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22.2: Bacterial Infections of the Respiratory Tract - Biology

22.2: Bacterial Infections of the Respiratory Tract - Biology


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Learning Objectives

  • Identify the most common bacteria that can cause infections of the upper and lower respiratory tract
  • Compare the major characteristics of specific bacterial diseases of the respiratory tract

The respiratory tract can be infected by a variety of bacteria, both gram positive and gram negative. Although the diseases that they cause may range from mild to severe, in most cases, the microbes remain localized within the respiratory system. Fortunately, most of these infections also respond well to antibiotic therapy.

Streptococcal Infections

A common upper respiratory infection, streptococcal pharyngitis (strep throat) is caused by Streptococcus pyogenes. This gram-positive bacterium appears as chains of cocci, as seen in Figure (PageIndex{1}). Rebecca Lancefieldserologically classified streptococci in the 1930s using carbohydrate antigens from the bacterial cell walls. S. pyogenes is the sole member of the Lancefield group A streptococci and is often referred to as GAS, or group A strep.

Similar to streptococcal infections of the skin, the mucosal membranes of the pharynx are damaged by the release of a variety of exoenzymes and exotoxins by this extracellular pathogen. Many strains of S. pyogenes can degrade connective tissues by using hyaluronidase, collagenase and streptokinase. Streptokinase activates plasmin, which leads to degradation of fibrin and, in turn, dissolution of blood clots, which assists in the spread of the pathogen. Released toxins include streptolysins that can destroy red and white blood cells. The classic signs of streptococcal pharyngitis are a fever higher than 38 °C (100.4 °F); intense pharyngeal pain; erythema associated with pharyngeal inflammation; and swollen, dark-red palatine tonsils, often dotted with patches of pus; and petechiae (microcapillary hemorrhages) on the soft or hard palate (roof of the mouth) (Figure (PageIndex{2})). The submandibular lymph nodes beneath the angle of the jaw are also often swollen during strep throat.

Some strains of group A streptococci produce erythrogenic toxin. This exotoxin is encoded by a temperate bacteriophage (bacterial virus) and is an example of phage conversion (see The Viral Life Cycle). The toxin attacks the plasma membranes of capillary endothelial cells and leads to scarlet fever (or scarlatina), a disseminated fine red rash on the skin, and strawberry tongue, a red rash on the tongue (Figure (PageIndex{2})). Severe cases may even lead to streptococcal toxic shock syndrome (STSS), which results from massive superantigen production that leads to septic shock and death.

S. pyogenes can be easily spread by direct contact or droplet transmission through coughing and sneezing. The disease can be diagnosed quickly using a rapid enzyme immunoassay for the group A antigen. However, due to a significant rate of false-negative results (up to 30%1), culture identification is still the gold standard to confirm pharyngitis due to S. pyogenes. pyogenes can be identified as a catalase-negative, beta hemolytic bacterium that is susceptible to 0.04 units of bacitracin. Antibiotic resistance is limited for this bacterium, so most β-lactams remain effective; oral amoxicillin and intramuscular penicillin G are those most commonly prescribed.

Sequelae of S. pyogenes Infections

One reason strep throat infections are aggressively treated with antibiotics is because they can lead to serious sequelae, later clinical consequences of a primary infection. It is estimated that 1%–3% of untreated S. pyogenes infections can be followed by nonsuppurative (without the production of pus) sequelae that develop 1–3 weeks after the acute infection has resolved. Two such sequelae are acute rheumatic fever and acute glomerulonephritis.

Acute rheumatic fever can follow pharyngitis caused by specific rheumatogenic strains of S. pyogenes (strains 1, 3, 5, 6, and 18). Although the exact mechanism responsible for this sequela remains unclear, molecular mimicry between the M protein of rheumatogenic strains of S. pyogenes and heart tissue is thought to initiate the autoimmune attack. The most serious and lethal clinical manifestation of rheumatic fever is damage to and inflammation of the heart (carditis). Acute glomerulonephritis also results from an immune response to streptococcal antigens following pharyngitis and cutaneous infections. Acute glomerulonephritis develops within 6–10 days after pharyngitis, but can take up to 21 days after a cutaneous infection. Similar to acute rheumatic fever, there are strong associations between specific nephritogenic strains of S. pyogenes and acute glomerulonephritis, and evidence suggests a role for antigen mimicry and autoimmunity. However, the primary mechanism of acute glomerulonephritis appears to be the formation of immune complexes between S. pyogenes antigens and antibodies, and their deposition between endothelial cells of the glomeruli of kidney. Inflammatory response against the immune complexes leads to damage and inflammation of the glomeruli (glomerulonephritis).

Exercise (PageIndex{1})

  1. What are the symptoms of strep throat?
  2. What is erythrogenic toxin and what effect does it have?
  3. What are the causes of rheumatic fever and acute glomerulonephritis?

Acute Otitis Media

An infection of the middle ear is called acute otitis media (AOM), but often it is simply referred to as an earache. The condition is most common between ages 3 months and 3 years. In the United States, AOM is the second-leading cause of visits to pediatricians by children younger than age 5 years, and it is the leading indication for antibiotic prescription.2

AOM is characterized by the formation and accumulation of pus in the middle ear. Unable to drain, the pus builds up, resulting in moderate to severe bulging of the tympanic membrane and otalgia (ear pain). Inflammation resulting from the infection leads to swelling of the eustachian tubes, and may also lead to fever, nausea, vomiting, and diarrhea, particularly in infants. Infants and toddlers who cannot yet speak may exhibit nonverbal signs suggesting AOM, such as holding, tugging, or rubbing of the ear, as well as uncharacteristic crying or distress in response to the pain.

AOM can be caused by a variety of bacteria. Among neonates, S. pneumoniae is the most common cause of AOM, but Escherichia coli, Enterococcus spp., and group B Streptococcus species can also be involved. In older infants and children younger than 14 years old, the most common bacterial causes are S. pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis. Among S. pneumoniae infections, encapsulated strains are frequent causes of AOM. By contrast, the strains of H. influenzae and M. cattarhalis that are responsible for AOM do not possess a capsule. Rather than direct tissue damage by these pathogens, bacterial components such as lipopolysaccharide (LPS) in gram-negative pathogens induce an inflammatory response that causes swelling, pus, and tissue damage within the middle ear (Figure (PageIndex{3})).

Any blockage of the eustachian tubes, with or without infection, can cause fluid to become trapped and accumulate in the middle ear. This is referred to as otitis media with effusion (OME). The accumulated fluid offers an excellent reservoir for microbial growth and, consequently, secondary bacterial infections often ensue. This can lead to recurring and chronic earaches, which are especially common in young children. The higher incidence in children can be attributed to many factors. Children have more upper respiratory infections, in general, and their eustachian tubes are also shorter and drain at a shallower angle. Young children also tend to spend more time lying down than adults, which facilitates drainage from the nasopharynx through the eustachian tube and into the middle ear. Bottle feeding while lying down enhances this risk because the sucking action on the bottle causes negative pressure to build up within the eustachian tube, promoting the movement of fluid and bacteria from the nasopharynx.

Diagnosis is typically made based on clinical signs and symptoms, without laboratory testing to determine the specific causative agent. Antibiotics are frequently prescribed for the treatment of AOM. High-dose amoxicillin is the first-line drug, but with increasing resistance concerns, macrolides and cephalosporins may also be used. The pneumococcal conjugate vaccine (PCV13) contains serotypes that are important causes of AOM, and vaccination has been shown to decrease the incidence of AOM. Vaccination against influenza has also been shown to decrease the risk for AOM, likely because viral infections like influenza predispose patients to secondary infections with S. pneumoniae. Although there is a conjugate vaccine available for the invasive serotype B of H. influenzae, this vaccine does not impact the incidence of H. influenzae AOM. Because unencapsulated strains of H. catarrhalis are involved in AOM, vaccines against bacterial cellular factors other than capsules will need to be developed.

Bacterial Rhinosinusitis

The microbial community of the nasopharynx is extremely diverse and harbors many opportunistic pathogens, so it is perhaps not surprising that infections leading to rhinitis and sinusitis have many possible causes. These conditions often occur as secondary infections after a viral infection, which effectively compromises the immune defenses and allows the opportunistic bacteria to establish themselves. Bacterial sinusitis involves infection and inflammation within the paranasal sinuses. Because bacterial sinusitis rarely occurs without rhinitis, the preferred term is rhinosinusitis. The most common causes of bacterial rhinosinusitis are similar to those for AOM, including S. pneumoniae, H. influenzae, and M. catarrhalis.

Exercise (PageIndex{2})

  1. What are the usual causative agents of acute otitis media?
  2. What factors facilitate acute otitis media with effusion in young children?
  3. What factor often triggers bacterial rhinosinusitis?

Diphtheria

The causative agent of diphtheria, Corynebacterium diphtheriae, is a club-shaped, gram-positive rod that belongs to the phylum Actinobacteria. Diphtheroids are common members of the normal nasopharyngeal microbiota. However, some strains of C. diphtheriae become pathogenic because of the presence of a temperate bacteriophage-encoded protein—the diphtheria toxin. Diphtheria is typically a respiratory infection of the oropharynx but can also cause impetigo-like lesions on the skin. Although the disease can affect people of all ages, it tends to be most severe in those younger than 5 years or older than 40 years. Like strep throat, diphtheria is commonly transmitted in the droplets and aerosols produced by coughing. After colonizing the throat, the bacterium remains in the oral cavity and begins producing the diphtheria toxin. This protein is an A-B toxin that blocks host-cell protein synthesis by inactivating elongation factor (EF)-2 (see Virulence Factors of Bacterial and Viral Pathogens). The toxin’s action leads to the death of the host cells and an inflammatory response. An accumulation of grayish exudate consisting of dead host cells, pus, red blood cells, fibrin, and infectious bacteria results in the formation of a pseudomembrane. The pseudomembrane can cover mucous membranes of the nasal cavity, tonsils, pharynx, and larynx (Figure (PageIndex{4})). This is a classic sign of diphtheria. As the disease progresses, the pseudomembrane can enlarge to obstruct the fauces of the pharynx or trachea and can lead to suffocation and death. Sometimes, intubation, the placement of a breathing tube in the trachea, is required in advanced infections. If the diphtheria toxin spreads throughout the body, it can damage other tissues as well. This can include myocarditis (heart damage) and nerve damage that may impair breathing.

The presumptive diagnosis of diphtheria is primarily based on the clinical symptoms (i.e., the pseudomembrane) and vaccination history, and is typically confirmed by identifying bacterial cultures obtained from throat swabs. The diphtheria toxin itself can be directly detected in vitro using polymerase chain reaction (PCR)-based, direct detection systems for the diphtheria tox gene, and immunological techniques like radial immunodiffusion or Elek’s immunodiffusion test.

Broad-spectrum antibiotics like penicillin and erythromycin tend to effectively control C. diphtheriae infections. Regrettably, they have no effect against preformed toxins. If toxin production has already occurred in the patient, antitoxins (preformed antibodies against the toxin) are administered. Although this is effective in neutralizing the toxin, the antitoxins may lead to serum sickness because they are produced in horses (see Hypersensitivities).

Widespread vaccination efforts have reduced the occurrence of diphtheria worldwide. There are currently four combination toxoid vaccines available that provide protection against diphtheria and other diseases: DTaP, Tdap, DT, and Td. In all cases, the letters “d,” “t,” and “p” stand for diphtheria, tetanus, and pertussis, respectively; the “a” stands for acellular. If capitalized, the letters indicate a full-strength dose; lowercase letters indicate reduced dosages. According to current recommendations, children should receive five doses of the DTaP vaccine in their youth and a Td booster every 10 years. Children with adverse reactions to the pertussis vaccine may be given the DT vaccine in place of the DTaP.

Exercise (PageIndex{3})

  1. What effect does diphtheria toxin have?
  2. What is the pseudomembrane composed of?

Bacterial Pneumonia

Pneumonia is a general term for infections of the lungs that lead to inflammation and accumulation of fluids and white blood cells in the alveoli. Pneumonia can be caused by bacteria, viruses, fungi, and other organisms, although the vast majority of pneumonias are bacterial in origin. Bacterial pneumonia is a prevalent, potentially serious infection; it caused more 50,000 deaths in the United States in 2014.3 As the alveoli fill with fluids and white blood cells (consolidation), air exchange becomes impaired and patients experience respiratory distress (Figure (PageIndex{5})). In addition, pneumonia can lead to pleurisy, an infection of the pleural membrane surrounding the lungs, which can make breathing very painful. Although many different bacteria can cause pneumonia under the right circumstances, three bacterial species cause most clinical cases: Streptococcus pneumoniae, H. influenzae, and Mycoplasma pneumoniae. In addition to these, we will also examine some of the less common causes of pneumonia.

Pneumococcal Pneumonia

The most common cause of community-acquired bacterial pneumonia is Streptococcus pneumoniae. This gram-positive, alpha hemolytic streptococcus is commonly found as part of the normal microbiota of the human respiratory tract. The cells tend to be somewhat lancet-shaped and typically appear as pairs (Figure (PageIndex{6})). The pneumococci initially colonize the bronchioles of the lungs. Eventually, the infection spreads to the alveoli, where the microbe’s polysaccharide capsule interferes with phagocytic clearance. Other virulence factors include autolysins like Lyt A, which degrade the microbial cell wall, resulting in cell lysis and the release of cytoplasmic virulence factors. One of these factors, pneumolysin O, is important in disease progression; this pore-forming protein damages host cells, promotes bacterial adherence, and enhances pro-inflammatory cytokine production. The resulting inflammatory response causes the alveoli to fill with exudate rich in neutrophils and red blood cells. As a consequence, infected individuals develop a productive cough with bloody sputum.

Pneumococci can be presumptively identified by their distinctive gram-positive, lancet-shaped cell morphology and diplococcal arrangement. In blood agar cultures, the organism demonstrates alpha hemolytic colonies that are autolytic after 24 to 48 hours. In addition, S. pneumoniae is extremely sensitive to optochin and colonies are rapidly destroyed by the addition of 10% solution of sodium deoxycholate. All clinical pneumococcal isolates are serotyped using the quellung reaction with typing antisera produced by the CDC. Positive quellung reactions are considered definitive identification of pneumococci.

Antibiotics remain the mainstay treatment for pneumococci. β-Lactams like penicillin are the first-line drugs, but resistance to β-lactams is a growing problem. When β-lactam resistance is a concern, macrolides and fluoroquinolones may be prescribed. However, S. pneumoniae resistance to macrolides and fluoroquinolones is increasing as well, limiting the therapeutic options for some infections. There are currently two pneumococcal vaccines available: pneumococcal conjugate vaccine (PCV13) and pneumococcal polysaccharide vaccine (PPSV23). These are generally given to the most vulnerable populations of individuals: children younger than 2 years and adults older than 65 years.

Haemophilus Pneumonia

Encapsulated strains of Haemophilus influenzae are known for causing meningitis, but nonencapsulated strains are important causes of pneumonia. This small, gram-negative coccobacillus is found in the pharynx of the majority of healthy children; however, Haemophilus pneumonia is primarily seen in the elderly. Like other pathogens that cause pneumonia, H. influenzae is spread by droplets and aerosols produced by coughing. A fastidious organism, H. influenzae will only grow on media with available factor X (hemin) and factor V (NAD), like chocolate agar (Figure (PageIndex{7})). Serotyping must be performed to confirm identity of H. influenzae isolates.

Infections of the alveoli by H. influenzae result in inflammation and accumulation of fluids. Increasing resistance to β-lactams, macrolides, and tetracyclines presents challenges for the treatment of Haemophilus pneumonia. Resistance to the fluoroquinolones is rare among isolates of H. influenzae but has been observed. As discussed for AOM, a vaccine directed against nonencapsulated H. influenzae, if developed, would provide protection against pneumonia caused by this pathogen.

WHY ME?

Tracy is a 6-year old who developed a serious cough that would not seem to go away. After 2 weeks, her parents became concerned and took her to the pediatrician, who suspected a case of bacterial pneumonia. Tests confirmed that the cause was Haemophilus influenzae. Fortunately, Tracy responded well to antibiotic treatment and eventually made a full recovery.

Because there had been several other cases of bacterial pneumonia at Tracy’s elementary school, local health officials urged parents to have their children screened. Of the children who were screened, it was discovered that greater than 50% carried H. influenzae in their nasal cavities, yet all but two of them were asymptomatic.

Why is it that some individuals become seriously ill from bacterial infections that seem to have little or no effect on others? The pathogenicity of an organism—its ability to cause host damage—is not solely a property of the microorganism. Rather, it is the product of a complex relationship between the microbe’s virulence factors and the immune defenses of the individual. Preexisting conditions and environmental factors such as exposure to secondhand smoke can make some individuals more susceptible to infection by producing conditions favorable to microbial growth or compromising the immune system. In addition, individuals may have genetically determined immune factors that protect them—or not—from particular strains of pathogens. The interactions between these host factors and the pathogenicity factors produced by the microorganism ultimately determine the outcome of the infection. A clearer understanding of these interactions may allow for better identification of at-risk individuals and prophylactic interventions in the future.

Mycoplasma Pneumonia (Walking Pneumonia)

Primary atypical pneumonia is caused by Mycoplasma pneumoniae. This bacterium is not part of the respiratory tract’s normal microbiota and can cause epidemic disease outbreaks. Also known as walking pneumonia, mycoplasmapneumonia infections are common in crowded environments like college campuses and military bases. It is spread by aerosols formed when coughing or sneezing. The disease is often mild, with a low fever and persistent cough. These bacteria, which do not have cell walls, use a specialized attachment organelle to bind to ciliated cells. In the process, epithelial cells are damaged and the proper function of the cilia is hindered (Figure (PageIndex{8})).

Mycoplasma grow very slowly when cultured. Therefore, penicillin and thallium acetate are added to agar to prevent the overgrowth by faster-growing potential contaminants. Since M. pneumoniae does not have a cell wall, it is resistant to these substances. Without a cell wall, the microbial cells appear pleomorphic. M. pneumoniae infections tend to be self-limiting but may also respond well to macrolide antibiotic therapy. β-lactams, which target cell wall synthesis, are not indicated for treatment of infections with this pathogen.

Chlamydial Pneumonias and Psittacosis

Chlamydial pneumonia can be caused by three different species of bacteria: Chlamydophila pneumoniae (formerly known as Chlamydia pneumoniae), Chlamydophila psittaci (formerly known as Chlamydia psittaci), and Chlamydia trachomatis. All three are obligate intracellular pathogens and cause mild to severe pneumonia and bronchitis. Of the three, Chlamydophila pneumoniae is the most common and is transmitted via respiratory droplets or aerosols. C. psittaci causes psittacosis, a zoonotic disease that primarily affects domesticated birds such as parakeets, turkeys, and ducks, but can be transmitted from birds to humans. Psittacosis is a relatively rare infection and is typically found in people who work with birds. Chlamydia trachomatis, the causative agent of the sexually transmitted disease chlamydia, can cause pneumonia in infants when the infection is passed from mother to baby during birth.

Diagnosis of chlamydia by culturing tends to be difficult and slow. Because they are intracellular pathogens, they require multiple passages through tissue culture. Recently, a variety of PCR- and serologically based tests have been developed to enable easier identification of these pathogens. Tetracycline and macrolide antibiotics are typically prescribed for treatment.

Health Care-Associated Pneumonia

A variety of opportunistic bacteria that do not typically cause respiratory disease in healthy individuals are common causes of health care-associated pneumonia. These include Klebsiella pneumoniae, Staphylococcus aureus, and proteobacteria such as species of Escherichia, Proteus, and Serratia. Patients at risk include the elderly, those who have other preexisting lung conditions, and those who are immunocompromised. In addition, patients receiving supportive therapies such as intubation, antibiotics, and immunomodulatory drugs may also be at risk because these interventions disrupt the mucociliary escalator and other pulmonary defenses. Invasive medical devices such as catheters, medical implants, and ventilators can also introduce opportunistic pneumonia-causing pathogens into the body.4

Pneumonia caused by K. pneumoniae is characterized by lung necrosis and “currant jelly sputum,” so named because it consists of clumps of blood, mucus, and debris from the thick polysaccharide capsule produced by the bacterium. K. pneumoniae is often multidrug resistant. Aminoglycoside and cephalosporin are often prescribed but are not always effective. Klebsiella pneumonia is frequently fatal even when treated.

Pseudomonas Pneumonia

Pseudomonas aeruginosa is another opportunistic pathogen that can cause serious cases of bacterial pneumonia in patients with cystic fibrosis (CF) and hospitalized patients assisted with artificial ventilators. This bacterium is extremely antibiotic resistant and can produce a variety of exotoxins. Ventilator-associated pneumonia with P. aeruginosa is caused by contaminated equipment that causes the pathogen to be aspirated into the lungs. In patients with CF, a genetic defect in the cystic fibrosis transmembrane receptor (CFTR) leads to the accumulation of excess dried mucus in the lungs. This decreases the effectiveness of the defensins and inhibits the mucociliary escalator. P. aeruginosa is known to infect more than half of all patients with CF. It adapts to the conditions in the patient’s lungs and begins to produce alginate, a viscous exopolysaccharide that inhibits the mucociliary escalator. Lung damage from the chronic inflammatory response that ensues is the leading cause of mortality in patients with CF.5

Exercise (PageIndex{4})

  1. What three pathogens are responsible for the most prevalent types of bacterial pneumonia?
  2. Which cause of pneumonia is most likely to affect young people?
  3. In what contexts does Pseudomonas aeruginosa cause pneumonia?

clinical focus - part 2

John’s chest radiograph revealed an extensive consolidation in the right lung, and his sputum cultures revealed the presence of a gram-negative rod. His physician prescribed a course of the antibiotic clarithromycin. He also ordered the rapid influenza diagnostic tests (RIDTs) for type A and B influenza to rule out a possible underlying viral infection. Despite antibiotic therapy, John’s condition continued to deteriorate, so he was admitted to the hospital.

Exercise (PageIndex{5})

What are some possible causes of pneumonia that would not have responded to the prescribed antibiotic?

Tuberculosis

Tuberculosis (TB) is one of the deadliest infectious diseases in human history. Although tuberculosis infection rates in the United States are extremely low, the CDC estimates that about one-third of the world’s population is infected with Mycobacterium tuberculosis, the causal organism of TB, with 9.6 million new TB cases and 1.5 million deaths worldwide in 2014.6

M. tuberculosis is an acid-fast, high G + C, gram-positive, nonspore-forming rod. Its cell wall is rich in waxy mycolic acids, which make the cells impervious to polar molecules. It also causes these organisms to grow slowly. tuberculosis causes a chronic granulomatous disease that can infect any area of the body, although it is typically associated with the lungs. tuberculosis is spread by inhalation of respiratory droplets or aerosols from an infected person. The infectious dose of M. tuberculosis is only 10 cells.7

After inhalation, the bacteria enter the alveoli (Figure (PageIndex{9})). The cells are phagocytized by macrophages but can survive and multiply within these phagocytes because of the protection by the waxy mycolic acid in their cell walls. If not eliminated by macrophages, the infection can progress, causing an inflammatory response and an accumulation of neutrophils and macrophages in the area. Several weeks or months may pass before an immunological response is mounted by T cells and B cells. Eventually, the lesions in the alveoli become walled off, forming small round lesions called tubercles. Bacteria continue to be released into the center of the tubercles and the chronic immune response results in tissue damage and induction of apoptosis (programmed host-cell death) in a process called liquefaction. This creates a caseous center, or air pocket, where the aerobic M. tuberculosis can grow and multiply. Tubercles may eventually rupture and bacterial cells can invade pulmonary capillaries; from there, bacteria can spread through the bloodstream to other organs, a condition known as miliary tuberculosis. The rupture of tubercles also facilitates transmission of the bacteria to other individuals via droplet aerosols that exit the body in coughs. Because these droplets can be very small and stay aloft for a long time, special precautions are necessary when caring for patients with TB, such as the use of face masks and negative-pressure ventilation and filtering systems.

Eventually, most lesions heal to form calcified Ghon complexes. These structures are visible on chest radiographs and are a useful diagnostic feature. But even after the disease has apparently ended, viable bacteria remain sequestered in these locations. Release of these organisms at a later time can produce reactivation tuberculosis (or secondary TB). This is mainly observed in people with alcoholism, the elderly, or in otherwise immunocompromised individuals (Figure (PageIndex{9})).

Because TB is a chronic disease, chemotherapeutic treatments often continue for months or years. Multidrug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of M. tuberculosis are a growing clinical concern. These strains can arise due to misuse or mismanagement of antibiotic therapies. Therefore, it is imperative that proper multidrug protocols are used to treat these infections. Common antibiotics included in these mixtures are isoniazid, rifampin, ethambutol, and pyrazinamide.

A TB vaccine is available that is based on the so-called bacillus Calmette-Guérin (BCG) strain of M. bovis commonly found in cattle. In the United States, the BCG vaccine is only given to health-care workers and members of the military who are at risk of exposure to active cases of TB. It is used more broadly worldwide. Many individuals born in other countries have been vaccinated with BCG strain. BCG is used in many countries with a high prevalence of TB, to prevent childhood tuberculous meningitis and miliary disease.

The Mantoux tuberculin skin test (Figure (PageIndex{10})) is regularly used in the United States to screen for potential TB exposure (see Hypersensitivities). However, prior vaccinations with the BCG vaccine can cause false-positive results. Chest radiographs to detect Ghon complex formation are required, therefore, to confirm exposure.

Exercise (PageIndex{6})

  1. What characteristic of Mycobacterium tuberculosis allows it to evade the immune response?
  2. What happens to cause miliary tuberculosis?
  3. Explain the limitations of the Mantoux tuberculin skin test.

Pertussis (Whooping Cough)

The causative agent of pertussis, commonly called whooping cough, is Bordetella pertussis, a gram-negative coccobacillus. The disease is characterized by mucus accumulation in the lungs that leads to a long period of severe coughing. Sometimes, following a bout of coughing, a sound resembling a “whoop” is produced as air is inhaled through the inflamed and restricted airway—hence the name whooping cough. Although adults can be infected, the symptoms of this disease are most pronounced in infants and children. Pertussis is highly communicable through droplet transmission, so the uncontrollable coughing produced is an efficient means of transmitting the disease in a susceptible population.

Following inhalation, B. pertussis specifically attaches to epithelial cells using an adhesin, filamentous hemagglutinin. The bacteria then grow at the site of infection and cause disease symptoms through the production of exotoxins. One of the main virulence factors of this organism is an A-B exotoxin called the pertussis toxin (PT). When PT enters the host cells, it increases the cyclic adenosine monophosphate (cAMP) levels and disrupts cellular signaling. PT is known to enhance inflammatory responses involving histamine and serotonin. In addition to PT, B. pertussis produces a tracheal cytotoxin that damages ciliated epithelial cells and results in accumulation of mucus in the lungs. The mucus can support the colonization and growth of other microbes and, as a consequence, secondary infections are common. Together, the effects of these factors produce the cough that characterizes this infection.

A pertussis infection can be divided into three distinct stages. The initial infection, termed the catarrhal stage, is relatively mild and unremarkable. The signs and symptoms may include nasal congestion, a runny nose, sneezing, and a low-grade fever. This, however, is the stage in which B. pertussis is most infectious. In the paroxysmal stage, mucus accumulation leads to uncontrollable coughing spasms that can last for several minutes and frequently induce vomiting. The paroxysmal stage can last for several weeks. A long convalescence stage follows the paroxysmal stage, during which time patients experience a chronic cough that can last for up to several months. In fact, the disease is sometimes called the 100-day cough.

In infants, coughing can be forceful enough to cause fractures to the ribs, and prolonged infections can lead to death. The CDC reported 20 pertussis-related deaths in 2012,9 but that number had declined to five by 2015.10

During the first 2 weeks of infection, laboratory diagnosis is best performed by culturing the organism directly from a nasopharyngeal (NP) specimen collected from the posterior nasopharynx. The NP specimen is streaked onto Bordet-Gengou medium. The specimens must be transported to the laboratory as quickly as possible, even if transport media are used. Transport times of longer than 24 hours reduce the viability of B. pertussis significantly.

Within the first month of infection, B. pertussis can be diagnosed using PCR techniques. During the later stages of infection, pertussis-specific antibodies can be immunologically detected using an enzyme-linked immunosorbent assay (ELISA).

Pertussis is generally a self-limiting disease. Antibiotic therapy with erythromycin or tetracycline is only effective at the very earliest stages of disease. Antibiotics given later in the infection, and prophylactically to uninfected individuals, reduce the rate of transmission. Active vaccination is a better approach to control this disease. The DPT vaccine was once in common use in the United States. In that vaccine, the P component consisted of killed whole-cell B. pertussis preparations. Because of some adverse effects, that preparation has now been superseded by the DTaP and Tdap vaccines. In both of these new vaccines, the “aP” component is a pertussis toxoid.

Widespread vaccination has greatly reduced the number of reported cases and prevented large epidemics of pertussis. Recently, however, pertussis has begun to reemerge as a childhood disease in some states because of declining vaccination rates and an increasing population of susceptible children.

This web page contains an audio clip of the distinctive “whooping” sound associated with pertussis in infants.

This interactive map shows outbreaks of vaccine preventable diseases, including pertussis, around the world.

Exercise (PageIndex{7})

  1. What accounts for the mucus production in a pertussis infection?
  2. What are the signs and symptoms associated with the three stages of pertussis?
  3. Why is pertussis becoming more common in the United States?

Legionnaires Disease

An atypical pneumonia called Legionnaires disease (also known as legionellosis) is caused by an aerobic gram-negative bacillus, Legionella pneumophila. This bacterium infects free-living amoebae that inhabit moist environments, and infections typically occur from human-made reservoirs such as air-conditioning cooling towers, humidifiers, misting systems, and fountains. Aerosols from these reservoirs can lead to infections of susceptible individuals, especially those suffering from chronic heart or lung disease or other conditions that weaken the immune system.

When L. pneumophila bacteria enter the alveoli, they are phagocytized by resident macrophages. However, L. pneumophila uses a secretion system to insert proteins in the endosomal membrane of the macrophage; these proteins prevent lysosomal fusion, allowing L. pneumophila to continue to proliferate within the phagosome. The resulting respiratory disease can range from mild to severe pneumonia, depending on the status of the host’s immune defenses. Although this disease primarily affects the lungs, it can also cause fever, nausea, vomiting, confusion, and other neurological effects.

Diagnosis of Legionnaires disease is somewhat complicated. L. pneumophila is a fastidious bacterium and is difficult to culture. In addition, since the bacterial cells are not efficiently stained with the Gram stain, other staining techniques, such as the Warthin-Starry silver-precipitate procedure, must be used to visualize this pathogen. A rapid diagnostic test has been developed that detects the presence of Legionella antigen in a patient’s urine; results take less than 1 hour, and the test has high selectivity and specificity (greater than 90%). Unfortunately, the test only works for one serotype of L. pneumophila (type 1, the serotype responsible for most infections). Consequently, isolation and identification of L. pneumophila from sputum remains the defining test for diagnosis.

Once diagnosed, Legionnaire disease can be effectively treated with fluoroquinolone and macrolide antibiotics. However, the disease is sometimes fatal; about 10% of patients die of complications.11 There is currently no vaccine available.

Exercise (PageIndex{8})

  • Why is Legionnaires disease associated with air-conditioning systems?
  • How does Legionella pneumophila circumvent the immune system?

Q Fever

The zoonotic disease Q fever is caused by a rickettsia, Coxiella burnetii. The primary reservoirs for this bacterium are domesticated livestock such as cattle, sheep, and goats. The bacterium may be transmitted by ticks or through exposure to the urine, feces, milk, or amniotic fluid of an infected animal. In humans, the primary route of infection is through inhalation of contaminated farmyard aerosols. It is, therefore, largely an occupational disease of farmers. Humans are acutely sensitive to C. burnetii—the infective dose is estimated to be just a few cells.12 In addition, the organism is hardy and can survive in a dry environment for an extended time. Symptoms associated with acute Q fever include high fever, headache, coughing, pneumonia, and general malaise. In a small number of patients (less than 5%13), the condition may become chronic, often leading to endocarditis, which may be fatal.

Diagnosing rickettsial infection by cultivation in the laboratory is both difficult and hazardous because of the easy aerosolization of the bacteria, so PCR and ELISA are commonly used. Doxycycline is the first-line drug to treat acute Q fever. In chronic Q fever, doxycycline is often paired with hydroxychloroquine.

BACTERIAL DISEASES OF THE RESPIRATORY TRACT

Numerous pathogens can cause infections of the respiratory tract. Many of these infections produce similar signs and symptoms, but appropriate treatment depends on accurate diagnosis through laboratory testing. The tables in Figure (PageIndex{11}) and Figure (PageIndex{12}) summarize the most important bacterial respiratory infections, with the latter focusing specifically on forms of bacterial pneumonia.

Key Concepts and Summary

  • A wide variety of bacteria can cause respiratory diseases; most are treatable with antibiotics or preventable with vaccines.
  • Streptococcus pyogenes causes strep throat, an infection of the pharynx that also causes high fever and can lead to scarlet fever, acute rheumatic fever, and acute glomerulonephritis.
  • Acute otitis media is an infection of the middle ear that may be caused by several bacteria, including Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The infection can block the eustachian tubes, leading to otitis media with effusion.
  • Diphtheria, caused by Corynebacterium diphtheriae, is now a rare disease because of widespread vaccination. The bacteria produce exotoxins that kill cells in the pharynx, leading to the formation of a pseudomembrane; and damage other parts of the body.
  • Bacterial pneumonia results from infections that cause inflammation and fluid accumulation in the alveoli. It is most commonly caused by S. pneumoniae or H. influenzae. The former is commonly multidrug resistant.
  • Mycoplasma pneumonia results from infection by Mycoplasma pneumoniae; it can spread quickly, but the disease is mild and self-limiting.
  • Chlamydial pneumonia can be caused by three pathogens that are obligate intracellular parasites. Chlamydophila pneumoniae is typically transmitted from an infected person, whereas C. psittaci is typically transmitted from an infected bird. Chlamydia trachomatis, may cause pneumonia in infants.
  • Several other bacteria can cause pneumonia in immunocompromised individuals and those with cystic fibrosis.
  • Tuberculosis is caused by Mycobacterium tuberculosis. Infection leads to the production of protective tuberclesin the alveoli and calcified Ghon complexes that can harbor the bacteria for a long time. Antibiotic-resistant forms are common and treatment is typically long term.
  • Pertussis is caused by Bordetella pertussis. Mucus accumulation in the lungs leads to prolonged severe coughing episodes (whooping cough) that facilitate transmission. Despite an available vaccine, outbreaks are still common.
  • Legionnaires disease is caused by infection from environmental reservoirs of the Legionella pneumophila bacterium. The bacterium is endocytic within macrophages and infection can lead to pneumonia, particularly among immunocompromised individuals.
  • Q fever is caused by Coxiella burnetii, whose primary hosts are domesticated mammals (zoonotic disease). It causes pneumonia primarily in farm workers and can lead to serious complications, such as endocarditis.

Multiple Choice

Which of the following does not involve a bacterial exotoxin?

A. diphtheria
B. whooping cough
C. scarlet fever
D. Q fever

D

What disease is caused by Coxiella burnetii?

A. Q fever
B. tuberculosis
C. diphtheria
D. walking pneumonia

A

In which stage of pertussis is the characteristic whooping sound made?

A. convalescence
B. catarrhal
C. paroxysmal
D. prodromal

C

What is the causative agent of Q fever?

A. Coxiella burnetii
B. Chlamydophila psittaci
C. Mycoplasma pneumoniae
D. Streptococcus pyogenes

A

Which of these microbes causes “walking pneumonia”?

A. Klebsiella pneumoniae
B. Streptococcus pneumoniae
C. Chlamydophila pneumoniae

C

Fill in the Blank

Calcified lesions called _______ form in the lungs of patients with TB.

Ghon complexes

An inflammation of the middle ear is called _______.

otitis media

The _______ is used to serologically identify Streptococcus pneumoniae isolates.

quellung reaction

_______ is a zoonotic infection that can be contracted by people who handle birds.

Psittacosis

The main virulence factor involved in scarlet fever is the _______.

erythrogenic toxin

Short Answer

Name three bacteria that commonly cause pneumonia. Which is the most common cause?

How does smoking make an individual more susceptible to infections?

How does the diphtheria pathogen form a pseudomembrane?

Critical Thinking

Why might β-lactam antibiotics be ineffective against Mycoplasma pneumoniae infections?

Why is proper antibiotic therapy especially important for patients with tuberculosis?

Footnotes

  1. 1 WL Lean et al. “Rapid Diagnostic Tests for Group A Streptococcal Pharyngitis: A Meta-Analysis.” Pediatrics 134, no. 4 (2014):771–781.
  2. 2 G. Worrall. “Acute Otitis Media.” Canadian Family Physician 53 no. 12 (2007):2147–2148.
  3. 3 KD Kochanek et al. “Deaths: Final Data for 2014.” National Vital Statistics Reports 65 no 4 (2016).
  4. 4 SM Koenig et al. “Ventilator-Associated Pneumonia: Diagnosis, Treatment, and Prevention.” Clinical Microbiology Reviews 19 no. 4 (2006):637–657.
  5. 5 R. Sordé et al. “Management of Refractory Pseudomonas aeruginosa Infection in Cystic Fibrosis.” Infection and Drug Resistance 4 (2011):31–41.
  6. 6 Centers for Disease Control and Prevention. “Tuberculosis (TB). Data and Statistics.” http://www.cdc.gov/tb/statistics/default.htm
  7. 7 D. Saini et al. “Ultra-Low Dose of Mycobacterium tuberculosis Aerosol Creates Partial Infection in Mice.” Tuberculosis 92 no. 2 (2012):160–165.
  8. 8 G. Kaplan et al. “Mycobacterium tuberculosis Growth at the Cavity Surface: A Microenvironment with Failed Immunity.” Infection and Immunity 71 no.12 (2003):7099–7108.
  9. 9 Centers for Disease Control and Prevention. “2012 Final Pertussis Surveillance Report.” 2015. http://www.cdc.gov/pertussis/downloa...eport-2012.pdf. Accessed July 6, 2016.
  10. 10 Centers for Disease Control and Prevention. “2015 Provisional Pertussis Surveillance Report.” 2016. http://www.cdc.gov/pertussis/downloa...rovisional.pdf. Accessed July 6, 2016.
  11. 11 Centers for Disease Control and Prevention. “Legionella (Legionnaires’ Disease and Pontiac Fever: Diagnosis, Treatment, and Complications).” http://www.cdc.gov/legionella/about/diagnosis.html. Accessed Sept 14, 2016.
  12. 12 WD Tigertt et al. “Airborne Q Fever.” Bacteriological Reviews 25 no. 3 (1961):285–293.
  13. 13 Centers for Disease Control and Prevention. “Q fever. Symptoms, Diagnosis, and Treatment.” 2013. http://www.cdc.gov/qfever/symptoms/index.html. Accessed July 6, 2016.

Bacterial battles in chronic respiratory infection

Applications are invited from candidates with a background in Microbiology, Bioinformatics, Biochemistry, Biomedical Sciences or a related discipline, for a 3 year PhD studentship to investigate the mechanisms underlying the virulence mechanisms of Pseudomonas aeruginosa. A background in Microbial Genomics and Molecular Microbiology would be helpful.

Project description

Pseudomonas aeruginosa is ranked 2nd on the critical priority list by the World Health Organisation (WHO) for pathogens requiring new antibiotics/treatment strategies. This is due to its ability to cause severe and often deadly infections, its intrinsically high level of antimicrobial resistance to a broad range of antibiotics and capacity to form biofilms. These infections occur at a range of sites including the respiratory tract, urinary tract, gastrointestinal tract, eyes, ears and bloodstream. In addition, this bacterium causes chronic infections in cystic fibrosis (CF) patients that have few treatment options and is one of the primary causes of mortality for those patients. Over time P. aeruginosa tends to dominate and outcompete organisms in these niches through competition and high resistance to certain antibiotics. An exciting PhD position exists to investigate mechanisms and factors that enable this to occur and will build on the momentum of recent high impact publications (https://www.pnas.org/content/114/29/7707.short?rss=1, https://www.pnas.org/content/115/49/12519) in Proceedings of the National Academy of Sciences

and https://www.atsjournals.org/doi/pdf/10.1164/rccm.202009-3639LE in the American Journal of Respiratory and Critical Care Medicine. This post will be co-supervised by Dr Luke Allsopp and Professor Jane Davies enabling strong real world links and access to diverse clinical isolates. This cutting edge molecular microbiological project requires an enthusiastic candidate with a passion for investigating microbial interactions. Candidates with strengths or demonstrable interests in Bioinformatics and Microbial Genomics are particularly encouraged to apply.

Imperial College London provides excellent opportunities for research students' training. All students benefit from a full programme of training in research and transferable skills organised through the Graduate School, the quality of which has been recognised several times at the Times Higher Education (THE) Awards.

The student will be based in the National Heart and Lung Institute which provides an exciting environment, with state of the art facilities and excellent opportunities for PhD student training including research seminars and journal clubs. In addition, the institute provides extensive collaborative opportunities with other research groups. Prof Davies holds a clinical position at our partner institution, the Royal Brompton & Harefield Hospital, one of the largest cystic fibrosis centres in Europe, providing unparalleled access to patient samples and clinical expertise. The project will be embedded within the Strategic Research Centre for Pseudomonas Infection in Cystic Fibrosis, a multi-centre collaborative of scientists and research-active clinical and allied health professionals. The position will be based in central London in a fully equipped class 2 biosafety laboratory and nestled in the vibrant research groups of Luke Allsopp and Jane Davies.

Eligibility

Applicants must hold a first or upper second-class undergraduate degree or UK equivalent. A Masters degree (merit or above) in an appropriate subject and from a recognised academic institution is strongly desirable (or expect to obtain) but not necessary. Strong candidates who are nearing Masters completion after July are invited to apply and make it clear they wish to be considered for this and future opportunities.

How to Apply

To apply please send a CV, a one page personal statement, and the names and addresses of at least two academic referees to Dr Luke Allsopp by email on [Email Address Removed]. Please note that candidates must fulfil College admissions criteria.

Application deadline: 3 rd of May

Proposed virtual interview date: 14 and 17 th of May.

Committed to equality and valuing diversity. We are also an Athena SWAN Silver Award winner, a Stonewall Diversity Champion, a Disability Confident Employer and are working in partnership with GIRES to promote respect for trans people.


Upper Respiratory Infections

Infections of the respiratory tract are grouped according to their symptomatology and anatomic involvement. Acute upper respiratory infections (URI) include the common cold, pharyngitis, epiglottitis, and laryngotracheitis (Fig. 93-1). These infections are usually benign, transitory and self-limited, altho ugh epiglottitis and laryngotracheitis can be serious diseases in children and young infants. Etiologic agents associated with URI include viruses, bacteria, mycoplasma and fungi (Table 93-1). Respiratory infections are more common in the fall and winter when school starts and indoor crowding facilitates transmission.

Figure 93-1

Upper and lower respiratory tract infections.

Common Cold

Etiology

Common colds are the most prevalent entity of all respiratory infections and are the leading cause of patient visits to the physician, as well as work and school absenteeism. Most colds are caused by viruses. Rhinoviruses with more than 100 serotypes are the most common pathogens, causing at least 25% of colds in adults. Coronaviruses may be responsible for more than 10% of cases. Parainfluenza viruses, respiratory syncytial virus, adenoviruses and influenza viruses have all been linked to the common cold syndrome. All of these organisms show seasonal variations in incidence. The cause of 30% to 40% of cold syndromes has not been determined.

Pathogenesis

The viruses appear to act through direct invasion of epithelial cells of the respiratory mucosa (Fig. 93-2), but whether there is actual destruction and sloughing of these cells or loss of ciliary activity depends on the specific organism involved. There is an increase in both leukocyte infiltration and nasal secretions, including large amounts of protein and immunoglobulin, suggesting that cytokines and immune mechanisms may be responsible for some of the manifestations of the common cold (Fig. 93-3).

Figure 93-2

Pathogenesis of viral and bacterial mucosal respiratory infections.

Figure 93-3

Pathogenesis of upper respiratory tract infections.

Clinical Manifestations

After an incubation period of 48� hours, classic symptoms of nasal discharge and obstruction, sneezing, sore throat and cough occur in both adults and children. Myalgia and headache may also be present. Fever is rare. The duration of symptoms and of viral shedding varies with the pathogen and the age of the patient. Complications are usually rare, but sinusitis and otitis media may follow.

Microbiologic Diagnosis

The diagnosis of a common cold is usually based on the symptoms (lack of fever combined with symptoms of localization to the nasopharynx). Unlike allergic rhinitis, eosinophils are absent in nasal secretions. Although it is possible to isolate the viruses for definitive diagnosis, that is rarely warranted.

Prevention and Treatment

Treatment of the uncomplicated common cold is generally symptomatic. Decongestants, antipyretics, fluids and bed rest usually suffice. Restriction of activities to avoid infecting others, along with good hand washing, are the best measures to prevent spread of the disease. No vaccine is commercially available for cold prophylaxis.

Sinusitis

Sinusitis is an acute inflammatory condition of one or more of the paranasal sinuses. Infection plays an important role in this affliction. Sinusitis often results from infections of other sites of the respiratory tract since the paranasal sinuses are contiguous to, and communicate with, the upper respiratory tract.

Etiology

Acute sinusitis most often follows a common cold which is usually of viral etiology. Vasomotor and allergic rhinitis may also be antecedent to the development of sinusitis. Obstruction of the sinusal ostia due to deviation of the nasal septum, presence of foreign bodies, polyps or tumors can predispose to sinusitis. Infection of the maxillary sinuses may follow dental extractions or an extension of infection from the roots of the upper teeth. The most common bacterial agents responsible for acute sinusitis are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Other organisms including Staphylococcus aureus, Streptococcus pyogenes, gram-negative organisms and anaerobes have also been recovered. Chronic sinusitis is commonly a mixed infection of aerobic and anaerobic organisms.

Pathogenesis

Infections caused by viruses or bacteria impair the ciliary activity of the epithelial lining of the sinuses and increased mucous secretions. This leads to obstruction of the paranasal sinusal ostia which impedes drainage. With bacterial multiplication in the sinus cavities, the mucus is converted to mucopurulent exudates. The pus further irritates the mucosal lining causing more edema, epithelial destruction and ostial obstruction. When acute sinusitis is not resolved and becomes chronic, mucosal thickening results and the development of mucoceles and polyps may ensue.

Clinical Manifestations

The maxillary and ethmoid sinuses are most commonly involved in sinusitis. The frontal sinuses are less often involved and the sphenoid sinuses are rarely affected. Pain, sensation of pressure and tenderness over the affected sinus are present. Malaise and low grade fever may also occur. Physical examination usually is not remarkable with no more than an edematous and hyperemic nasal mucosa.

In uncomplicated chronic sinusitis, a purulent nasal discharge is the most constant finding. There may not be pain nor tenderness over the sinus areas. Thickening of the sinus mucosa and a fluid level are usually seen in x-ray films or magnetic resonance imaging.

Microbiologic Diagnosis

For acute sinusitis, the diagnosis is made from clinical findings. A bacterial culture of the nasal discharge can be taken but is not very helpful as the recovered organisms are generally contaminated by the resident flora from the nasal passage. In chronic sinusitis, a careful dental examination, with sinus x-rays may be required. An antral puncture to obtain sinusal specimens for bacterial culture is needed to establish a specific microbiologic diagnosis.

Prevention and Treatment

Symptomatic treatment with analgesics and moist heat over the affected sinus pain and a decongestant to promote sinus drainage may suffice. For antimicrobial therapy, a beta-lactamase resistant antibiotic such as amoxicillin-clavulanate or a cephalosporin may be used. For chronic sinusitis, when conservative treatment does not lead to a cure, irrigation of the affected sinus may be necessary. Culture from an antral puncture of the maxillary sinus can be performed to identify the causative organism for selecting antimicrobial therapy. Specific preventive procedures are not available. Proper care of infectious and/or allergic rhinitis, surgical correction to relieve or avoid obstruction of the sinusal ostia are important. Root abscesses of the upper teeth should receive proper dental care to avoid secondary infection of the maxillary sinuses.

Otitis

Infections of the ears are common events encountered in medical practice, particularly in young children. Otitis externa is an infection involving the external auditory canal while otitis media denotes inflammation of the middle ear.

Etiology

For otitis externa, the skin flora such as Staphylococcus epidermidis, Staphylococcus aureus, diphtheroids and occasionally an anaerobic organism, Propionibacterium acnes are major etiologic agents. In a moist and warm environment, a diffuse acute otitis externa (Swimmer's ear) may be caused by Pseudomonas aeruginosa, along with other skin flora. Malignant otitis externa is a severe necrotizing infection usually caused by Pseudomonas aeruginosa.

For otitis media, the commonest causative bacteria are Streptococcus pneumoniae, Hemophilus influenzae and beta-lactamase producing Moraxella catarrhalis. Respiratory viruses may play a role in otitis media but this remains uncertain. Mycoplasma pneumoniae has been reported to cause hemorrhagic bullous myringitis in an experimental study among nonimmune human volunteers inoculated with M pneumoniae. However, in natural cases of M pneumoniae infection, clinical bullous myringitis or otitis media is uncommon.

Pathogenesis

The narrow and tortuous auditory canal is lined by a protective surface epithelium. Factors that may disrupt the natural protective mechanisms, such as high temperature and humidity, trauma, allergy, tissue maceration, removal of cerumen and an alkaline pH environment, favor the development of otitis externa. Prolonged immersion in a swimming pool coupled with frequent ear cleansing increases the risk of otitis externa.

Acute otitis media commonly follows an upper respiratory infection extending from the nasopharynx via the eustachian tube to the middle ear. Vigorous nose blowing during a common cold, sudden changes of air pressure, and perforation of the tympanic membrane also favor the development of otitis media. The presence of purulent exudate in the middle ear may lead to a spread of infection to the inner ear and mastoids or even meninges

Clinical Manifestations

Otitis externa

Furuncles of the external ear, similar to those in skin infection, can cause severe pain and a sense of fullness in the ear canal. When the furuncle drains, purulent otorrhea may be present. In generalized otitis externa, itching, pain and tenderness of the ear lobe on traction are present. Loss of hearing may be due to obstruction of the ear canal by swelling and the presence of purulent debris.

Malignant otitis externa tends to occur in elderly diabetic patients. It is characterized by severe persistent earache, foul smelling purulent discharge and the presence of granulation tissue in the auditory canal. The infection may spread and lead to osteomyelitis of the temporal bone or externally to involve the pinna with osteochondritis.

Otitis media

Acute otitis media occurs most commonly in young children. The initial complaint usually is persistent severe earache (crying in the infant) accompanied by fever, and, and vomiting. Otologic examination reveals a bulging, erythematous tympanic membrane with loss of light reflex and landmarks. If perforation of the tympanic membrane occurs, serosanguinous or purulent discharge may be present. In the event of an obstruction of the eustachian tube, accumulation of a usually sterile effusion in the middle ear results in serous otitis media. Chronic otitis media frequently presents a permanent perforation of the tympanic membrane. A central perforation of the pars tensa is more benign. On the other hand, an attic perforation of the pars placcida and marginal perforation of the pars tensa are more dangerous and often associated with a cholesteatoma.

Diagnosis

The diagnosis of both otitis externa and otitis media can be made from history, clinical symptomatology and physical examinations. Inspection of the tympanic membrane is an indispensable skill for physicians and health care workers. All discharge, ear wax and debris must be removed and to perform an adequate otoscopy. In the majority of patients, routine cultures are not necessary, as a number of good bacteriologic studies have shown consistently the same microbial pathogens mentioned in the section of etiology. If the patient is immunocompromised or is toxic and not responding to initial antimicrobial therapy tympanocentesis (needle aspiration) to obtain middle ear effusion for microbiologic culture is indicated.

Prevention and Treatment

Otitis externa

Topical therapy is usually sufficient and systemic antimicrobials are seldom needed unless there are signs of spreading cellulitis and the patient appears toxic. A combination of topical antibiotics such as neomycin sulfate, polymyxin B sulfate and corticosteroids used as eardrops, is a preferred therapy. In some cases, acidification of the ear canal by applying a 2% solution of acetic acid topically may also be effective. If a furuncle is present in the external canal, the physician should allow it to drain spontaneously.

Otitis media

Amoxicillin is an effective and preferred antibiotic for treatment of acute otitis media. Since beta-lactamase producing H influenzae and M catarrhalis can be a problem in some communities, amoxicillin-clavulanate is used by many physicians. Oral preparations of trimethoprim/sulfamethoxazole, second and third generation cephalosporins, tetracyclines and macrolides can also be used. When there is a large effusion, tympanocentesis may hasten the resolution process by decreasing the sterile effusion. Patients with chronic otitis media and frequent recurrences of middle ear infections may be benefitted by chemoprophylaxis with once daily oral amoxicillin or trimethoprim/sulfamethoxazole during the winter and spring months. In those patients with persistent effusion of the middle ear, surgical interventions with myringotomy, adenoidectomy and the placement of tympanotomy tubes has been helpful.

Use of polyvalent pneumococcal vaccines has been evaluated for the prevention of otitis media in children. However, children under two years of age do not respond satisfactorily to polysaccharide antigens further, no significant reduction in the number of middle ear infections was demonstrable. Newer vaccines composed of pneumococcal capsular polysaccharides conjugated to proteins may increase the immunogenicity and are currently under clinical investigation for efficacy and safety.

Pharyngitis

Etiology

Pharyngitis is an inflammation of the pharynx involving lymphoid tissues of the posterior pharynx and lateral pharyngeal bands. The etiology can be bacterial, viral and fungal infections as well as noninfectious etiologies such as smoking. Most cases are due to viral infections and accompany a common cold or influenza. Type A coxsackieviruses can cause a severe ulcerative pharyngitis in children (herpangina), and adenovirus and herpes simplex virus, although less common, also can cause severe pharyngitis. Pharyngitis is a common symptom of Epstein-Barr virus and cytomegalovirus infections.

Group A beta-hemolytic streptococcus or Streptococcus pyogenes is the most important bacterial agent associated with acute pharyngitis and tonsillitis. Corynebacterium diphtheriae causes occasional cases of acute pharyngitis, as do mixed anaerobic infections (Vincent's angina), Corynebacterium haemolyticum, Neisseria gonorrhoeae, and Chlamydia trachomatis. Outbreaks of Chlamydia pneumoniae (TWAR agent) causing pharyngitis or pneumonitis have occurred in military recruits. Mycoplasma pneumoniae and Mycoplasma hominis have been associated with acute pharyngitis. Candida albicans, which causes oral candidiasis or thrush, can involve the pharynx, leading to inflammation and pain.

Pathogenesis

As with common cold, viral pathogens in pharyngitis appear to invade the mucosal cells of the nasopharynx and oral cavity, resulting in edema and hyperemia of the mucous membranes and tonsils (Fig 93-2). Bacteria attach to and, in the case of group A beta-hemolytic streptococci, invade the mucosa of the upper respiratory tract. Many clinical manifestations of infection appear to be due to the immune reaction to products of the bacterial cell. In diphtheria, a potent bacterial exotoxin causes local inflammation and cell necrosis.

Clinical Manifestations

Pharyngitis usually presents with a red, sore, or “scratchy” throat. An inflammatory exudate or membranes may cover the tonsils and tonsillar pillars. Vesicles or ulcers may also be seen on the pharyngeal walls. Depending on the pathogen, fever and systemic manifestations such as malaise, myalgia, or headache may be present. Anterior cervical lymphadenopathy is common in bacterial pharyngitis and difficulty in swallowing may be present.

Microbiologic Diagnosis

The goal in the diagnosis of pharyngitis is to identify cases that are due to group A beta-hemolytic streptococci, as well as the more unusual and potentially serious infections. The various forms of pharyngitis cannot be distinguished on clinical grounds. Routine throat cultures for bacteria are inoculated onto sheep blood and chocolate agar plates. Thayer-Martin medium is used if N gonorrhoeae is suspected. Viral cultures are not routinely obtained for most cases of pharyngitis. Serologic studies may be used to confirm the diagnosis of pharyngitis due to viral, mycoplasmal or chlamydial pathogens. Rapid diagnostic tests with fluorescent antibody or latex agglutination to identify group A streptococci from pharyngeal swabs are available. Gene probe and polymerase chain reaction can be used to detect unusual organisms such as M pneumoniae, chlamydia or viruses but these procedures are not routine diagnostic methods.

Prevention and Treatment

Symptomatic treatment is recommended for viral pharyngitis. The exception is herpes simplex virus infection, which can be treated with acyclovir if clinically warranted or if diagnosed in immunocompromised patients. The specific antibacterial agents will depend on the causative organism, but penicillin G is the therapy of choice for streptococcal pharyngitis. Mycoplasma and chlamydial infections respond to erythromycin, tetracyclines and the new macrolides.

Epiglottitis and Laryngotracheitis

Etiology

Inflammation of the upper airway is classified as epiglottitis or laryngotracheitis (croup) on the basis of the location, clinical manifestations, and pathogens of the infection. Haemophilus influenzae type b is the most common cause of epiglottitis, particularly in children age 2 to 5 years. Epiglottitis is less common in adults. Some cases of epiglottitis in adults may be of viral origin. Most cases of laryngotracheitis are due to viruses. More serious bacterial infections have been associated with H influenzae type b, group A beta-hemolytic streptococcus and C diphtheriae. Parainfluenza viruses are most common but respiratory syncytial virus, adenoviruses, influenza viruses, enteroviruses and Mycoplasma pneumoniae have been implicated.

Pathogenesis

A viral upper respiratory infection may precede infection with H influenzae in episodes of epiglottitis. However, once H influenzae type b infection starts, rapidly progressive erythema and swelling of the epiglottis ensue, and bacteremia is usually present. Viral infection of laryngotracheitis commonly begins in the nasopharynx and eventually moves into the larynx and trachea. Inflammation and edema involve the epithelium, mucosa and submucosa of the subglottis which can lead to airway obstruction.

Clinical Manifestations

The syndrome of epiglottitis begins with the acute onset of fever, sore throat, hoarseness, drooling, dysphagia and progresses within a few hours to severe respiratory distress and prostration. The clinical course can be fulminant and fatal. The pharynx may be inflamed, but the diagnostic finding is a 𠇌herry-red” epiglottis.

A history of preceding cold-like symptoms is typical of laryngotracheitis, with rhinorrhea, fever, sore throat and a mild cough. Tachypnea, a deep barking cough and inspiratory stridor eventually develop. Children with bacterial tracheitis appear more ill than adults and are at greater risk of developing airway obstruction.

Haemophilus influenzae type b is isolated from the blood or epiglottis in the majority of patients with epiglottis therefore a blood culture should always be performed. Sputum cultures or cultures from pharyngeal swabs may be used to isolate pathogens in patients with laryngotracheitis. Serologic studies to detect a rise in antibody titers to various viruses are helpful for retrospective diagnosis. Newer, rapid diagnostic techniques, using immunofluorescent-antibody staining to detect virus in sputum, pharyngeal swabs, or nasal washings, have been successfully used. Enzyme-linked immunosorbent assay (ELISA), DNA probe and polymerase chain reaction procedures for detection of viral antibody or antigens are now available for rapid diagnosis.

Prevention and Treatment

Epiglottitis is a medical emergency, especially in children. All children with this diagnosis should be observed carefully and be intubated to maintain an open airway as soon as the first sign of respiratory distress is detected. Antibacterial therapy should be directed at H influenzae. Patients with croup are usually successfully managed with close observation and supportive care, such as fluid, humidified air, and racemic epinephrine. For prevention, Haemophilus influenzae type b conjugated vaccine is recommended for all pediatric patients, as is immunization against diphtheria.


Colonization and infection of the respiratory tract: What do we know?

Distinguishing colonization from infection is an important factor in making the correct diagnosis in a wide variety of paediatric conditions. For example, in this issue of Paediatrics & Child Health, Al-Mutairi and Kirk ( pages 25�) describe the difficulty in distinguishing bacterial tracheitis from other causes of upper airway obstruction. Part of this difficulty is that growth of bacteria from the trachea can occur because of contamination of specimens by organisms that are colonizing the upper respiratory tract.

Colonization implies that the patient has a sufficiently high concentration of organisms at a site that they can be detected, yet the organism is causing no signs or symptoms. This differs from contamination, where the organism was never present in the site from which it has been detected, but was introduced into the specimen from another site or from contamination in the laboratory. A carrier is a person who is colonized with an organism and may transmit the organism to other people. Colonization can persist for days to years, with resolution influenced by the immune response to the organism, competition at the site from other organisms and, sometimes, use of antimicrobials.

Table 1 summarizes the most common organisms isolated from the respiratory tract and their significance. The most important factor in determining if a patient is colonized or infected with an organism is the clinical picture. For example, in the upper respiratory tract, up to 20% of children are colonized with group A streptococcus (GAS) (1), with the highest concentration of organisms being in the oropharynx. Throat cultures should only be done in children who have symptoms of GAS pharyngitis (sore throat in the absence of cough, rhinitis or laryngitis) because culturing all children with sore throats results in these carriers being treated with antibiotics that will not improve their symptoms. Antibiotics are far less successful in eradicating the organism in the carrier state than in a patient with symptomatic GAS pharyngitis (1) therefore, their use cannot be justified as a measure to reduce transmission of the organism.

TABLE 1

The most common organisms isolated from the respiratory tract and their significance

- colonization of the nasal cavity occurs in about 30% of children and adults

- this sometimes leads to impetigo in the nasal cavity, but otherwise is a benign condition

- increases the risk of indwelling venous catheter or wound infections with S aureus

- a patient with colonization but no infection may require isolation if the organism is methicillin-resistant

- present in up to 20% of children (1)

- most commonly represents colonization, which is a benign condition

- less commonly causes pharyngitis or local suppurative infections, with rheumatic fever being a rare sequelae if untreated

- the relationship between Group A Streptococcus invasive disease and pharyngeal colonization is not clear

- most commonly represents colonization, but can cause single cases and outbreaks of symptomatic pharyngitis

- poststreptococcal glomerulonephritis has been described, but it is not clear that antibiotics decrease the incidence of this rare complication, so treatment is only recommended in the face of persistent symptoms

- present in a large percentage of infants and toddlers (range of 6% to 100% [3])

- persists for weeks to months

- growth from the oropharynx/nasopharynx is of no significance, but about 15% of children will develop clinical infections within one month of acquiring a new strain (2)

- a child who develops a viral upper respiratory tract infection while colonized may develop pneumococcal acute otitis media or sinusitis

- colonization can be followed by bacteremia, with the risk being highest soon after acquisition of a new strain

- bacteremia may resolve spontaneously but can lead to serious invasive disease (pneumonia, meningitis, septic arthritis)

- present in a large percentage of infants and toddlers

- growth from the oropharynx/nasopharynx is of no significance, but a child who develops a viral upper respiratory tract infection while colonized may develop acute otitis media or sinusitis with these organisms

- present in about 5% to 15% of individuals in nonendemic areas (higher in endemic areas or during an epidemic) (4)

- growth from the oropharynx/nasopharynx is of no significance, but colonization can be followed by bacteremia, which may resolve spontaneously but more commonly leads to serious invasive disease (septic shock, meningitis, septic arthritis)

- can result in vesicular lesions in the oropharynx, but asymptomatic shedding is more common

- shedding occurs at times of stress, and therefore is often isolated from the mouth or pharynx of intensive care patients

- growth in the absence of lesions is only of significance in an immunocompromised host or in an infant less than 30 days of age

- results in thrush if concentration is too high

- not thought to be a cause of upper respiratory tract disease except in the immunocompromised host, where laryngitis and tracheitis can occur

- despite colonization of the pharynx being common, it is rare for lower respiratory tract specimens to be contaminated therefore, isolation from the lower respiratory tract should usually be treated

- common contaminants from the upper respiratory tract (where they are colonizing organisms), but also common causes of lower respiratory tract disease

- significance depends on the clinical picture and the results of investigations ( Figure 1 )

- common contaminants from the upper respiratory tract (where they are colonizing organisms) and rare causes of lower respiratory tract disease in children

- usually require no treatment

- usually a pathogen if isolated from the lower respiratory tract, therefore, it should be treated

- colonize the upper respiratory tract in children who have been in intensive care or have received frequent courses of antibiotics, so often contaminate lower respiratory tract specimens

- however, can cause pneumonia, especially in ventilated patients

- significance depends on the clinical picture and the results of investigations ( Figure 1 )

- a colonized patient may require isolation if the organism is resistant to multiple antibiotics

- rare cause of lower respiratory tract disease, even in immunocompromised hosts

- if grown from lower respiratory tract specimens, usually originated in the upper respiratory tract

- usually not from the lower respiratory tract (might originate in the oropharynx, or in the esophagus in an immunocompromised host with fungal esophagitis)

- can cause lower respiratory tract disease in immunocompromised hosts

- can be contaminants, but can result in rapidly progressive invasive disease therefore, lung biopsy is often done if the clinical picture fits

- part of normal upper respiratory tract flora and not thought to be causes of lower respiratory tract disease

- most laboratories would not report because therapy is not indicated

- usually a pathogen, but can occasionally be detected in well normal hosts

- usually a pathogen, but carrier state has been described

- can cause sinusitis, bronchitis or pneumonia but can also be part of normal flora

- significance depends on clinical picture

- a carrier state has not been described, so identification of these organisms by any means is probably always indicative of active or recent infection, although the spectrum of disease can vary widely

Bacterial pharyngitis in the developed world is usually due to beta-hemolytic streptococci. However, many other organisms can be present in the pharynx. Infants and toddlers commonly become colonized with Streptococcus pneumoniae, nontypeable Haemophilus influenzae, Neisseria meningitidis and/or Moraxella catarrhalis, with the highest concentration of organisms usually being in the nasopharynx (2). Colonization with these organisms occurs earlier in life if children attend a childcare centre or live in overcrowded conditions (2). Clearance of one serotype of S pneumoniae is often followed by colonization with another serotype, and it has been suggested that children may be colonized with multiple serotypes simultaneously, with the predominant type growing in cultures (3). A small percentage of children will develop invasive disease following colonization with S pneumoniae or N meningitidis. This risk appears to be highest immediately after colonization, likely because the patient has not yet produced antibodies to the organism (2). An inflamed nasopharynx (such as that which occurs with influenza or smoking) may increase the risk of invasive disease following colonization with N meningitidis (4). If a child develops an upper respiratory tract infection while colonized with S pneumoniae, H influenzae or M catarrhalis, they may develop acute otitis media or sinusitis with the colonizing strain. Antibiotics may eradicate the strain, but they may also increase the risk of the child being colonized with a different organism that is resistant to the antibiotic that was chosen (2).

Approximately one-third of adults are persistently colonized with Staphylococcus aureus (5), with the highest concentration of organisms in the respiratory tract being in the nasopharynx. Colonization of the skin and the nasophaynx can occur shortly after birth. Colonization with S aureus precedes most invasive diseases caused by S aureus (osteomyelitis, cellulitis or pneumonia), but such conditions are so rare that eradication could never be justified in an attempt to prevent them. Eradication may be useful in preventing infection of indwelling venous catheters or wound infections in patients undergoing invasive procedures (6). Other possible indications for eradication are if a health care worker is a carrier of methicillin-resistant S aureus (MRSA), or if an MRSA carrier has a chronic severe disease and is therefore likely to spend long periods in strict isolation if they remain colonized. However, the reason to attempt eradication is for infection control purposes, because the risk of invasive disease is very low.

Viruses and fungi can also be detected in the upper respiratory tract. Traditional respiratory viruses (adenovirus, influenza, parainfluenza virus, respiratory syncytial virus and rhinovirus) are almost always pathogens if detected anywhere in the respiratory tract, although they sometimes result in only minor signs or symptoms. Furthermore, there is some evidence that parainfluenza virus can persist for weeks following acute infection (7). Herpes simplex virus can cause stomatitis, but reactivation of this and other herpes viruses, such as Epstein Barr virus and cytomegalovirus, can result in asymptomatic shedding in the pharynx and mouth, which is of no significance. Candida species can be part of normal gastrointestinal flora from mouth to anus, with thrush occuring when the concentration of organisms in the mouth is high. Growth of Candida from the upper respiratory tract usually implies that a sample is contaminated with mouth flora.

It is thought that the lower respiratory tract should be sterile and, therefore, any organism detected there is a pathogen. However, molecular techniques allow for the detection of much lower concentrations of organisms than traditional culture techniques (8), and it may eventually become evident that asymptomatic colonization also occurs in the lower respiratory tract. When attempts are made to obtain samples from the lower respiratory tract, it is always possible for them to be contaminated with organisms that are causing colonization in the upper respiratory tract. Sputum is difficult to obtain from paediatric patients, and adult studies have shown the sensitivity and specificity to be sufficiently low such that some experts recommend obtaining sputum from adults with suspected bacterial pneumonia only in selected circumstances (9). For patients with severe pneumonia, there is no consensus on the relative value of samples obtained by endotracheal aspirate, bronchoalveolar lavage or a protected brush specimen (10), but all are superior to sputum. Direct lung aspiration is rarely done but has been described as a useful technique in children with bacterial pneumonia (11). The ultimate way to determine if an organism is a pathogen is to obtain a lung biopsy, because contamination of such specimens is exceedingly rare. However, even that may not confirm a diagnosis if the patient has already received antimicrobials, or if cultures are not requested for the correct organism (such as mycobacteria). One problem with all techniques is that the sensitivity is limited by the fact that the area that is sampled may not be the one with the highest concentration of organisms. However, one basic principle that applies to specimens obtained from all lower respiratory sites is that an organism that is seen on a Gram stain is present in higher concentration than one detected only on culture therefore, it is more likely to be a pathogen. Also, because polymicrobial lower respiratory tract infections are rare, it is more likely that the true pathogen has been identified if there is heavy growth of a single organism than if there is mixed growth.

The significance of specific organisms detected in the lower respiratory tract is shown in Table 1 . Most of the time, one must put together the pieces of the puzzle that constitute the clinical picture ( Figure 1 ) to determine if antimicrobials should be started.

Diagnosis of lower respiratory tract infection in the paediatric patient. All the factors shown in the puzzle must be considered in deciding if a child has a lower respiratory tract infection and in choosing antimicrobial agents

The difficulty in detecting anaerobes means that clinicians are seldom faced with determining the significance of these isolates in respiratory tract samples. A more common dilemma is whether to add empiric anaerobic coverage for respiratory tract disease. When proper isolation techniques are employed, anaerobes are isolated from persistent otitis media (12), sinusitis (12), bacterial tracheitis (13) and paediatric pneumonia (14). It remains unclear when anaerobes are pathogens and when they are contaminants or colonizing organisms, but treatment should be considered if they are the predominant organism grown from a sterile or lower respiratory site.

In summary, to determine the significance of an organism isolated from the respiratory tract, one must consider the site from which the organism was isolated, the method of obtaining the sample, the Gram stain results, the other organisms isolated from the same site and, most importantly, the clinical picture.


2 CONCLUSION

Respiratory viruses such as SARS-CoV-2 are well-characterized to cause severe disorders and pneumonia, particularly in individuals with serious medical comorbidities and aged populations. Additionally, respiratory virus infection could usually lead to enhanced susceptibility to secondary bacterial infections. However, the mechanisms responsible for bacterial-SARS-CoV-2 co-infection require further study. It has been noted that an elicited adaptive immune reaction toward viral infection fails the reaction of the host innate immunity against bacterial infection. This situation can explain why bacterial co-infections occur when the virus starts to be eradicated from the lungs of patients with COVID-19. This is accompanied by a shift in phagocytic activity of lung cells that mediate basal levels of innate protection via phagocytosis and pro-inflammatory cytokines formation to cells better attuned to antigen presentation and stimulation of adaptive immune reactions. Additionally, recently it has been found the microbiome diversity shapes our immune system. In line with this, the depletion of the gut microbiome hinders the immune system's ability to create a humoral response against viruses like the flu virus. However, this novel paradigm ultimately allows the development of new immune intervention approaches for the prevention and management of viral-bacterial co-infections in COVID-19 patients. The COVID-19 pandemic reinforces the importance of preventative measures such as vaccination and antimicrobial treatments in maintaining human health.


The Microbiology of Respiratory System Infections

The Microbiology of Respiratory System Infections reviews modern approaches in the diagnosis, treatment, and prophylaxis of respiratory system infections. The book is very useful for researchers, scientists, academics, medical practitioners, graduate and postgraduate students, and specialists from pharmaceutical and laboratory diagnostic companies. The book has been divided into three sections according to the types of respiratory pathogens.

The first section contains reviews on the most common and epidemiologically important respiratory viruses, such as influenza virus, severe acute respiratory system coronavirus, and recently discovered Middle East respiratory syndrome coronavirus.

The second section is devoted to bacterial and fungal pathogens, which discusses etiology and pathogenesis including infections in patients with compromised immune system, and infections caused by fungal pathogens, such as Aspergillus and Pneumocystis.

The third section incorporates treatment approaches against different types of bacterial infections of the lower respiratory tract. This section reviews classical antimicrobial and phytomedical approaches as well as the application of nanotechnology against respiratory pathogens.

The Microbiology of Respiratory System Infections reviews modern approaches in the diagnosis, treatment, and prophylaxis of respiratory system infections. The book is very useful for researchers, scientists, academics, medical practitioners, graduate and postgraduate students, and specialists from pharmaceutical and laboratory diagnostic companies. The book has been divided into three sections according to the types of respiratory pathogens.

The first section contains reviews on the most common and epidemiologically important respiratory viruses, such as influenza virus, severe acute respiratory system coronavirus, and recently discovered Middle East respiratory syndrome coronavirus.

The second section is devoted to bacterial and fungal pathogens, which discusses etiology and pathogenesis including infections in patients with compromised immune system, and infections caused by fungal pathogens, such as Aspergillus and Pneumocystis.

The third section incorporates treatment approaches against different types of bacterial infections of the lower respiratory tract. This section reviews classical antimicrobial and phytomedical approaches as well as the application of nanotechnology against respiratory pathogens.


Defense Mechanisms of the Respiratory System

The average person who is moderately active during the daytime breathes about 20,000 liters (more than 5,000 gallons) of air every 24 hours. Inevitably, this air (which would weigh more than 20 kilograms [44 pounds]) contains potentially harmful particles and gases. Particles, such as dust and soot, mold, fungi, bacteria, and viruses deposit on airway and alveolar surfaces. Fortunately, the respiratory system has defense mechanisms to clean and protect itself. Only extremely small particles, less than 3 to 5 microns (0.000118 to 0.000196 inches) in diameter, penetrate to the deep lung.

Cilia, tiny muscular, hair-like projections on the cells that line the airway, are one of the respiratory system's defense mechanisms. Cilia propel a liquid layer of mucus that covers the airways.

The mucus layer traps pathogens (potentially infectious microorganisms) and other particles, preventing them from reaching the lungs.

Cilia beat more than 1,000 times a minute, moving the mucus that lines the trachea upwards about 0.5 to 1 centimeter per minute (0.197 to 0.4 inch per minute). Pathogens and particles that are trapped on the mucus layer are coughed out or moved to the mouth and swallowed.

Alveolar macrophages, a type of white blood cell on the surface of alveoli, are another defense mechanism for the lungs. Because of the requirements of gas exchange, alveoli are not protected by mucus and cilia—mucus is too thick and would slow movement of oxygen and carbon dioxide. Instead, alveolar macrophages seek out deposited particles, bind to them, ingest them, kill any that are living, and digest them. When the lungs are exposed to serious threats, additional white blood cells in the circulation, especially neutrophils, can be recruited to help ingest and kill pathogens. For example, when the person inhales a great deal of dust or is fighting a respiratory infection, more macrophages are produced and neutrophils are recruited.


​Electron Transport System

The electron transport system (ETS) is the last component involved in the process of cellular respiration it comprises a series of membrane-associated protein complexes and associated mobile accessory electron carriers (Figure 1). Electron transport is a series of chemical reactions that resembles a bucket brigade in that electrons from NADH and FADH2 are passed rapidly from one ETS electron carrier to the next. These carriers can pass electrons along in the ETS because of their redox potential. For a protein or chemical to accept electrons, it must have a more positive redox potential than the electron donor. Therefore, electrons move from electron carriers with more negative redox potential to those with more positive redox potential. The four major classes of electron carriers involved in both eukaryotic and prokaryotic electron transport systems are the cytochromes, flavoproteins, iron-sulfur proteins, and the quinones.

​In aerobic respiration, the final electron acceptor (i.e., the one having the most positive redox potential) at the end of the ETS is an oxygen molecule (O2) that becomes reduced to water (H2O) by the final ETS carrier. This electron carrier, cytochrome oxidase, differs between bacterial types and can be used to differentiate closely related bacteria for diagnoses. For example, the gram-negative opportunist Pseudomonas aeruginosaand the gram-negative cholera-causing Vibrio choleraeuse cytochrome c oxidase, which can be detected by the oxidase test, whereas other gram-negative Enterobacteriaceae, likeE. coli, are negative for this test because they produce different cytochrome oxidase types.

There are many circumstances under which aerobic respiration is not possible, including any one or more of the following:

  • The cell lacks genes encoding an appropriate cytochrome oxidase for transferring electrons to oxygen at the end of the electron transport system.
  • The cell lacks genes encoding enzymes to minimize the severely damaging effects of dangerous oxygen radicals produced during aerobic respiration, such as hydrogen peroxide (H2O2) or superoxide (O2 – ).
  • The cell lacks a sufficient amount of oxygen to carry out aerobic respiration.

​One possible alternative to aerobic respiration is anaerobic respiration, using an inorganic molecule other than oxygen as a final electron acceptor. There are many types of anaerobic respiration found in bacteria and archaea. Denitrifiers are important soil bacteria that use nitrate ( NO 3 – ) and nitrite ( NO 2 – ) as final electron acceptors, producing nitrogen gas (N2). Many aerobically respiring bacteria, including E. coli, switch to using nitrate as a final electron acceptor and producing nitrite when oxygen levels have been depleted.

​Microbes using anaerobic respiration commonly have an intact Krebs cycle, so these organisms can access the energy of the NADH and FADH2 molecules formed. However, anaerobic respirers use altered ETS carriers encoded by their genomes, including distinct complexes for electron transfer to their final electron acceptors. Smaller electrochemical gradients are generated from these electron transfer systems, so less ATP is formed through anaerobic respiration.


Results

During the study period, 7196 samples from 4958 patients were included in the analysis. In accordance with our definition and exclusion of repeat samples, no sample obtained, within 30 days, from the same area of the respiratory tract of a given patient as the initial sample was included. The median interval between two included samples from the same respiratory tract area of the same patient was 133 days (interquartile range [IQR] = 63–223). Median patient age was 59.9 years [IQR = 48.0–70.0] and 40.0% [IC95 = 38.2–41.4%] of the patients were female. Overall, 2098 (29.2%, [IC95 = 28.1–30.2]) samples tested positive for a respiratory virus and there were 118 viral co-infections (1.6%, [IC95 = 1.3–2.0%]). The groups of viruses identified were, in descending order of prevalence: picornavirus (n = 762, 34.3% [IC95 = 32.2–36.3%]), influenza (n = 592, 26.6% [IC95 = 24.8–28.5%]), coronavirus (n = 260, 11.7% [IC95 = 10.4–13.1%]), respiratory syncytial virus (RSV) (n = 215, 9.7% [IC95 = 8.5–11.0%]), parainfluenza (n = 179, 8.1% [IC95 = 7.0–9.3%]), metapneumovirus (n = 126, 5.7% IC95 = 4.7–6.7%]), adenovirus (n = 61, 2.7% [IC95 = 2.1–3.5%]) and bocavirus (n = 28, 1.3% [IC95 = 0.8–1.8%]).

The overall distribution of viruses, from year to year, is depicted in Fig 1 and S1 Fig The variation between years was driven mostly by influenza epidemic variations and by a specific variation in the summer of 2012. The number of tests performed annually increased steadily over the study period, from 634 to 1640 samples (Table 1).

For each group of viruses, the monthly number of identifications is shown for the entire study period.

Seasonality

The monthly cumulative results and viral distribution across the study period are depicted in Table 2 and Fig 1. The cumulative numbers of samples and the frequency of positive tests were higher from November to April, a period extending from late fall to early spring. We refer to this period as the “winter period” here. The frequency of positive tests for viruses was higher during the winter period than during the summer period (33.3 vs. 22.2%, p<0.0001), and, as expected, the distribution of viruses differed between these two periods (Fig 2, p<0.0001). The distribution of influenza followed the typical seasonal pattern observed in Europe, with active circulation from January to March. RSV, coronavirus, metapneumovirus and bocavirus were also more frequently detected in the winter, with a period of circulation extending from November to May. Two groups of viruses circulated throughout the year: picornavirus and parainfluenza virus. Together, these two groups accounted for 78.6% of all viruses detected during the summer period and picornaviruses constituted the second most frequent group of viruses during the winter (26.1%), after influenza (34.4%). Adenovirus had a more complex seasonal pattern over the years covered by this study. It was very rare in adults during 2011 and 2014, circulated throughout the year in 2012 and caused an epidemic exclusively during the winter in 2013.

Respiratory tract area

The area of the respiratory tract sampled was indicated for 6594 samples (91.6%): 58.6 of these samples came from the URT and 41.4% came from the LRT. The results are depicted in Fig 2 and Table 2.

The URT was more frequently sampled during the winter (77.5% vs. 55.5% for the LRT, p<0.0001) and the positivity rate was higher for the URT than for the LRT (32.0% [IC95 = 31.1–34.1] vs. 25.3% [20.9–23.5], p<0.0001). The distribution of viral groups differed significantly between the URT and LRT (p<0.0001). The three most frequent viral groups in URT were influenza (32.0%, [IC95 = 29.4–34.6]), picornavirus (30.0%, [27.5, 32.6]) and coronavirus (13.1%, [11.7–15.5]). The most frequent viral groups in the LRT were picornavirus (41.3%, [37.6–44.9]), parainfluenza (11.7%, [9.8–14.7]), influenza (16.3%, [13.4–18.8]) and RSV (10.5%, [8.1–12.7]).

Medical units

The results obtained for the 6532 (90.7%) samples for which the type of medical unit was recorded are depicted in Fig 2 and Table 2. Positivity rates differed significantly between types of medical unit (p<0.001), ranging from 23.8 in ICUs to 34.0% in lung transplantation units. The distribution of viruses also differed between types of medical units (p<0.001): picornaviruses were the most frequent group in ICUs (37.6%, [IC95 = 33.2–42.0]), pneumology units (43.9%, [IC95 = 36.9–51.0]) and lung transplantation units (40.5%, [IC95 = 36.5–44.6]). By contrast, in other medical units, influenza and picornavirus accounted for 39.4% [IC95 = 36.0–42.9] and 27.4% [IC95 = 24.4–30.7] of all viruses, respectively. The proportion of samples obtained during the winter differed significantly between types of medical unit: 58.9% [56.5–61.4] for lung transplantation units, 65.1% [63.0–67.2] for ICUs, 72.3% [69.1–75.4] for pneumology units and 75.6% [74.0–77.4] for other medical units (p<0.001), consistent with the greater focus on virus identification during influenza epidemics.

Infections of the LRT area are more representative of severe infections and rates of sampling for this part of the respiratory tract are constant through the year. We therefore analyzed the viral distribution between types of medical unit separately for LRT samples (S2 Fig). For LRT samples, positivity rates were 20.6% [IC95 = 18.0–23.3] for ICUs, 24.0% [19.3–29.2] for pneumology units, 33.0% [30.0–36.1] for lung transplantation units and 21.5% [18.1–25.2] for other units. The prevalence of influenza was higher for ICUs than for all the other types of units (24.6% [18.9–31.2], versus 11.3% [8.8–14.3], p<0.001). The picornavirus group was the most frequent viral group in all units, at frequencies ranging from 41.4% [34.5-48-5] of all viruses detected in ICUs to 43.0% [34.0–52.3] in lung transplantation and the other medical units group.

Positivity rates did not differ significantly between age groups, ranging from 27.2% to 31.8% (p = 0.61). However, the distribution of viruses differed between age groups (Fig 2 and Table 2, p<0.0001). Picornaviruses accounted for between 20.1% [IC95 = 15.1–25.9] and 40.1% [IC95 = 34.5–46.0] of the viruses identified in all age groups. This group of viruses was the most frequent in all but the most extreme age groups considered (20–30 and >80 years), in which influenza was the most frequent (35.6% [IC95 = 27.0–44.9] and 40.6% [IC95 = 34.2–47.3], respectively).

An analysis of URT and LRT samples separately (S3 Fig) showed influenza to be less frequent in the LRT than in the URT for all age groups. The picornavirus group was the group of viruses most frequently detected in LRT samples, for all age groups.

Viral co-infections

Only 118 of the 7196 samples tested (1.6%, [IC95 = 1.3–2.0%]) corresponded to viral co-infections, six of which were co-infections involving three viruses. The proportion of viruses identified in co-infections differed between groups: 7.3% [IC95 = 5.3–9.7] for influenza, 9.4% [7.5, 11.8] for picornavirus, 10.6% [6.5–16.1] for parainfluenza, 11.9% [6.8–18.9] for metapneumovirus, 13.6% [9.3–18.9] for RSV, 15.8% [11.6–20.8] for coronavirus, 24.6% [14.5–37.3] for adenovirus and 28.6% [13.2–48.7] for bocavirus (p<0.001). All groups of viruses seemed to be able to exist alongside other viruses in co-infections, and no preferential association between respiratory viruses was identified.


Methods

Study population

A total of 64 subjects, including 35 laboratory-confirmed COVID-19 patients, 10 SARS-CoV-2 negative patients with various diseases (non-COVID-19) and 19 healthy adults were enrolled in this study. COVID-19 was diagnosed in adult patients according to the National Guidelines for Diagnosis and Treatment of COVID-19. The virus RNA was extracted from all samples using a Mag-Bind RNA Extraction Kit (MACCURA, Sichuan, China) according to the manufacturer’s instructions. Then the ORFlab and N genes of SARS-CoV-2 was detected using a Novel Coronavirus (2019-nCoV) Real Time RT-PCR Kit (Liferiver, Shanghai, China) according to the manufacturer’s instructions. Only the individuals who had at least two consecutive throat swabs been positive for both ORFlab and N genes of SARS-CoV-2 were defined as COVID-19 patients. All positive specimens of COVID-19 patients were confirmed by Nantong Center for Disease Control and Prevention (CDC) using recommended real-time RT-PCR assay by China CDC. Mild and moderate cases are defined as having clinical symptoms (e.g., fever, cough, etc.) with and without the pneumonia on lung imaging. Severe COVID-19 (adult) is defined as the presence of any one of the following: respiratory rate ≥30 breaths/minute, arterial oxygen saturation ≤93% at rest PaO2/FiO2 ≤ 300 mm Hg. The COVID-19 patients were hospitalized at Nantong Third Hospital Affiliated to Nantong University. Among 35 COVID-19 patients, 34 were mild or moderate cases, and only one (P09) was severe case.

Demographic and clinical characteristics of the COVID-19 patients were provided in Supplementary Data 2 and 3 43 . Specimens including throat swabs and anal swabs were collected from the COVID-19 patients at different timepoints during their hospitalization (10–40 days). Sampling was performed using flexible, sterile, dry swabs, which can reach the posterior oropharynx and anus easily (

2 inches) by the professionals at the hospital. At least two throat swabs at different days were available for 32 of 35 COVID-19 patients (Supplementary Fig S1).

Non-COVID-19 control patients were selected from patients hospitalized at the same hospital during the COVID-19 pandemic due to other diseases, and healthy controls were selected from adults who came for routine physical examination and showed no symptoms. Throat swabs of non-COVID-19 patients and healthy controls were collected during their hospital visit.

The study was approved by Nantong Third Hospital Ethics Committee (EL2020006: 28 February 2020). Written informed consents were obtained from each of the involved individuals. All experiments were performed in accordance with relevant guidelines and regulations.

16S-rRNA gene sequencing

Bacterial DNA was extracted from the swabs using a QIAamp DNA Microbiome Kit (QIAGEN, Düsseldorf, Germany) according to the manufacturer’s instructions, and eluted with Nuclease-free water and stored at −80 °C until use. The V4 hypervariable region (515–806 nt) of the 16 S rRNA gene was amplified universal bacterial primers 44 . To pool and sort multiple samples in a single tube of reactions, two rounds of PCR amplifications were performed using a novel triple-index amplicon sequencing strategy as described previously 45 . The first round of the PCR (PCR1) amplification was performed with a reaction mixture containing 8 μL Nuclease-free water, 0.5 μL KOD-Plus-Neo (TOYOBO, Osaka Boseki, Japan), 2.5 μL of 1 μM PCR1 forward primer, 2.5 μL of 1 μM PCR1 reverse primer, and 5 μL DNA template. The products of the PCR1 reactions were verified using a 1.5% agarose gel, purified using Monarch DNA Gel Extraction Kit (New England Biolabs, Ipswich, MA, USA), and quantified by a Qubit® 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Equal amounts of purified PXR1 products were pooled, and subjected to the secondary round of PCR (PCR2) amplification. The PCR2 was performed with a reaction mix containing 21 μL Nuclease-free water, 1 μL KOD-Plus-Neo (TOYOBO, Osaka Boseki, Japan), 5 μL of 1 μM PCR2 forward primer, 5 μL of 1 μM PCR2 reverse primer, and 5 μL pooled PCR1 products. The PCR2 products were verified using a 2% agarose gel, purified using the same Gel Extraction Kit and qualified using the Qubit® 4.0 Fluorometer. The amounts of the specific product bands were further qualified by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Equal molars of specific products were pooled and purified after mixing with AMPure XP beads (Beckman Coulter, Pasadena, CA, USA) in a ratio of 0.8:1. Purified amplicons were paired-end sequenced (2 × 250) using Illumina-P250 sequencer.

Bioinformatic analysis of 16S-rRNA gene sequence data

Sequenced forward and reverse reads were merged using USEARCH11 software 46 , then demultiplexed according to known barcodes using FASTX-Toolkit 47 . After trimming barcode, adapter and primer sequences using USEARCH11, 19,096,003 sequences were retained with an average of 105508 sequences per sample. After excluding the samples with sequences <1000, 157 samples from 35 COVID-19 patients, 10 non-COVID-19 patients and 13 healthy individuals were subjected to the following analysis.

Because traditional OTU (operational taxonomic units) picking based on a 97% sequence similarity threshold may miss subtle and real biological sequence variation 48 , several novel methods such as DADA2 49 and Deblur 50 were developed to resolve sequence data into single-sequence variants. Here, the DADA2 was employed to perform quality control, dereplicate, chimeras remove on Qiime2 platform 51 with default settings except for truncating sequence length to 250 bp. Finally, an amplicon sequence variant (ASV) table, equivalent to OTU table, was generated and then spitted into gut ASV table (2348 ASVs) and throat ASV table (4050 ASVs). The taxonomic classification of ASV representative sequences was conducted by using the RDP Naive Bayesian Classifier algorithm 52 based on the Ribosomal Database project (RDP) 16 S rRNA training set (v16) database 53 . To eliminate sequencing bias across all samples, both the gut ASV table and throat ASV table were subsampled at an even depth of 4700 and 3000 sequences per sample, respectively. The ASV coverage of 82.6% (gut) and 77.2% (throat) were sufficient to capture microbial diversity of both sites.

Identification and characterization of microbial community types

Dirichlet multinomial mixtures (DMM) 54 is an algorithm that can efficiently cluster samples based on microbial composition, its sensitivity, reliability, and accuracy had been confirmed in many microbiome studies 55,56,57 . DMM clustering were conducted with bacterial genus abundance from throat and gut microbiota using the command “get.communitytype” introduced by v1.44.1 of mothur 58 . The appropriate microbial community type numbers (DMM clusters) were determined based on the lowest Laplace approximation index. According to sample counts per cluster, the fisher exact test was applied to discover significant associations between each cluster and host conditions (such as healthy controls, COVID-19 patients, and Non-COVID-19 patients) under P values that are below 0.05 adjusted by the False Discovery Rate (FDR). Conjugated with the Analysis of Similarities (ANOSIM), the reliability of DMM clustering was further validated and then visualized by the Nonmetric multidimensional scaling (NMDS) based on the Bray–Curtis distance under bacterial genus level. The ANOSIM statistic “R” compares the mean of ranked dissimilarities between groups to the mean of ranked dissimilarities within groups. An R value close to “1.0” indicates dissimilarity between groups, whereas an R value close to “0” indicates an even distribution of high and low ranks within and between groups”. The ANOSIM statistic R always ranges between −1 to 1. The positive R values closer to 1 suggest more similarity within sites than between sites, and that close to 0 represent no difference between sites or within sites 59 . ANOSIM p values that are lower than 0.05 imply a higher similarity within sites. Richness (Observed OTUs/ASVs) and Pielou’s evenness for each community type were calculated for estimating the difference of alpha-diversity. The analyses of alpha-diversity, NMDS and ANOSIM were performed using R package “vegan” v2.5-6. Dynamic change of community types was showed according to collected dates of specimens with ‘pheatmap’ package in R. Furthermore, to compensate for the effects of sample size, the Margalef’s index was calculated by dividing the number of species in a sample by the natural log of the number of organisms collected 15 . For association between community types and potential confounding factors such as sex, age, virus existence, and antibiotic use, the fisher exact test based on sample count was performed and the association with FDR-corrected p value < 0.05 was considered significant.

Indicator analysis in throat and gut community types

According to the definition given by the United Nations Environment Programme (1996), the indicator species are a group of species whose status provides information on the overall condition of the ecosystem and of other species in that ecosystem, reflecting the quality and changes in environmental conditions as well as aspects of community composition. To obtain the reliable indicator genus that is specific to each community type, we performed the Indicator Species Analysis using the indicspecies package (ver.1.7.8) 60 in R platform with top 30 genus contributing to DMM clustering in both throat (accounting for 66% cumulative difference) and gut (68% cumulative difference). Dynamic changes of indicator genera corresponding to each throat community type were showed in all COVID-19 patients using the pheatmap package in R and only gut indicator genera with indicator values that were above 0.05 were presented in the patients.

Co-occurrence network analysis of a crosstalk between throat and gut microbiota

Based on microbial genus abundances normalized by the centered log ratio transformation of both throat and gut samples collected from 13 COVID-19 patients at the same time point, we calculated the Pearson Correlation Coefficient (Pearson’s r) among the throat and gut microbial genera. The Pearson’s r with P values that were below 0.05 after the FDR adjustment were considered significant correlations. Co-occurrence network of significantly correlated microbial genus pairs was visualized using Cytoscape v3.8.0 61 .

Statistics and reproducibility

Raw sequences were analyzed on Linux (Red Hat 4.8.5-36) and Windows10 environment. Software under Linux environment include USEARCH11, FASTX-Toolkit, DADA2 and Deblur, both of which were integrated in Qiime2 (v2019.10) and RDP Naive Bayesian Classifier algorithm. Software under Windows10 including Dirichlet multinomial mixtures integrated in Mothur v1.44.1, RStudio v1.2.1335. Data analysis and plotting were performed in RStudio with R v3.6.1 and R packages including pheatmap (v1.0.12), vegan (v2.5-6), permute (v0.9-5), lattice (v0.20-38), ggplot2 (v3.3.0), RColorBrewer (v1.1-2), viridis (v0.5.1), indicspecies (v 1.7.9), ade4 (v 1.7-15), ggalluvial (v 0.11.3), and grid. To promote reproducibility, we provided the analyses scripts/code of the correlation analysis R package as supplemental file 1. Detailed information on statistics and comparisons are provided in Method and/or figure legends.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.


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