Relationship between Azithromycin Susceptibility and Administration Efficacy for Nontypeable Haemophilus influenzae Respiratory Infection

ABSTRACTNontypeableHaemophilus influenzae(NTHI) is an opportunistic pathogen that is an important cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). COPD is an inflammatory disease of the airways, and exacerbations are acute inflammatory events superimposed on this background of chronic inflammation. Azithromycin (AZM) is a macrolide antibiotic with antibacterial and anti-inflammatory properties and a clinically proven potential for AECOPD prevention and management. Relationships between AZM efficacy and resistance by NTHI and between bactericidal and immunomodulatory effects on NTHI respiratory infection have not been addressed. In this study, we employed two pathogenic NTHI strains with different AZM susceptibilities (NTHI 375 [AZM susceptible] and NTHI 353 [AZM resistant]) to evaluate the prophylactic and therapeutic effects of AZM on the NTHI-host interplay. At the cellular level, AZM was bactericidal toward intracellular NTHI inside alveolar and bronchial epithelia and alveolar macrophages, and it enhanced NTHI phagocytosis by the latter cell type. These effects correlated with the strain MIC of AZM and the antibiotic dose. Additionally, the effect of AZM on NTHI infection was assessed in a mouse model of pulmonary infection. AZM showed both preventive and therapeutic efficacies by lowering NTHI 375 bacterial counts in lungs and bronchoalveolar lavage fluid (BALF) and by reducing histopathological inflammatory lesions in the upper and lower airways of mice. Conversely, AZM did not reduce bacterial loads in animals infected with NTHI 353, in which case a milder anti-inflammatory effect was also observed. Together, the results of this work link the bactericidal and anti-inflammatory effects of AZM and frame the efficacy of this antibiotic against NTHI respiratory infection.

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Molecular Characterization of Fluoroquinolone Resistance in Nontypeable Haemophilus influenzae Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04005-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 461-466 ◽

Cited By ~ 24

Author(s):

Carmen Puig ◽

José Manuel Tirado-Vélez ◽

Laura Calatayud ◽

Fe Tubau ◽

Junkal Garmendia ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Nalidixic Acid ◽

Respiratory Infections ◽

Pulse Field ◽

Fluoroquinolone Resistance ◽

Chronic Obstructive ◽

Nontypeable Haemophilus Influenzae ◽

High Genetic Diversity ◽

Content Type ◽

Pulse Field Gel Electrophoresis

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a common cause of respiratory infections in adults, who are frequently treated with fluoroquinolones. The aims of this study were to characterize the genotypes of fluoroquinolone-resistant NTHi isolates and their mechanisms of resistance. Among 7,267H. influenzaeisolates collected from adult patients from 2000 to 2013, 28 (0.39%) were ciprofloxacin resistant according to Clinical and Laboratory Standards Institute (CLSI) criteria. In addition, a nalidixic acid screening during 2010 to 2013 detected five (0.23%) isolates that were ciprofloxacin susceptible but nalidixic acid resistant. Sequencing of their quinolone resistance-determining regions and genotyping by pulse-field gel electrophoresis and multilocus sequence typing of the 25 ciprofloxacin-resistant isolates available and all 5 nalidixic acid-resistant isolates were performed. In the NTHi isolates studied, two mutations producing changes in two GyrA residues (Ser84, Asp88) and/or two ParC residues (Ser84, Glu88) were associated with increased fluoroquinolone MICs. Strains with one or two mutations (n= 15) had ciprofloxacin and levofloxacin MICs of 0.12 to 2 μg/ml, while those with three or more mutations (n= 15) had MICs of 4 to 16 μg/ml. Long persistence of fluoroquinolone-resistant strains was observed in three chronic obstructive pulmonary disease patients. High genetic diversity was observed among fluoroquinolone-resistant NTHi isolates. Although fluoroquinolones are commonly used to treat respiratory infections, the proportion of resistant NTHi isolates remains low. The nalidixic acid disk test is useful for detecting the first changes in GyrA or in GyrA plus ParC among fluoroquinolone-susceptible strains that are at a potential risk for the development of resistance under selective pressure by fluoroquinolone treatment.

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Closed Complete Genome Sequences of Two Nontypeable Haemophilus influenzae Strains Containing Novel modA Alleles from the Sputum of Patients with Chronic Obstructive Pulmonary Disease

Microbiology Resource Announcements ◽

10.1128/mra.00821-18 ◽

2018 ◽

Vol 7 (2) ◽

Cited By ~ 6

Author(s):

John M. Atack ◽

Timothy F. Murphy ◽

Lauren O. Bakaletz ◽

Kate L. Seib ◽

Michael P. Jennings

Keyword(s):

Chronic Obstructive Pulmonary Disease ◽

Haemophilus Influenzae ◽

Pulmonary Disease ◽

Complete Genome ◽

Chronic Obstructive ◽

Phase Variable ◽

Nontypeable Haemophilus Influenzae ◽

Genome Sequences ◽

Obstructive Pulmonary Disease ◽

Content Type

Nontypeable Haemophilus influenzae (NTHi) is an important bacterial pathogen that causes otitis media and exacerbations of chronic obstructive pulmonary disease (COPD). Here, we report the complete genome sequences of NTHi strains 10P129H1 and 84P36H1, isolated from COPD patients, which contain the phase-variable epigenetic regulators ModA15 and ModA18, respectively.

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A P5 Peptide That Is Homologous to Peptide 10 of OprF from Pseudomonas aeruginosa Enhances Clearance of Nontypeable Haemophilus influenzae from Acutely Infected Rat Lung in the Absence of Detectable Peptide-Specific Antibody

Infection and Immunity ◽

10.1128/iai.68.1.377-381.2000 ◽

2000 ◽

Vol 68 (1) ◽

pp. 377-381 ◽

Cited By ~ 15

Author(s):

Dianne C. Webb ◽

Allan W. Cripps

Keyword(s):

Pseudomonas Aeruginosa ◽

Haemophilus Influenzae ◽

Measles Virus ◽

Opportunistic Pathogen ◽

Lavage Fluid ◽

Antibody Responses ◽

Rat Lung ◽

Nontypeable Haemophilus Influenzae ◽

Vaccine Potential ◽

Humoral Responses

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with otitis media and the exacerbation of chronic bronchitis. This study reports the vaccine potential of three peptides representing conserved regions of the NTHi P5 outer membrane protein which have been fused to a promiscuous measles virus F protein T-cell eptitope (MVF). The peptides correspond to a region in surface loop one (MVF/L1A), the central region of loop four (MVF/L4), and a C-terminal region homologous to peptide 10 of OprF from Pseudomonas aeruginosa (MVF/H3). Immunization of rats with MVF/H3 was the most efficacious in significantly reducing the number of viable NTHi in both the broncho-alveolar lavage fluid (74%) and lung homogenates (70%), compared to control rats. Importantly, despite significantly increased rates of clearance, immunization with MVF/H3 elicited poor antibody responses, suggesting that cell-mediated rather than humoral responses play an important role in the enhanced clearance of NTHi in this model.

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Ferric Uptake Regulator and Its Role in the Pathogenesis of Nontypeable Haemophilus influenzae

Infection and Immunity ◽

10.1128/iai.01227-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1221-1233 ◽

Cited By ~ 26

Author(s):

Alistair Harrison ◽

Estevan A. Santana ◽

Blake R. Szelestey ◽

David E. Newsom ◽

Peter White ◽

...

Keyword(s):

Gene Expression ◽

Haemophilus Influenzae ◽

Iron Homeostasis ◽

Expression Patterns ◽

Opportunistic Pathogen ◽

Nontypeable Haemophilus Influenzae ◽

Bacterial Persistence ◽

Content Type ◽

Regulatory Pathways ◽

Iron Utilization

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a commensal microorganism of the human nasopharynx, and yet is also an opportunistic pathogen of the upper and lower respiratory tracts. Host microenvironments influence gene expression patterns, likely critical for NTHi persistence. The host sequesters iron as a mechanism to control microbial growth, and yet iron limitation influences gene expression and subsequent production of proteins involved in iron homeostasis. Careful regulation of iron uptake, via theferricuptakeregulator Fur, is essential in multiple bacteria, including NTHi. We hypothesized therefore that Fur contributes to iron homeostasis in NTHi, is critical for bacterial persistence, and likely regulates expression of virulence factors. Toward this end,furwas deleted in the prototypic NTHi clinical isolate, 86-028NP, and we assessed gene expression regulated by Fur. As expected, expression of the majority of genes that encode proteins with predicted roles in iron utilization was repressed by Fur. However, 14 Fur-regulated genes encode proteins with no known function, and yet may contribute to iron utilization or other biological functions. In a mammalian model of human otitis media, we determined that Fur was critical for bacterial persistence, indicating an important role for Fur-mediated iron homeostasis in disease progression. These data provide a profile of genes regulated by Fur in NTHi and likely identify additional regulatory pathways involved in iron utilization. Identification of such pathways will increase our understanding of how this pathogen can persist within host microenvironments, as a common commensal and, importantly, as a pathogen with significant clinical impact.

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Draft Genome Sequence of an Isolate of Nontypeable Haemophilus influenzae from an Acute Exacerbation of Chronic Obstructive Pulmonary Disease in Tasmania

Microbiology Resource Announcements ◽

10.1128/mra.00375-20 ◽

2020 ◽

Vol 9 (19) ◽

Author(s):

Rajendra KC ◽

Kelvin W. C. Leong ◽

Belinda McEwan ◽

Julia Lachowicz ◽

Nicholas M. Harkness ◽

...

Keyword(s):

Chronic Obstructive Pulmonary Disease ◽

Haemophilus Influenzae ◽

Pulmonary Disease ◽

Draft Genome ◽

Chronic Obstructive ◽

Nontypeable Haemophilus Influenzae ◽

Obstructive Pulmonary Disease ◽

Content Type ◽

Acute Exacerbations ◽

Human Illness

Nontypeable Haemophilus influenzae (NTHi) is an important cause of human illness, including pneumonia and acute exacerbations of chronic obstructive pulmonary disease (COPD). We report here the draft genome of an isolate of NTHi collected from the sputum of a patient presenting with COPD in Tasmania, Australia.

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Antibody Binding and Complement-Mediated Killing of Invasive Haemophilus influenzae Isolates from Spain, Portugal, and the Netherlands

Infection and Immunity ◽

10.1128/iai.00454-20 ◽

2020 ◽

Vol 88 (10) ◽

Author(s):

Elena Dudukina ◽

Laura de Smit ◽

Giel J. A. Verhagen ◽

Arie van de Ende ◽

José María Marimón ◽

...

Keyword(s):

The Netherlands ◽

Haemophilus Influenzae ◽

Human Serum ◽

Opportunistic Pathogen ◽

Invasive Disease ◽

Chronic Obstructive ◽

Bacterial Survival ◽

Upper Respiratory Tract ◽

Content Type ◽

Bacterial Surface

ABSTRACT Haemophilus influenzae is a Gram-negative bacterium that can be classified into typeable (types a through f) and nontypeable (NTHi) groups. This opportunistic pathogen asymptomatically colonizes the mucosal epithelium of the upper respiratory tract, from where it spreads to other neighboring regions, potentially leading to disease. Infection with NTHi can cause otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and pneumonia, but it is increasingly causing invasive disease, including bacteremia and meningitis. Invasive NTHi strains are more resistant to complement-mediated killing. However, the mechanisms of complement resistance have never been studied in large numbers of invasive NTHi strains. In this study, we determined the relationship between binding of IgG or IgM and the bacterial survival in normal human serum for 267 invasive H. influenzae strains from Spain, Portugal, and the Netherlands, of which the majority (200 [75%]) were NTHi. NTHi bacteria opsonized with high levels of IgM had the lowest survival in human serum. IgM binding to the bacterial surface, but not IgG binding, was shown to be associated with complement-mediated killing of NTHi strains. We conclude that evasion of IgM binding by NTHi strains increases survival in blood, thereby potentially contributing to their ability to cause severe invasive diseases.

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Early-Life Intranasal Colonization with Nontypeable Haemophilus influenzae Exacerbates Juvenile Airway Disease in Mice

Infection and Immunity ◽

10.1128/iai.01539-15 ◽

2016 ◽

Vol 84 (7) ◽

pp. 2022-2030 ◽

Cited By ~ 10

Author(s):

Jessica R. McCann ◽

Stanley N. Mason ◽

Richard L. Auten ◽

Joseph W. St. Geme ◽

Patrick C. Seed

Keyword(s):

Haemophilus Influenzae ◽

Early Life ◽

Inflammatory Responses ◽

Airway Disease ◽

Lavage Fluid ◽

Nontypeable Haemophilus Influenzae ◽

Human Infants ◽

Mucus Production ◽

Content Type ◽

Increased Risk

Accumulating evidence suggests a connection between asthma development and colonization with nontypeableHaemophilus influenzae(NTHi). Specifically, nasopharyngeal colonization of human infants with NTHi within 4 weeks of birth is associated with an increased risk of asthma development later in childhood. Monocytes derived from these infants have aberrant inflammatory responses to common upper respiratory bacterial antigens compared to those of cells derived from infants who were not colonized and do not go on to develop asthma symptoms in childhood. In this study, we hypothesized that early-life colonization with NTHi promotes immune system reprogramming and the development of atypical inflammatory responses. To address this hypothesis in a highly controlled model, we tested whether colonization of mice with NTHi on day of life 3 induced or exacerbated juvenile airway disease using an ovalbumin (OVA) allergy model of asthma. We found that animals that were colonized on day of life 3 and subjected to induction of allergy had exacerbated airway disease as juveniles, in which exacerbated airway disease was defined as increased cellular infiltration into the lung, increased amounts of inflammatory cytokines interleukin-5 (IL-5) and IL-13 in lung lavage fluid, decreased regulatory T cell-associatedFOXP3gene expression, and increased mucus production. We also found that colonization with NTHi amplified airway resistance in response to increasing doses of a bronchoconstrictor following OVA immunization and challenge. Together, the murine model provides evidence for early-life immune programming that precedes the development of juvenile airway disease and corroborates observations that have been made in human children.

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The Nonantibiotic Macrolide EM703 Improves Survival in a Model of Quinolone-Treated Pseudomonas aeruginosa Airway Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02761-16 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 4

Author(s):

Gopinath Kasetty ◽

Ravi K. V. Bhongir ◽

Praveen Papareddy ◽

Heiko Herwald ◽

Arne Egesten

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Resistance ◽

Inflammatory Responses ◽

Lavage Fluid ◽

Macrolide Antibiotics ◽

Chronic Obstructive ◽

Gram Negative ◽

Content Type ◽

Airway Infections ◽

Anti Inflammatory

ABSTRACT Macrolide antibiotics are used as anti-inflammatory agents, e.g., for prevention of exacerbations in chronic obstructive pulmonary disease and cystic fibrosis. Several studies have shown improved outcomes after the addition of macrolides to β-lactam antibiotics for treatment of severe community-acquired pneumonia. However, a beneficial effect of macrolides in treating Gram-negative bacterial airway infections, e.g., those caused by Pseudomonas aeruginosa, remains to be shown. Macrolide antibiotics have significant side effects, in particular, motility-stimulating activity in the gastrointestinal tract and promotion of bacterial resistance. In this study, EM703, a modified macrolide lacking antibiotic and motility-stimulating activities but with retained anti-inflammatory properties, was used as an adjunct treatment for experimental P. aeruginosa lung infection, in combination with a conventional antibiotic. Airway infections in BALB/cJRj mice were induced by nasal instillation of P. aeruginosa; this was followed by treatment with the quinolone levofloxacin in the absence or presence of EM703. Survival, inflammatory responses, and cellular influx to the airways were monitored. Both pretreatment and simultaneous administration of EM703 dramatically improved survival in levofloxacin-treated mice with P. aeruginosa airway infections. In addition, EM703 reduced the levels of proinflammatory cytokines, increased the numbers of leukocytes in bronchoalveolar lavage fluid, and reduced the numbers of neutrophils present in lung tissue. In summary, the findings of this study show that the immunomodulatory properties of the modified macrolide EM703 can be important when treating Gram-negative pneumonia, as exemplified by P. aeruginosa infection in this study.

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Shielding of a Lipooligosaccharide IgM Epitope Allows Evasion of Neutrophil-Mediated Killing of an Invasive Strain of Nontypeable Haemophilus influenzae

mBio ◽

10.1128/mbio.01478-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 22

Author(s):

Jeroen D. Langereis ◽

Jeffrey N. Weiser

Keyword(s):

Haemophilus Influenzae ◽

Immune Cells ◽

Inner Core ◽

Inflammatory Diseases ◽

White Blood Cells ◽

Human Neutrophils ◽

Chronic Obstructive ◽

Phase Variable ◽

Nontypeable Haemophilus Influenzae ◽

Content Type

ABSTRACTNontypeableHaemophilus influenzaeis a frequent cause of noninvasive mucosal inflammatory diseases but may also cause invasive diseases, such as sepsis and meningitis, especially in children and the elderly. Infection by nontypeableHaemophilus influenzaeis characterized by recruitment of neutrophilic granulocytes. Despite the presence of a large number of neutrophils, infections with nontypeableHaemophilus influenzaeare often not cleared effectively by the antimicrobial activity of these immune cells. Herein, we examined how nontypeableHaemophilus influenzaeevades neutrophil-mediated killing. Transposon sequencing (Tn-seq) was used on an isolate resistant to neutrophil-mediated killing to identify genes required for its survival in the presence of human neutrophils and serum, which provided a source of complement and antibodies. Results show that nontypeableHaemophilus influenzaeprevents complement-dependent neutrophil-mediated killing by expression of surface galactose-containing oligosaccharide structures. These outer-core structures block recognition of an inner-core lipooligosaccharide epitope containing glucose attached to heptose HepIII-β1,2-Glc by replacement with galactose attached to HepIII or through shielding HepIII-β1,2-Glc by phase-variable attachment of oligosaccharide chain extensions. When the HepIII-β1,2-Glc-containing epitope is expressed and exposed, nontypeableHaemophilus influenzaeis opsonized by naturally acquired IgM generally present in human serum and subsequently phagocytosed and killed by human neutrophils. Clinical nontypeableHaemophilus influenzaeisolates containing galactose attached to HepIII that are not recognized by this IgM are more often found to cause invasive infections.IMPORTANCENeutrophils are white blood cells that specialize in killing pathogens and are recruited to sites of inflammation. However, despite the presence of large numbers of neutrophils in the middle ear cavity and lungs of patients with otitis media or chronic obstructive pulmonary disease, respectively, the bacterium nontypeableHaemophilus influenzaeis often not effectively cleared from these locations by these immune cells. In order to understand how nontypeableHaemophilus influenzaeis able to cause inflammatory diseases in the presence of neutrophils, we determined the mechanism that underlies resistance to neutrophil-mediated killing. We have shown that nontypeableHaemophilus influenzaeprevents binding of antibodies of the IgM subtype through changes in their surface lipooligosaccharide structure, thereby preventing complement activation and clearance by human neutrophils.

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Overlapping and Complementary Oxidative Stress Defense Mechanisms in Nontypeable Haemophilus influenzae

Journal of Bacteriology ◽

10.1128/jb.01973-14 ◽

2014 ◽

Vol 197 (2) ◽

pp. 277-285 ◽

Cited By ~ 10

Author(s):

Alistair Harrison ◽

Beth D. Baker ◽

Robert S. Munson

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Haemophilus Influenzae ◽

Fenton Reaction ◽

Defense Mechanisms ◽

Reactive Oxygen ◽

Chronic Obstructive ◽

Oxygen Species ◽

Nontypeable Haemophilus Influenzae ◽

Content Type

The Gram-negative commensal bacterium nontypeableHaemophilus influenzae(NTHI) can cause respiratory tract diseases that include otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During colonization and infection, NTHI withstands oxidative stress generated by reactive oxygen species produced endogenously, by the host, and by other copathogens and flora. These reactive oxygen species include superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals, whose killing is amplified by iron via the Fenton reaction. We previously identified genes that encode proteins with putative roles in protection of the NTHI isolate strain 86-028NP against oxidative stress. These include catalase (HktE), peroxiredoxin/glutaredoxin (PgdX), and a ferritin-like protein (Dps). Strains were generated with mutations inhktE,pgdX, anddps. ThehktEmutant and apgdX hktEdouble mutant were more sensitive than the parent to killing by H2O2. Conversely, thepgdXmutant was more resistant to H2O2due to increased catalase activity. Supporting the role of killing via the Fenton reaction, binding of iron by Dps significantly mitigated the effect of H2O2-mediated killing. NTHI thus utilizes several effectors to resist oxidative stress, and regulation of free iron is critical to this protection. These mechanisms will be important for successful colonization and infection by this opportunistic human pathogen.

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