Bacteroides, Prevotella, Porphyromonas, and Fusobacterium Species (and Other Medically Important Anaerobic Gram-Negative Bacilli)

Overview

The genera Bacteroides, Porphyromonas, Prevotella, and Fusobacterium account for most infections caused by gram-negative anaerobic rods (GNARs). Bilophila and Sutterella also cause human infections, although they are less frequently encountered in clinical practice. These obligately anaerobic gram-negative bacteria colonize the oropharynx, gastrointestinal tract, and urogenital tract of humans. Several species from some of these genera are useful symbiotic bacteria, facilitating host metabolism and favorably shaping immune responses. However, many of these microbes act opportunistically, causing infections when they gain access to otherwise sterile tissues. When tissue barriers are breached, GNARs have a predilection for abscess formation, with the most common sites being the oropharynx, abdominal cavity, lungs, and female genital tract. These bacterial species also present clinical challenges because they are often resistant to commonly used antibiotics. A few strains from the Fusobacterium, Bilophila, and Sutterella genera have been associated with either inflammatory bowel disease or colon cancer, although causative roles remain to be robustly established outside of preclinical models.

History

The end of the 19th century was a fertile time for the discovery of GNARs. The recognition of GNARs as important pathogens first occurred in animals in 1884 and is attributed to Loeffler. Schmorl is credited with identifying GNARs as opportunists in humans in 1891. Identification of GNARs as members of the human microbiota dates back to 1898, when Halle published his discovery of Fusobacterium colonizing the female genital tract. At the same time, GNARs were also isolated by Veillon and Zuber from clinical material from pelvic, appendiceal, and brain abscesses.

The Bacteroides spp. as a group were first described in the late 1890s, and for many years Bacteroides was a physiologically disparate genus of pleomorphic, obligately anaerobic, gram-negative rods. Until the early 1960s, competing taxonomic systems and confusion regarding nomenclature made it difficult to determine the role of specific members of this group of organisms during infectious processes. Holdeman and colleagues brought some conformity to the phenotypic classification of this group of organisms, which allowed other investigators to identify Bacteroides fragilis as a clinically significant abscess-inducing pathogen. With the advent of uniform taxonomic classification and the use of 16S ribosomal RNA phylogenetic-driven taxonomic classifications, several species were reclassified. Bacteroides has been further subdivided into two additional genera— Porphyromonas and Prevotella —both of which primarily colonize the oral cavity. The genus Fusobacterium was identified in the early 20th century and is well documented in studies by microbiologists at the Virginia Polytechnic Institute and Pasteur Institute.

Microbiology

Bacteroides

Bacteroides is a genus of gram-negative, non–spore-forming, obligately anaerobic, rod-shaped bacteria. More than 30 species of Bacteroides have been recognized. The strictest taxonomic definition of Bacteroides limits this census to less than a dozen separate species. Within the Bacteroidaceae family, Bacteroides organisms are distinctive in their DNA guanine-cytosine composition—40 to 48 mol%. The principal products of their saccharolytic metabolism are acetate, succinate, and isovalerate. The saturated anteiso-methyl and isomethyl branched-chain fatty acids are used for their long-chain fatty acid–based identification. Bacteroides organisms express hexose monophosphate shunt–pentose phosphate pathway enzymes. Their sphingolipid-rich membranes also possess menaquinones, particularly MK-10 and MK-11. Bacteroides peptidoglycan contains meso-diaminopimelic acid. Several Bacteroides spp. express numerous capsular polysaccharides. These glycoantigens are of interest biologically for their immunomodulatory potential, particularly in the case of B. fragilis polysaccharide A.

GNARs are differentiated from one another in the clinical laboratory by use of standard techniques: colony morphology, Gram stain, pigment production visualized in natural light and as fluorescence emission after exposure to ultraviolet light, and numerous biochemical tests. Short-chain fatty acid analysis can also be used for species-level Bacteroides discrimination. The B. fragilis group is of special medical importance for several reasons. It is often the predominant GNAR in polymicrobial infections, and members of this group have the potential to express β-lactamase. B. fragilis group bacteremias are also associated with a high mortality rate of 27%. Consequently, identification of the B. fragilis group by the clinical laboratory is often critical for appropriate therapeutic intervention. On blood agar, B. fragilis forms circular, entire, white or gray, 2- to 3-mm colonies that are shiny and smooth. The B. fragilis group can be pleomorphic on Gram stain, forming straight rods of varying length as well as coccobacilli ( Fig. 247.1A ). When grown in liquid medium, cells develop bipolar vacuoles and show a characteristic “safety pin” appearance. A useful characteristic of the B. fragilis group is bile tolerance compared with other GNARs, enabling its growth on Bacteroides bile-esculin agar. In addition, B. fragilis is highly resistant to the antibiotics kanamycin, vancomycin, and colistin. The use of a simple disk diffusion assay for these antibiotics is often part of the identification process for GNARs ( Table 247.1 ).

FIG. 247.1, Gram stains of selected gram-negative anaerobic rods.

TABLE 247.1

Techniques and Properties to Differentiate Medically Important Gram-Negative Anaerobic Rods

	ANTIBIOTIC SENSITIVITY (PENCILLIN [P], VANCOMYCIN [V], COLISTIN [C], KANAMYCIN [K])	GROWTH IN 20% BILE	PIGMENT	BRICK RED FLUORESCENCE
Bacteroides fragilis group	P ^R V ^R C ^R K ^R	Y	N	N
Prevotella	P ^S V ^R C ^V K ^R	N	Y	V
Porphyromonas	P ^S V ^S C ^R K ^R	N	Y	V
Fusobacterium	P ^S V ^R C ^S K ^S	V	N	N

N, No; R, resistant; S, sensitive; V, variable; Y, yes.

Prevotella and Porphyromonas

Both Prevotella and Porphyromonas were previously considered to be part of the genus Bacteroides. These pigmented GNARs can be distinguished from one another metabolically as the saccharolytic Prevotella spp. and the asaccharolytic Porphyromonas spp. Approximately 20 Prevotella spp. have been implicated in causing human disease. Prevotella forms circular, convex, 1- to 2-mm, shiny, gray colonies. On Gram stain, they form short gram-negative rods and may assume coccobacilli forms ( Fig. 247.1B ). Prevotella grows well on laked blood agar with kanamycin and vancomycin (LKV) and has variable resistance to colistin. Although Prevotella spp. are largely regarded as pigmented GNARs, they can be nonpigmented as well. Pigmented Prevotella spp. form brown or black colonies after a week of growth on LKV. Before this brown or black pigment develops, Prevotella may fluoresce a dark red on exposure to a Wood lamp (long-wave ultraviolet light). By colony morphology and Gram stain, Porphyromonas spp. tend to form smaller colonies and appear as shorter rods or coccobacilli on Gram stain but can be difficult to distinguish from Prevotella. Porphyromonas usually grows as pigmented colonies, initially forming gray colonies that darken to black colonies within a week after plating on laked blood agar. Porphyromonas does not grow on LKV media because of its sensitivity to vancomycin, but it is resistant to colistin.

Fusobacterium

Fusobacterium is a genus of obligately anaerobic filamentous gram-negative rods that are members of the phylum Fusobacteria, in contrast to the Bacteroides, Prevotella, and Porphyromonas genera, which are members of the phylum Bacteroidetes. On blood agar, Fusobacterium forms pinpoint colonies that can be circular or irregular, with some species, such as Fusobacterium nucleatum, forming umbonate “fried egg” colonies after 3 to 5 days of incubation. Depending on the strain, they can be hemolytic, and some strains hemagglutinate. Fusobacterium spp. can be variable on Gram stain and display a range of cellular morphologies from coccoid, pleomorphic spherules (Fusobacterium necrophorum) to rod shaped. Rods can be short with rounded ends or long and thin with pointed ends (F. nucleatum) , arrayed end to end ( Fig. 247.1C ). As a genus, Fusobacterium is sensitive to both kanamycin and colistin and resistant to vancomycin. It can be distinguished by its bile sensitivity and metabolism of threonine to propionate. Most species are indole positive and produce butyric acid during the fermentation of glucose.

Symbiosis

The human microbiome is dominated by anaerobes. GNARs colonize the mucosal surfaces of the oropharynx, gastrointestinal tract, and female urogenital tract, and Bacteroides and Prevotella are among the most abundant genera present. Mutualism and opportunism are important features of the symbiotic relationship between human hosts and colonizing species of the Bacteroides, Prevotella, Porphyromonas, and Fusobacterium genera.

Gastrointestinal Tract

The human colon is colonized by 10 trillion to 100 trillion bacteria, making it the largest repository for bacteria in the body. The Bacteroides genus constitutes 30% of the total colonic bacteria. Bacteroides vulgatus, Bacteroides thetaiotaomicron, B. fragilis, and Bacteroides ovatus are the most commonly encountered Bacteroides spp. in the human colon. There is significant variability among humans regarding the colonic colonization, with different B. fragilis group members as assessed by stool culture and 16S ribosomal RNA gene amplicon sequencing (Comstock and Onderdonk, unpublished). The colonization of the gastrointestinal tract occurs during descent through the vaginal canal or postnatally for infants delivered by cesarean section. In particular, children born by cesarean section appear to have a delay in reaching their full Bacteroides complement. Both breast milk and exposure to environmental factors are important forces in shaping colonization. Many studies over the past several decades have been undertaken to examine this process in detail, with genomic-based technology taking center stage more recently.

Symbiosis and Mutualism in Immunity and Metabolism

There has been an explosion of interest in clinical correlations between microbiota profiles including the presence or absence of specific species and human health and disease. The pioneering work of Björkstén and others examined correlations between infectious diseases and atopic and allergic diseases for many decades. One study suggested that B. fragilis colonization at younger than 3 weeks of age increases the risk of asthma later in life. The basic science behind these clinical observations rests in the hypothesis that certain bacterial factors not only instruct immune cell development but also tune immune responsiveness. A large body of work in preclinical models over the past decade supports that the B. fragilis zwitterionic polysaccharide A (PSA) reinforces homeostatic immune function both in the gut and systemically despite earlier work that focused on the abscessogenic potential of B. fragilis . Investigators have demonstrated that PSA is a symbiosis factor that balances T-cell subset population size and function and may be a beneficial microbe for the treatment of chronic inflammatory diseases such as inflammatory bowel disease and multiple sclerosis.

The immunomodulatory effects of Bacteroides spp. are not exclusive to B. fragilis; B. thetaiotaomicron also has immunomodulatory properties with the potential to dampen proinflammatory responses to commensal, intestinal bacteria. B. thetaiotaomicron targets the RelA subunit of the transcription factor nuclear factor kappa B, a master regulator of proinflammatory immune responses. This process is dependent on a nuclear receptor and transcription factor called peroxisome proliferator-activated receptor gamma and is independent of the nuclear export receptor CRM1. However, the mechanism by which B. thetaiotaomicron drives this process remains to be fully understood. Strains of B. thetaiotaomicron with sulfatase activity have also been shown to contribute to intestinal inflammation in genetically susceptible mice. Bacteroides and other intestinal commensals also contribute to host immunity through the process of colonization resistance, the concept that the entrenched presence of gut symbionts provides protection from invading pathogens. The most central and well-understood function of Bacteroides spp. involves the mutualistic function of metabolism. The human gut at its core is a bioreactor. The saccharolytic Bacteroides spp. process diverse dietary and host polysaccharides for their own metabolic needs and in doing so aid in human digestion and nutrient liberation for their human hosts. A well-nuanced understanding of the biochemistry and genomics of glycan foraging by B. thetaiotaomicron has emerged over the past several years. In addition, numerous studies support the potential role of these bacteria in human energy balance with implications for the human obesity epidemic.

Female Urogenital Tract

The female urogenital tract, particularly the vagina, is densely colonized by anaerobes. Although gram-positive anaerobic lactobacilli are the predominant colonizers, GNARs may also be present in significant numbers. Vaginal colonization is dynamic, with variations not only intrinsic to the female reproductive life cycle from the premenarchal through postmenopausal years but also with significant shift over a given menstrual cycle. Pregnancy and parturition result in substantial microbiota population shifts in both the urogenital tract and the gastrointestinal tract as well.

There is also intraindividual genus-level diversity in colonization with Bacteroides, Fusobacterium, Prevotella, and Porphyromonas genera. In addition, distinct microbial community differences exist among the labia, the urethral vestibular region, the length of the vagina, and the cervix. Frequent isolates from the vaginal vault include Bacteroides urealyticus and members of the B. fragilis group; Prevotella bivia, Prevotella disiens, Prevotella buccalis, Prevotella melaninogenica, and Prevotella corporis; Porphyromonas asaccharolytica; and F. nucleatum. Most research on the vaginal microbiota and symbiotic effects has focused on Lactobacillus, with the view that GNARs may contribute to vaginal dysbiosis, negative consequences for fetal outcomes, and host mycotic and bacterial infections.

Oropharynx

The oropharynx is a diverse niche for both aerobes and anaerobes. Within the mouth, the teeth, gingival crevices, saliva, and posterior pharyngeal structures provide distinctive milieus with diverse pH and oxygen tensions. Gingival scrapings are particularly dense in bacteria, with estimated concentrations on the order of 10 ^11–12 CFU/mL. Similar to the colonization of the lower gastrointestinal tract, colonization of the oropharynx starts at birth. Among the anaerobes, Lactobacillus and Peptostreptococcus are the earliest colonizers. Fusobacterial populations emerge with the eruption of the first teeth and increase with establishment of full juvenile dentition. Colonization with Prevotella and Porphyromonas spp. emerges after colonization by Fusobacterium spp . Of the GNARs, Porphyromonas gingivalis and P. endodontalis; F. nucleatum; Prevotella intermedia, P. melaninogenica, Prevotella denticola, and Prevotella loescheii; and Tannerella forsythia all commonly populate dental plaque. Poor dentition, gingivitis, and other periodontal diseases correlate with increased numbers of GNARs, as do hospitalization, residence in a long-term care facility, and multiple medical comorbidities.

Opportunism

Symbiosis should be viewed as a host-microbe relationship spanning the spectrum from mutualism through opportunism. Most, if not all, symbiotic GNARs have pathogenic potential. B. fragilis warrants special consideration because although B. fragilis itself may not be the most abundant GNAR cultured from the gastrointestinal tract, it is the most common GNAR identified in clinical isolates from both blood and abscesses. Multiple virulence factors underpin this observation and account for the observed opportunism of Prevotella, Porphyromonas, Fusobacterium, and Bilophila. Virulence factors produced by these bacteria include capsular polysaccharides, outer membrane proteins, lipopolysaccharide (LPS) endotoxins, attachment factors (fimbriae/pili/adhesins), toxins, and numerous enzymes. Synergism is another important concept that explains the presence of GNARs in many polymicrobial infections. Several facultative anaerobes and aerobes can provide favorable environments that can promote the growth of GNAR. For example, the production of superoxide dismutase by facultative species protects obligate anaerobes that do not produce this enzyme constitutively against the highly lethal superoxide anion. Without the presence of these other microbes, the oxygen tension would not be favorable for GNARs. In many polymicrobial infections, facultative anaerobes and aerobes function mutualistically for GNARs, providing strong synergy for their expansion, especially for B. fragilis. In turn, the B. fragilis group with its frequent β-lactamase activity can provide a protective environment for normally β-lactam-sensitive facultative anaerobes and aerobes. Such cooperativity can also translate into an interdependency that may explain why antimicrobial therapy that is not effective against every member of a polymicrobial infection is nevertheless able to successfully cure some infections.

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