and 2,485 nasopharynx samples of 3,160 Dutch individuals 0-87 years of age, participating in a cross-sectional population-wide study (PIENTER-3) using 16S-rRNA sequencing. The microbiota composition was strongly related to age, especially in the nasopharynx, with maturation occurring throughout childhood and adolescence. Clear niche- and age-specific associations were found between the microbiota composition and host/environmental factors and health outcomes. Among others, social interaction, sex, and season were associated with the nasopharyngeal microbial community. By contrast, the oral microbiota was more related to antibiotics, tobacco, and alcohol use. We present an atlas of the URT microbiota across the lifespan in association with environment and health, establishing a baseline for future research.

Lower respiratory tract microbiota in patients with clinically suspected nontuberculous mycobacterial pulmonary disease according to the presence of gastroesophageal reflux.

Although gastroesophageal reflux has been recognized as one of the risk factors of nontuberculous mycobacterial pulmonary disease (NTM-PD) progression, the effect of reflux on the lower respiratory tract microbiota has not been studied in detail. We investigated the composition of the lower respiratory tract microbiota in patients with clinically suspected NTM-PD, comparing them based on the presence of reflux. Forty-seven patients suspected of having NTM-PD were enrolled and assigned according to presence of reflux (n = 22) and non- reflux (n = 25). We performed a pepsin ELISA assay to identify the presence of reflux and 16S ribosomal RNA gene amplicon sequencing to evaluate the microbiota in bronchoalveolar lavage fluid. There were no significant differences in the diversity or composition of the lower respiratory microbiota between the NTM-PD and non-NTM-PD groups. Bacterial richness was observed in the non-reflux group than in the reflux group [P = 0.03] and a cluster in the reflux group was observed. The reflux group showed a predominance for Pseudomonas aeruginosa or Staphylococcus aureus among the NTM-PD group and for P. aeruginosa, Haemophilus influenzae, Klebsiella pneumoniae, or Eikenella species among the non-NTM-PD group. The non-reflux groups presented diverse patterns. A linear discriminant analysis and volcano plot demonstrated that P. aeruginosa, H. haemolyticus, Selenomonas artemidis, and Dolosigranulum pigrum were specifically associated with the NTM-PD reflux group, while P. aeruginosa was specifically associated with the non-NTM-PD reflux group. These observations confirm that the lower respiratory microbiota is consistently altered by reflux but not in NTM-PD.

Deposition of airborne cyanobacteria and microalgae in the human respiratory tract (Baltic Sea coastal zone, Poland).

Scientific research confirms the harmful effects of airborne cyanobacteria and microalgae. However, determining human exposure to these microorganisms remains a challenge. The six-stage Tisch impactor was used to collect bioaerosols from April to September 2020 in the coastal zone of the southern Baltic. The MPPD model was used for estimation of regional, lobar, and generation deposition of microorganisms in human respiratory tract. The mass deposition fraction of cyanobacteria and microalgae in the head region gradually increased with the aerosol size. The maximum deposition fractions in the trachea, bronchial, and the pulmonary region were found for particles between 2.1 and 3.3 µm. The contribution of cyanobacteria and microalgae was the highest in the head region. The majority of microorganisms found in pulmonary region dominated in particles smaller than 2.1 µm. Exposure to the ambient bioaerosols may have an adverse impact on the human health in the region of southern Baltic Sea.

Assessment and examination of the respiratory system.

This article aims to outline the fundamental principles of consultations with and clinical assessments of patients with symptoms that may be indicative of respiratory system pathology. The article explores how to perform a respiratory system-focused patient history and physical examination. An evaluation of clinical 'red flags' to reduce the risk of omitting serious illness is also considered, alongside the exploration of features of respiratory pathology and evidence-based clinical decision-making tools that may be used to support clinical diagnosis.

Cytochrome oxidase requirements in Bordetella reveal insights into evolution towards life in the mammalian respiratory tract.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis, the causal agent of human whooping cough, and B. bronchiseptica, which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica, which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis, which evolved from a B. bronchiseptica-like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1, and cyoABCD1. To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo. No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis-conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens.