Anaerobic Bacteria: Clinical Concepts and the Microbiome in Health and Disease

Establishment and Composition of Normal Flora ^a

a See also the section “Microbiome in Health and Disease.”

Anaerobes are dominant in mucosal surfaces and the frequency of their recovery as normal flora varies by body site and age. Table 187.2 shows sites of colonization of clinically important anaerobic bacteria. Mucocutaneous surfaces in humans have complex indigenous flora composed of anaerobic, microaerophilic, facultative, and aerobic bacteria. ^{, ,} Molecular data suggest that microbial flora may exist prenatally and play a role in fetal maturation. Flora is remarkably predictable within days after birth, depending on the type of delivery, feeding, and receipt of antibiotics. Successive colonization of the gut simulates that in germ-free laboratory animals after exposure to peers. Facultative organisms, such as oral streptococci, intestinal Escherichia coli , and skin staphylococci, precede anaerobic species, but by the end of the first few weeks of life, flora is complex and predictable. Infants fed human milk exclusively have a fermentative gut flora with predominance of Bifidobacterium , fewer Bacteroides and facultative gram-negative bacilli, and Enterococcus spp., whereas infants fed cow’s milk have putrefactive flora similar to that of adults, with gram-negative anaerobic and facultative bacilli predominating. ^, Early colonization at <3 weeks with B. fragilis has been associated with the risk of asthma at a later age. Remarkable perturbation of gut flora is noted at weaning from human milk, and disruption of flora occurs with the use of antibiotics, especially in infancy.

Colonization of the mouth similarly is rapid and predictable. Viridans streptococci and Streptococcus salivarius are present and predominate, usually within 12 hours after birth, with successive representation by facultatively anaerobic Neisseria and Staphylococcus spp. and then anaerobic streptococci, Veillonella , and Bifidobacterium spp. on the second day. ^{, ,} Nasopharyngeal and oropharyngeal Streptococcus pneumoniae, nontypable Haemophilus influenzae , and Moraxella catarrhalis follow and remain as facultative flora throughout childhood. Prevotella melaninogenica becomes predominant at gingival crevices, seemingly under hormonal influences, at puberty. ^, Predominant oral flora organisms are Prevotella , Porphyromonas , Fusobacterium , and non -B. fragilis species. Culture-independent techniques have demonstrated the presence of a respiratory tract microbiota from the nasal passages to the alveoli.

Colonization of skin varies by site and age. ^, Coagulase-negative staphylococci (or Staphylococcus aureus in nurseries where it is prevalent) appear, followed by Enterobacteriaceae at diapered sites. Cutibacterium (formerly Propionibacterium) acnes becomes the dominant anaerobe, quantitatively accounting for almost one-half of culturable flora at most anatomic sites; anaerobic cocci also are prevalent. Anaerobic bacteria predominate in vaginal flora, even prepubertally, with gram-positive bacilli and cocci and Prevotella highly represented.

Clostridium ramosum and Clostridium perfringens are dominant clostridial species in intestinal flora. Clostridioides difficile (formerly Clostridium difficile ) is found in only 1%–4% of healthy adults and children >6 months of age. Asymptomatic C. difficile rates of young infants have varied vastly from 4%–71% across studies and reflect unusual nosocomial phenomena. ^,

The qualitative and quantitative complexity of flora and the difficulty of its study cannot be overestimated. Within the mouth, for example, the gingival crevice has an oxidation-reduction potential of −300 mV (similar to that in the colon); the ratio of anaerobes to aerobes is 1000:1, yet saliva and teeth have ratios closer to 1:1. The normal flora of the colon contains more than 500 obligate anaerobic species in a total concentration of 10 ¹¹ to 10 ¹² colony-forming units (CFU) per gram of feces. However, the majority of the bacteria in the human colon cannot be isolated in culture using current laboratory methods. The predominant clinically important gram-negative anaerobes are Bacteroides and Fusobacterium spp. Although <0.1% of normal flora consists of facultative or aerobic organisms (e.g., 10 ⁸ CFU/g of feces), ease of recovery from endogenous infections has led to overestimation of their importance in the microbial ecology of the gut. Similarly, an erroneous tendency is to focus on the clinically most significant anaerobic bacteria as critical in ecology. B. fragilis, for example, is quantitatively among the least important species of the Bacteroides group in normal flora. Anaerobes reach a concentration of 10 ⁶ organism/mL in vaginal secretions, with predominance of Prevotella , Bacteroides , Fusobacterium , and Clostridium spp. Anaerobes isolated most commonly from clinical specimens are Prevotella bivia and Prevotella disiens.

Observations on the Role of Indigenous Flora

Multiple observations made prior to the advent of molecular methods for study lead to the conclusion that indigenous organism, especially anaerobic flora, provide resistance to colonization and invasion by nonindigenous micro-organisms. A few such observations follow:

• 1.

  Infants in an intensive care nursery are at higher risk for pharyngeal colonization with potentially pathogenic organisms if they are not colonized initially with viridans streptococci.

• 2.

  Children who are colonized with group A Streptococcus (GAS) who have recurrent GAS tonsillitis or have recurrent otitis media or sinusitis are less likely to have inhibitory facultative (α-hemolytic streptococci) and anaerobic ( Prevotella and Peptostreptococcus spp.) oropharyngeal flora.

• 3.

  Cutibacterium ( Propionibacterium ) spp. hydrolyze triglycerides, thus producing free fatty acids that are inhibitory to GAS and S. aureus.

• 4.

  Primary metabolic byproducts of anaerobes, volatile fatty acids, inhibit the multiplication of nonindigenous organisms in the intestine.

• 5.

  Lactobacillus spp. produce hydrogen peroxide, which is bactericidal for P. bivia , and Gardnerella vaginalis presence in vaginal flora is inversely related to bacterial vaginosis.

• 6.

  Susceptibility of germ-free mice to colonization and outgrowth of Clostridium botulinum can be manipulated dramatically by selective colonization, especially with anaerobic flora.

• 7.

  Infant botulism occurs exclusively in the first year of life, especially at the time of perturbation of anaerobic gut flora at weaning.

• 8.

  C. difficile pseudomembranous colitis is associated with the use of antibiotics. The normal colonic microbiota plays an important role in protection against C. difficile- associated diarrhea or colitis. A modicum of protection from colonization or cure of C. difficile disease, as well as of ulcerative colitis and metabolic syndrome, can be afforded by intestinal administration of probiotics or fecal transplantion. ^,

• 9.

  Experimental infectivity of Salmonella or Shigella spp. is enhanced by pretreatment of animals with antibiotics; infectivity is reduced when germ-free animals are fed mixed cultures of pathogens with anaerobic bacteria.

• 10.

  Susceptibility of children <2 years of age to all enteric pathogens correlates with the relative ease of disruption of indigenous flora.

• 11.

  Prophylactic administration of a probiotic mixture of Lactobacillus acidophilus and Bifidobacterium infantis given to very low birth weight infants reduced the incidence of all cases of necrotizing enterocolitis as well as severe stage III necrotizing enterocolitis.

• 12.

  The ability of anaerobes to interfere with potential pathogens has been observed for B. fragilis , Prevotella oralis , Peptostreptococcus anaerobius , C. difficile , Veillonella spp., and Bifidobacterium spp. ^,

Mechanisms by which normal flora provide resistance to colonization have been reviewed. ^{, ,} They include (1) production of bacteriocins and other metabolic products (e.g., volatile fatty acids) that inhibit multiplication of nonindigenous flora at local conditions of pH and oxidation-reduction potential, (2) competition for limited nutrients, and (3) competition for available attachment sites. Host factors undoubtedly provide exquisitely specific microbial niches by means of the unique properties of epithelial surfaces, enzymes, secretory immunoglobulins, pH, nutrients, exfoliation of skin and mucous membranes, motility of the gastrointestinal tract, and mucociliary movement in the respiratory tract. Coliform organisms are present in the oropharyngeal or nasopharyngeal flora of only approximately 3% of healthy adults and children. Severe noninfectious illness, viral infection, malignant disease, diabetes, chemotherapy, antibiotic therapy, and antacid therapy affect changes in colonizing bacteria. Changes in attachment properties of epithelial cells in vitro correlate with changes in flora. A relative or absolute increase in nasopharyngeal S. pneumoniae , H. influenzae , or M. catarrhalis occurs in association with viral illness and acute otitis media, and it predicts the presence of concordant middle ear pathogens in acute otitis media. ^,

Organisms of the normal anaerobic intestinal flora also have broadly ranging metabolic effects, mainly through provision of catabolic enzymes for organic compounds that cannot be digested by enzymes of eukaryotic origin. The enzymes are necessary to (1) process diverse dietary and host polysaccharides, thus aiding human digestion ; (2) catabolize cholesterol, bile acids, and steroid hormones; (3) hydrolyze certain flavonoid glycosides of plant origin to anticarcinogens; and (4) detoxify certain carcinogens and drugs. Erythromycin or tetracycline treatment raises the serum digoxin level 50%, almost certainly by reducing gut Eubacterium lentum , which inactivates digoxin through hydrogenation of the double bond of its lactone ring.

Normal flora provides “education” of the host’s immune system from birth onward, and possibly earlier. Germ-free animals (and probably heavily antibiotic-exposed infants) have immune defects, such as diminished production of complement and immunoglobulins as well as reticuloendothelial and lymphoid tissues. B . fragilis zwitterionic polysaccharide A regulates an immunologic equilibrium in the intestine by balancing colonic helper T lymphocytes, subtypes 1 and 2, and promotes anti-inflammatory interleukin-10 production.

Clinical Effect of Antimicrobial Agents on Flora

The effect of antibiotics on normal flora is extremely complex and varies with dose and route of administration, spectrum of antimicrobial activity (breadth, potency, and specificity for anaerobes), and chemical and pharmacokinetic properties. The clinical impact depends on the duration of antibiotic use, the presence and severity of underlying conditions, exposure to nonindigenous pathogens ( Pseudomonas , C. difficile ), and host susceptibility to indigenous and nonindigenous microbes (underlying valvular heart disease, malignant disease, surgical procedures, inserted devices, and age). In general, agents that have a major impact on anaerobic flora cause the greatest disruption of homeostasis.

The main determinant of effect on oropharyngeal flora is secretion of the agent in saliva or from mucous membranes, thus correlating with lipophilicity. Clindamycin, erythromycin, and rifampin are present in high concentrations; penicillins and ampicillin, despite relatively low concentrations, have potent activity against oral facultative and anaerobic bacteria. The use of β-lactam antibiotics (even penicillin) may not supplant a species from its microbial niche but can select for organisms in the oropharynx and nasopharynx with reduced antimicrobial susceptibility ( S. pneumoniae , viridans streptococci) or β-lactamase production ( Moraxella, Prevotella spp.). The nasopharyngeal flora are depleted of organisms with protective potential by amoxicillin-clavulanate therapy more than by use of a narrower-spectrum agent.

The most profound effects on gut flora occur with administration of agents that are active against anaerobic bacteria, which are present in high density at the ileal-colonic mucosa. The pharmacodynamics of specific agents is important. Exquisite activity against anaerobes (clindamycin, amoxicillin-clavulanate), high biliary excretion (ceftriaxone, cefoperazone), or the use of oral nonabsorbable agents (neomycin, gentamicin) ensures an effect on flora, but high absorption of oral agents does not necessarily predict low colonic concentrations because antibiotics are delivered to the mucosal site by blood (amoxicillin). Outgrowth of Enterococcus and Candida spp. occurs rapidly when potent agents have selective nonactivity (aztreonam, ceftriaxone, cefoxitin) or poor activity (imipenem) against enterococci. The β-lactamases of certain facultative organisms (the SPACEY group: Serratia , Pseudomonas , Acinetobacter , Citrobacter , Enterobacter , and Yersinia spp.) can be induced by administration of cefoxitin and several third-generation cephalosporins ^, ; β-lactamase production by anaerobes can increase ; or resistant nosocomial pathogens can be acquired. The use of broad-spectrum, exquisitely active cephalosporins, carbapenems, and fluoroquinolones with profound effects on normal flora undeniably have changed the incidence, microbiology, and susceptibility of superinfecting microbes in children. ^{, ,} Attempts to reconstitute flora, as with lactobacilli, have an overall positive but uncertain impact. ^,