Presumptive (PID) Identifications of Clinically Significant Anaerobes A suggested scheme followed by many authorities in anaerobic clinical microbiology includes

es the inoculation of four different agar-containing PRAS media: Brucella Agar Phenyl-Ethyl Alcohol Aga Bacteroides Bile Esculin Agar Laked Kanamycin Vancomycin Agar The specimen can also be inoculated into Thioglycolate Broth or Chopped Meat Broth, but this is to be used only as an emergency back-up After 24 hour incubation, the primary plates (BRU, PEA, and BBE/LKV) are analyzed for colony morphologies/features/characteristics. Each morphotype seen on the primary plates is prepared for a Gram stain, for aerotolerance testing, and is subcultured onto a non-selective media (such as BRU) supplemented with specific special-potency disks (Gram stain reaction and microscopic examination determine which disks are used). Aerotolerance test uses CHOC Agar inoculated in a pie plate fashion, placed in a CO2 incubator (an aerobic atmosphere) at 35-37C for 24 hours. Colony types must be carefully noted and labels accurately placed on the plates and in the lab notebook. It is important not to commit an error in this step; it determines if an isolate is an obligate anaerobe or if an isolate is an aerotolerant/facultative anaerobe.

For gram-negative bacilli or coccobacillary isolates, use a Kanamycin 1000 g disk, a

Vancomycin 5 g disk, and a Colistin 10 g disk, as well as a Nitrate disk (Nitrate disk is added to each plate that is subcultured, no matter the Gram reaction or the bacterial cell morphology).

For a gram-negative coccus, Nitrate ( disk, most-likely PID is Veillonella spp. (parvula) ) For a gram-positive bacillus or coccobacillus, use a Nitrate disk, and if necessary, use a Vancomycin 5 g
disk. An appropriate situation to utilize a Vancomycin disk is when the cells in the Gram stain are of questionable gram-negative or gram-positive distinction. All truly gram-positive anaerobic rods and cocci are sensitive to Vancomycin, whether they stain appropriately so or otherwise. The only clinically significant gramnegative anaerobe sensitive to Vancomycin is Porphyromonas spp..

For gram-positive, spore-forming rods, an optional Egg Yolk Agar plate may be used (in addition to the BRU
plate with special-potency disks) to detect lipase or Lecithinase activity. Lipase ( reaction is noted by an opalescent sheen, mother of pearl sheen, or an oil on water type light ) refraction. Lecithinase () reaction is noted by a cloudy precipitate that uniformly forms around colonies, an increase in opacity, or a whitening of the EYA media.

For anaerobic, gram-positive cocci, place an SPS (Sodium Polyanethol Sulfonate) disk.

The use of this disk will lead to the PID of Peptostreptococcus anaerobius because it is the only anaerobic gram-positive coccus that is sensitive to this substance. A clear zone of inhibition around the SPS disk indicates that the culture is P. anaerobius. GNARs Gram-Negative Anaerobic Rods (and coccalbacilli) PIDs B.fragilis group KVC reaction is RRR, and shows good growth on BBE with colony size >1 mm in diameter. Blackening of BBE agar due to Esculin Hydrolysis (except B.vulgatus). The B.fragilis group is Nitrate (). * B.fragilis grows on LKV, is Catalase () and Indole () Bile resistance shown by growth support on BBE. KVC reaction is RRR (a strong indicator the PID will be Bacteroides fragilis).

* Bacteroides thetaiotamicron Same definitive parameters as B.fragilis, except for Indole. B.thetaiotamicron is Indole () * Bacteroides vulgatus Esculin Hydrolysis (), Indole (), and Catalase () * Bacteroides distasonis Bacteroides ureolyticus group Microaerophilic, non-pigmented, Bile-sensitive bacilli noted by a distinguishing pitting of the agar surface, and the requirement for growth stimulation by Formate/Fumarate. Several species of non-pigmented Prevotella are placed in this group (Prevotella buccae, P.oralis, P.bivia, P.disiens with the defining characteristics: KVC reaction is SRS, Nitrate Reduction () (this differentiates the isolate from Fusobacterium spp..) This group is Indole (), also separating it from most Fusobacteria. However, the spot Indole test is not usually performed if the Nitrate test is positive.. This group is also Catalase (), whereas Bilophila wadsworthia (the other Nitrate () GNAR with a KVC reaction of SRS) is Catalase (). * Bacteroides ureolyticus KVC reaction of SRS, Nitrate (), (Indole ()), pitting of the agar surface, and a sound PID comes from a Urease () test. B.ureolyticus is Urease (). * Campylobacter gracilis (formerly called Bacteroides gracilis) KVC reaction of SRS, Nitrate (), (Indole ()), may show a degree of Bile resistance, pitting of the agar surface, and the PID comes from a Urease () reaction . *Also, C.gracilis is Oxidase (), whereas B.ureolyticus is Oxidase (), as are most Campylobacter spp. * Wolinella spp. or other Campylobacter spp. KVC reaction of SRS, Nitrate (), (Indole ()), Urease (), and a PID comes from observing Motility on a hanging drop slide, as well as supported growth in a microaerophilic atmosphere. Wolinella species were formerly classified as anaerobic vibrios. They are associated with dental infections. Members are sensitive to Kanamycin and Colistin, resistant to vancomycin. They are also Catalase negative and sensitive to bile. Wolinella species can be distinguished from microaerophilic Campylobacter species on the basis of the Oxidase reaction; Wolinella are Oxidase () and Campylobacter species are Oxidase (). Other anaerobic curved rods include Butyrivibrio and Succinivibrio. Bilophila wadsworthia KVC reaction is SRS, Nitrate (), Bile-resistance shown by growth on BBE, Catalase (), no growth on LKV (due to Kanamycin sensitivity). The appearance of a fish-eye colony, a translucent colony with a black, opaque center (resulting from H2S production, not from Esculin hydrolysis), that is <1 mm in diameter soundly supports a PID of plate isolate to be B.wadsworthia *This organism also tests Urease (/), but this does not contribute to its PID. Fusobacterium spp. gram-negative, non spore-forming and non-motile bacilli. They can sometimes grow in the presence of up to 6% O2; most strains produce a greening of blood-containing agar when exposed to air due to H2O2 production (especially F.nucleatum.) Like most gram-negative bacteria, Fusobacterium spp. are resistant to Vancomycin, but it is sensitive to Kanamycin and Colistin. The name comes from fusus, a spindle, and bacterion, a small rod. The species name of nucleatum was given due to its nucleated appearance from the light microscope from their intracellular granules. Most of the bacteria are 5 to 10 m in length with pointed ends. They are weakly saccharolytic or non-fermentative; although the production of butyric acid from fermentation of glucose and peptone is unique among its family. Fusobacterium species reside in the genitourinary tract, mouth, and gastrointestinal tract as normal flora. Fusobacteria can be differentiated from other gram-negative, obligate anaerobes by its ability to produce significant amounts of butyric acid from glucose, giving cultured colonies a characteristic odor. Many Fusobacterium spp. exhibit a chartreuse fluorescence on BRU under UV light * Fusobacterium necrophorum KVC reaction of SRS, Nitrate (), Indole (), Lipase (), and sensitive to Bile. Also known as the Lipase Positive Fusobacterium, this species produces a "fried egg," off-white colony; on BRU this organism produces a chartreuse fluorescence. -hemolysis is often observed. Gram stain reveals a highly pleomorphic, pale staining organism, and long filamentous forms with occasional swollen areas.

* Fusobacterium nucleatum group KVC reaction of SRS, Nitrate (), Indole (), but Lipase (), and sensitive to Bile. F. nucleatum colonies often fluoresce chartreuse under UV light. Thin, gramnegative bacillus with pointed ends, may demonstrate a nucleated appearance due to intracellular granules. Several colony morphotypes may be observed; small (<1mm) breadcrumb-like colony, speckled looking (ground glass like), and smooth, convex. The different colony morphotypes probably represent different subspecies of F. nucleatum. Gram stain usually demonstrates pale staining rods with pointed ends (F. nucleatum ss. nucleatum is always fusiform). * Fusobacterium mortiferum-varium KVC reaction of SRS, Nitrate (), Indole (). This group differs from other members of the genus in that they are resistant to 20% bile. Members of this group often demonstrate irregular staining, bizarre forms called round bodies when Gram stained. To differentiate between F.mortiferum and F.varium, one should note that F.mortiferum is Indole () and positive for Esculin Hydrolysis, while F.varium is Indole () and is unable to hydrolyze Esculin. An ONPG BSS test may also be performed; F.mortiferum is ONPG () and F.varium is ONPG () Colonies of F. mortiferum-varium do not fluoresce. Porphyromonas spp. KVC reaction of RSR, representing the only gram-negative anaerobe that is truly sensitive to Vancomycin. Brown-black pigmented colonies (protoheme) on PEA (unable to grow on LKV because of its inherent sensitivity to Vancomycin,) asaccharolytic, Bile-sensitive, gram-negative bacilli. The Gram stain reveals pale-staining coccobacilli that may show a degree of pleomorphism; colonies are often mucoid. * Porphyromonas asaccharolytica KVC reaction of RSR, red fluorescence, Indole (), Lipase (), dark brown-black pigment (usually requires an extended incubation for pigment to develop, which consequently diminishes fluorescence), gram-stain may reveal shorter, almost spherical cells, with longer cells occasionally seen. * Porphyromonas gingivalis KVC reaction of RSR, no fluorescence, dark brown-black pigment (usually requires an extended incubation for pigment to develop) * Porphyromonas endodontalis KVC reaction of RSR, red fluorescence, Indole (), Lipase (), dark brown-black pigment (usually requires an extended incubation for pigment to develop, which consequently diminishes fluorescence). To differentiate from P.asaccharolytica, most strains of P.endodontalis produce colonies that adhere strongly to blood agar plates; gram-stain may reveal bacilli or coccobacilli. Pigmented Prevotella Pigmented brown to black colonies (protoporphyrin,) Brick-Red fluorescence (fluorescence weakens as pigment develops,) saccharolytic, Bile-sensitive, gram-negative, pale-staining coccobacilli. May be pleomorphic; young cultures may appear gram-variable. Pigment production may take up to two weeks (1/3 isolates show pigment within 2 days, another 1/3 within 7 days, and the last 1/3 within 14 days,) and is faster on laked rabbit blood. Pigmentation may vary from light tan to black; intensity of pigment is useful for identification of members within this group Prevotella intermedia Prevotella intermedia is a gram-negative, black-pigmented periodontal pathogen (6). Their possession of black pigments is the reason why these organisms are also known as Bacteroides melaninogenicus subsp. intermedius. These rod-shaped microbes are strict oral anaerobes, but a short oxygen exposure will not be sufficient to kill them (5). These organisms live in the periodontal pockets in between the teeth where they co-exist with other microbes, forming an oral microbiota (3). Prevotella melaninogenica Prevotella melaninogenica is generally found in the oral cavity causing opportunistic pathogen in humans and at the rumen Prevotella melaninogenica are gram-negative, non-spore forming, obligate anaerobic, coccobacilli that are commonly found in the oral cavity of humans; however, they are also found in other areas of the body where they can act as opportunistic pathogens. These bacteria are also found in the rumen of cattle and sheep, where they aid in the breaking down of proteins and carbohydrates. Originally discovered in 1921 by Oliver and Wherry, Prevotella melaninogenica was re-classified from Bacteroides

of cattle and sheep which helps to break down protein and carbohydrates food. Similar to all species of the genus Prevotella, it is strictly anaerobic, gram-negative bacterium with non-spore forming coccobacilli. When Prevotella melaninogenica grows on blood-containing media, it gives black pigment that can be easily seen in adults with rapidly progressing periodontitis lesions. This bacterium is nonmotile, therefore usually forms biofilm. In addition, P. melaninogenica includes those organisms which hydrolyze esculin but not starch and produce acid in peptone-yeast-glucose medium and peptoneyeast-mannose medium. It is catalase negative, indole negative, lipase negative and bile sensitive. It also liquefies gelatin and clots milk which shows this organism is very acidic. Because of its acidity it prevents the bovine disease of rumen acidosis; however, it affects milk production due to too much acid in stomach. The genus Prevotella used to be part of the genus Bacteroides, therefore, there are similarities that these two genera share. Prevotella denticola melaninogenicus, due to new information gathered from 16s rDNA sequencing, circa 2002. They are now classified as follows: Bacteria, Bacteroidetes, Bacteroidetes, Bacteroidales, Noctuoidea, Prevotella, Prevotella melaninogenica. P. melaninogenica has been associated with many types of infection including: oral abscesses, infections in the intestinal tract, the female genitalia tract, the upper and lower respiratory tracts and in the bone marrow. Pyomyositis (infection of the skeletal muscles) has also recently been associated with Prevotella melaninogenica. This microorganism has also been linked with infections caused by dog and cat bites. It causes infections by producing hydrogen sulfide, ammonia, and cytotoxic substances. This microbe will also inhibit phagocytosis and killing of other bacteria when the body attempts to remove the invaders. Furthermore, P. melaninogenica can cause the host immunes system to generate substances with tissue destroying potential. To further irritate the body, these microbes can generate hemolysin; therefore, they can lyse erythrocytes. However, these bacteria can also lyse other important cells like mast cells, neutrophils and polymorphonuclear cells. In addtion to causing infections, because Prevotella melaninogenica are nonmotile, they contribute to developing a biofilm, particularly on the teeth, that can lead to periodontitis - the progressive loss of bone and recession of the gingiva around the teeth, which can lead to tooth loss if left untreated. * Prevotella corporis Other Prevotella, Non-Pigmented (also known as the Prevotella oralis group): P. bivia, P. disiens, P. buccae, P. oralis, P. buccalis The genus Prevotella includes strictly anaerobic, gram-negative, moderately saccharolytic, bile-sensitive rods formerly belonging to the genus Bacteroides (33). These bacteria, which are part of the human oral, intestinal, and urogenital floras, maybe involved in various infections, including infections of the head and neck, respiratory tract, central nervous system, and abdominal and urogenital tracts, as well as bacteremia(19). Metronidazole is commonly used for the treatment of infections caused by anaerobic organisms. For a long time, it has been considered that acquired resistance to these antibiotics rare among anaerobes, despite its extensive use. However, recent studies have shown that this resistance is no longer uncommon among these organisms Reduced susceptibility to 5-nitroimidazole drugs is generally associated with the presence of a nitroimidazole reductase encoded by a nim gene. This enzyme converts 4- or 5-nitroimidazoleto 4- or 5-aminoimidazole, avoiding the formation of the toxic nitroso radicals that are essential for antimicrobial activity (4). Currently, seven nim genes, named nimA to nimG, which are either plasmid or chromosomally encoded, have been described (30), while a new nim gene, nimH (GenBank accession number FJ969397), has been described in Bacteroides fragilis. Prevotella spp. have only rarely been investigated for the presence of nim genes in studies concerning metronidazole susceptibility (20, 23, 27). This led us to investigate a large panel of clinical Prevotella strains that belong to different species and that were isolated from patients in two French tertiary-care teaching hospitals for the presence of nim
genes and for the type of nim genes that they carry

GNAC Gram-Negative Anaerobic Cocci There are three clinically

genera of GNAC. The most encountered are of the genus Veillonella, and the most common isolate is Veillonella parvula. The other two genera of clinically significant GNAC are Acidaminococcus and Megasphaera. * Veillonella parvula KVC reaction of SRS, Indole (), Bile-sensitive, small gram-negative cocci, Glucose (). Slow growing and may require prolonged incubation. Nitrate () and Catalase (/). Often exhibits a red fluorescence on BRU; however this fluorescence will fade relatively quickly upon exposure to air. * Acidaminococcus fermentans Nitrate (), Catalase (), Glucose () * Megasphaera elsdenii Nitrate (), Catalase (), Glucose ()