Actinobacillus & Others

TAXONOMY AND DESCRIPTION OF THE AGENTS Back to top

The taxonomy of the family Pasteurellaceae has changed in important ways (51, 52, 83).

The Pasteurellaceaeconsist of several genera, of which four are known to contain human

pathogens: Actinobacillus, Aggregatibacter, Haemophilus, and Pasteurella. Minimal standards

for the description of genera, species, and subspecies of the Pasteurellaceae have been

proposed and might create further changes (24). Aggregatibacterspp., formerly

named Haemophilus (106), are included in this chapter. The genus Haemophilus, as

presently constituted, is described in chapter 34. Neisseria spp. with rod forms, and CDC

group EF-4a, renamed Neisseria animaloris (137), are described in chapter 32 but are

included in a differentiation table in this chapter (seeTable 4).

Actinobacillus

The genus Actinobacillus in the family Pasteurellaceae consists of facultatively anaerobic,

nonmotile, gram-negative rods and comprises animal (A. equuli, A. lignieresii, and A.

suis) and exclusively human (A. hominis andA. suis) species but no longer

includes Actinobacillus actinomycetemcomitans (106). Subspeciation of A. equulicould not be

upheld by 16S rRNA and infB gene sequencing (105). The G+C content of the DNA

of Actinobacillusspp. is between 40 and 43 mol%.

Aggregatibacter

Earlier, 16S rRNA gene sequencing and DNA-DNA hybridization had placed Actinobacillus

actinomycetemcomitans closer to the genus Haemophilus than to the

genus Actinobacillus (108), and multilocus sequence analysis of A. actinomycetemcomitans,

Haemophilus aphrophilus, Haemophilus paraphrophilus, andHaemophilus segnis (106) had

suggested a monophyletic group. In addition, DNA-DNA relatedness between H.

aphrophilus and H. paraphrophilus was 77%, and DNA from H. aphrophilus was able to

transform H.paraphrophilus to have an NAD-independent phenotype (106). It was, therefore,

proposed to transfer A. actinomycetemcomitans, H. aphrophilus, H. paraphrophilus, and H.

segnis into a new genus, Aggregatibacter in the family Pasteurellaceae, with the species A.

aphrophilus comprising the organisms formerly named H. aphrophilus and H.

paraphrophilus, i.e., including V factor-dependent and -independent isolates (106). The G+C

content of the DNA of Aggregatibacter spp. is 42 to 44 mol%.

Capnocytophaga

The Capnocytophaga genus in the family Flavobacteriaceae (51) at present consists of nine

species (C. canimorsus, C. cynodegmi, C. ochracea, C. gingivalis, C. sputigena,

C. haemolytica, C. granulosa, C. leadbetteri, and genospecies AHN8471) of facultatively

anaerobic, nonmotile, gram-negative rods (43). The G+C content of the DNA of this genus is

34 to 40 mol%.

Cardiobacterium

The Cardiobacterium genus in the family Cardiobacteriaceae (51) consists of facultatively

anaerobic, nonmotile, gram-negative rods, with the species C. hominis and C.

valvarum (61). The G+C content of the DNA of these species is 59 to 60 mol%.

Chromobacterium

The Chromobacterium genus in the family Neisseriaceae (51) contains several facultatively

anaerobic, motile species, of which C. violaceum is at present the only agent of human

disease. Its DNA content is between 65 and 68 mol%. Chromobacterium

haemolyticum, recently isolated from a sputum culture, is so far represented by one strain

only (62) and will not be covered here.

Dysgonomonas

The genus Dysgonomonas in the family Porphyromonadaceae consists of facultatively

anaerobic, nonmotile, gram- negative rods (51). Four species have been described: D.

capnocytophagoides and D. gadei (69), D. mossii (91), and “D. hofstadii” (90). The G+C

content of the DNA of these species is around 38 mol%. The related “DF-3-like” bacteria (30)

have not been investigated further.

Eikenella

The genus Eikenella in the family Neisseriaceae (51) consists of facultatively anaerobic,

nonmotile, gram-negative rods. Thus far, only one species, E. corrodens, has been

recognized, but DNA-DNA hybridization, analysis of the composition of the organism’s

cellular carbohydrates, and the occurrence of biochemically aberrant (e.g., catalase-positive)

isolates suggest that there may be more than one genomospecies (77). The G+C content of

the DNA of Eikenella is between 56 and 58 mol%.

Kingella

The genus Kingella in the family Neisseriaceae consists of the facultatively anaerobic,

nonmotile species K. kingae, K. denitrificans, K. oralis, and K. potus (51). The G+C content

of the DNA of these species is between 47 and 58 mol%.

Pasteurella

The taxonomy of the genus Pasteurella in the family Pasteurellaceae (51) has been in flux for

some time (24).Pasteurella multocida can be separated into the subspecies multocida,

septica, and gallicida; Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis are

other species isolated from humans. In spite of the genotypical homogeneity of P.

multocida isolates, phenotypically diverse lineages have been observed, e.g., sucrosenegative

variants from infections from bite wounds made by large cats (22). The G+C

content of the DNA of Pasteurella species is between 38 and 46 mol%. New genera or

reclassifications may be necessary for the species discussed below that are preceded

by “(P.)” or “(Pasteurella)” (52, 66).

Simonsiella

The genus Simonsiella in the family Neisseriaceae (51) consists of several obligately aerobic

species which may show gliding motility (85). The only species isolated from humans

is Simonsiella muelleri. This genus has a G+C content in its DNA of 40 to 50 mol%.

Streptobacillus

The Streptobacillus genus in the family Fusobacteriaceae (51) consists of one facultatively

anaerobic, nonmotile species, S. moniliformis, with a G+C content in its DNA of 24 to 26

mol% (40).

Suttonella

The Suttonella genus in the family Cardiobacteriaceae (51) consists of facultatively

anaerobic, nonmotile, gram- negative rods and so far contains only one species, S.

indologenes (formerly Kingella indologenes) (35). The G+C content of its DNA is 49 mol%.

EPIDEMIOLOGY AND TRANSMISSION Back to top

Most bacteria in this group are part of the flora of the nasopharynx and/or the oral cavity of

animals and/or humans and are parasitic, with the only environmental species

being Chromobacterium. Transmission from animals occurs by contact (e.g., bites and licking

of wounds), from humans to humans by droplets (e.g., directly with Kingella spp. or by

paraphernalia or human bites with Eikenella spp.). They may cause infections anywhere in

the human body. Risk factors exist for certain types of septicemia (e.g., liver cirrhosis for P.

multocida, neutropenia for oxidase-negative Capnocytophaga spp., and chronic

granulomatous disease forChromobacterium violaceum). Endogenous infections occur as

well, e.g., HACEK (Haemophilus parainfluenzae,

Aggregatibacter spp., Cardiobacterium spp., Eikenella corrodens, and Kingella spp.)

endocarditis (see “Clinical Significance” below) (16).

Actinobacillus lignieresii (primary habitat in the oral cavities of sheep and

cattle), Actinobacillus equuli (in the oral cavities of horses and pigs), and Actinobacillus

suis (in the oral cavities of pigs) can be transmitted to humans by animal contact (21).

Exclusively human are Actinobacillus hominis and Actinobacillus ureae, whose normal habitat

is unknown (47, 81).

The habitat of Aggregatibacter spp. is the human oral cavity, including dental plaque (81).

Infections are endogenous.

The oxidase- and catalase-negative species Capnocytophaga ochracea, Capnocytophaga

gingivalis, Capnocytophaga sputigena, Capnocytophaga haemolytica, and Capnocytophaga

granulosa, as well as the recently described Capnocytophaga leadbetteri and genospecies

AHN8471 (43), are normal but not prominent members of the human oral flora. The first

three have been isolated from adults with periodontal disease but also from periodontitis-free

adults; the other four have been isolated from supragingival and subgingival plaque in

children and adults (43). Infections are endogenous. The oxidase- and catalase-positive

species C. canimorsusand C. cynodegmi reside in the oral cavities of healthy dogs (25% of

dogs have C. canimorsus as determined by culture and 85 to 100% of dogs have it as

determined by PCR) and cats (15%, as determined by culture) (10, 136).

The normal habitat of Cardiobacterium spp. is the human oral cavity and nasopharynx but

possibly also the gastrointestinal and urogenital tracts (129). Infections are endogenous.

Chromobacterium inhabits soil and water in tropical and subtropical climates between

latitudes of 35°N and 35°S (South Africa, Southeast Asia, Australia, southeastern United

States, and, rarely, South America) (93). The portal of entry is usually the skin, but oral

intake has also been reported (125).

Most Dysgonomonas capnocytophagoides strains have been isolated from stools of

immunocompromised patients, and a few strains have been isolated from other sources

(63, 95). The natural habitats of this and the other Dysgonomonas spp. are unknown.

The natural habitat of Eikenella corrodens is the oral cavities and possibly the

gastrointestinal tracts of humans and some mammals, from which it can be transmitted via

saliva (bites, syringes) to other individuals (112, 123,134). Endogenous infections prevail,

however.

The natural habitat of Kingella spp. is the upper respiratory tract and oral mucosa of humans

and possibly other primates. K. kingae colonizes the throat but not the nasopharynx of many

children aged 6 months to 4 years (142). The natural habitats of K. denitrificans and K.

potus are unknown. K. oralis has been isolated from the human mouth (18). Ribotyping and

pulsed-field gel electrophoresis have shown that K. kingae can be transmitted via respiratory

droplets (128), although most infections are endogenous.

Pasteurella spp. are widespread in healthy and diseased wild and domestic animals, including

rodents, dogs, and cats, inhabiting the nasopharynx and gingiva (139). Human isolates are

transmitted predominantly from animals by contact (bites or licking or scratching of

wounds). Of the “related” species, (Pasteurella) aerogenesoccurs primarily in pigs

(39), (Pasteurella) caballi in pigs and equines (23), and (Pasteurella) pneumotropica in

rodents and dogs (44); the natural habitat of (Pasteurella) bettyae is uncertain.

The natural habitat of Simonsiella muelleri is the oral cavities of humans (86).

Streptobacillus moniliformis occurs naturally in the upper respiratory tracts of up to 100% of

wild and laboratory rats and other rodents (mice, gerbils, squirrels, ferrets, weasels) and

occasionally of dogs and cats preying on rodents. Transmission to humans occurs either from

bites of those animals (rat bite fever) or from consumption of contaminated food or water

(Haverhill fever) (40).

The natural habitat of Suttonella indologenes is not known.

CLINICAL SIGNIFICANCE Back to top

Actinobacillus spp.

A. lignieresii causes actinobacillosis, a granulomatous disease in cattle and sheep in which,

as with actinomycosis, sulfur granules form in tissues (117). A few human soft tissue

infections after a cattle or sheep bite or other contacts have been reported (113). A.

equuli and A. suis have caused a variety of diseases in horses and pigs; human infections are

generally due to horse or pig bites or contact (5, 41). Both species have also been isolated,

albeit rarely, from the human upper respiratory tract (118, 140). A. ureae is most often a

commensal in the human respiratory tract, particularly in patients with lower respiratory

tract disease (81), but has also been found as an agent of meningitis following trauma or

surgery (32) and of other infections in immunocompromised patients (78). A. hominis has

also been isolated from such patients but has occurred as a commensal as well, albeit rarely

(47). Virulence factors belong to the pore-forming protein toxins of the RTX family; RTX

toxins have repeats in the structural toxin peptide and exhibit a cytotoxic and often also a

hemolytic activity. They are particularly widespread in species of the

family Pasteurellaceae (46).

Aggregatibacter spp.

A. actinomycetemcomitans is one of the major agents of juvenile and adult periodontitis

(104) and may occur together with Actinomyces spp. in actinomycotic sulfur granules (76).

Furthermore, it may cause HACEK endocarditis (16), soft tissue infections, and other

infections (76, 110). HACEK are the causes of approximately 1% of all cases of endocarditis.

HACEK endocarditis is characterized by a relatively long interval between first symptoms and

diagnosis (range, 2 weeks to 6 months), large vegetations on native or artificial valves of the

left side, and frequent embolizations. Prognosis is good with appropriate antibiotic treatment

(16).

Virulence factors are an RTX leukotoxin (46), a cytotoxic distending toxin (7), and the

adhesin EmaA (132), as well as fimbriae (67).

A. aphrophilus may cause systemic disease, particularly bone and joint infections,

spondylodiscitis, and endocarditis (29, 72, 111).

A. segnis, whose frequency may be underestimated due to apparent misdiagnoses, may

cause endocarditis and, rarely, other systemic infections (89, 130).

Capnocytophaga spp.

C. ochracea, C. gingivalis, C. sputigena, C. haemolytica, and C. granulosa have been

reported as agents of septicemia and other endogenous infections (endocarditis,

endometritis, osteomyelitis, soft tissue infections, peritonitis, keratitis, noma) (13, 112, 138)

in immunocompetent and immunosuppressed (mainly neutropenic) patients. They are able to

suppress neutrophilic chemotaxis and lymphocyte proliferation (107). The association with

periodontitis remains unclear (43).

Infections with C. canimorsus and C. cynodegmi are associated mainly with dog or cat bites

or contact. Patients infected with C. canimorsus most often present with septicemia and have

previously been splenectomized or are alcoholics. In fulminant cases with a poor prognosis,

disseminated intravascular coagulation, acute renal failure, respiratory distress syndrome,

and shock may develop (74). Hemolytic-uremic syndrome and thrombotic thrombocytopenic

purpura are other possible sequelae (82, 100). Meningitis (31), eye infections (13), and

endocarditis (119) have been reported as well. C. cynodegmi has been isolated more rarely,

mainly from localized or systemic infections (114). C. canimorsus resists phagocytosis by

macrophages and killing by complement and leukocytes; macrophages incubated with the

bacterium fail to produce several proinflammatory cytokines (124).

Cardiobacterium spp.

Disease caused by both Cardiobacterium species is mainly HACEK endocarditis (94); on rare

occasions, C. hominis has been isolated from other body sites (87, 112). In blood culturenegative

cases, the diagnosis has been made by broad-range PCR applied to valve tissue

(103).

Chromobacterium violaceum

Localized infections usually arise from contaminated wounds, and septicemia with multiple

organ abscesses may follow (93). They are significantly associated with neutrophil

dysfunction (glucose-6-phosphate dehydrogenase deficiency, chronic granulomatous

disease). Children without these conditions and those with bacteremia show a high fatality

rate (125). A number of virulence factors other than endotoxin, i.e., adhesins, invasins, and

cytolytic proteins, have been described (15).

Dysgonomonas spp.

Diarrhea was reported to have occurred in 10 of 20 patients with fecal isolates of C.

capnocytophagoides,whereas routine stool cultures yielded the organism in 1.1 to 2.3% of

cultures (95). Bacteremia occurs as well (63); one blood isolate was found to be identical by

ribotyping to one in the stool of the same patient (58). One strain of D. mossii was isolated

from intestinal juice of a patient with pancreatic carcinoma (96). D. gadeihas been isolated

from a human gallbladder (69) and from blood (3), and “D. hofstadii” has been isolated from

a wound (90).

Eikenella corrodens

E. corrodens is associated with juvenile and adult periodontitis (104) but is also an agent of

infections of the upper respiratory tract, pleura and lungs, abdomen, joints, bones, wounds

(e.g., from a human bite), and, rarely, other infections, like noma (71, 112, 123, 134).

These organisms are often indolent and found mixed with other members of the

oropharyngeal biota, particularly staphylococci and streptococci. Risk factors are dental

manipulations and intravenous drug abuse. Endocarditis is of the HACEK type if

monomicrobial, but polymicrobial non-HACEK cases are known (16). E. corrodens can trigger

a cascade of events that induce inflammation in periodontal tissue (145).

Kingella spp.

Infections with K. kingae show a predilection for bones and joints of previously healthy

children under 4 years of age (142). The use of culture and broad-range and K. kingaespecific

real-time PCR show it to be the most common cause of osteoarthritic infection in this

age group (20). Septic arthritis, discitis, and osteomyelitis of the lower extremities as well as

occult bacteremia are conspicuous. Stomatitis and/or upper respiratory infections may

precede systemic disease, suggesting entry through a damaged mucosa (142). In adults,

systemic infections occur in immunocompromised individuals (11) or may present as HACEK

endocarditis (16). Virulence factors are an RTX toxin (79) and type IV pili (80).

K. denitrificans has been reported as an agent of endocarditis (99). K. oralis has been

isolated from patients with periodontitis, but its relationship to the disease is unclear (18). K.

potus has caused a wound infection following a kinkajou bite (92).

Pasteurella spp.

Human isolates of P. multocida are mostly found in wound or soft tissue infections. Less

frequent are colonization or infection of the respiratory tract and (by the hematogenous or

contiguous route) systemic disease, such as meningitis, dialysis-associated peritonitis,

endocarditis, osteomyelitis, and septicemia, with cirrhosis of the liver being a particular risk

factor (139). The subspecies most frequently encountered is subsp.multocida, which is also

more frequent in respiratory infections and bacteremias than subsp. septica, which is most

often associated with wound infections (36). Infected cat bite wounds contain pasteurellae

significantly more often than infected dog bite wounds, reflecting a higher oropharyngeal

colonization rate in cats than in dogs (36). In the respiratory tract, colonization may

eventually lead to sinusitis or bronchitis as well as pneumonia and empyema, the latter two

mostly in patients with prior respiratory disease (101). Virulence factors are capsules (six

serotypes, of which A and D account for most human isolates) (36), lipopolysaccharide, an

RTX cytotoxin (46), surface adhesins, and iron acquisition proteins (64).

Cases of human infection with P. canis (from dogs), P. dagmatis, and P. stomatis (from dogs

or cats) are infrequent (2, 36, 59). In some cases of pasteurellosis, animal contact could not

be established. Double infections with two Pasteurella spp. have also been observed (70).

Of the “related” species, (P.) aerogenes has caused wound infections from pig and hamster

bites (39, 45), (P.) bettyae has been found in infections of newborns and in infections of the

male and female genital tracts (12,33), and (P.) caballi has been isolated from horse bites

(42); (P.) pneumotropica is a rare agent of systemic infection in humans (44). Reported

cases of human infection with Avibacterium gallinarum, Bibersteinia trehalosi, Gallibacterium

anatis, and Mannheimia haemolytica, all formerly in the genus Pasteurella, remain doubtful

when stricter identification criteria are employed (60) or require confirmation (52).

Simonsiella spp.

S. muelleri has not been found associated with disease (17, 141).

Streptobacillus moniliformis

Rat bite fever is a systemic illness beginning with fever and chills, followed by migratory,

sometimes even suppurative, polyarthritis and a maculopapular rash on the extremities.

Rare complications include endocarditis, myo- or pericarditis, pneumonia, septicemia, and

abscess formation (37, 40).

Suttonella indologenes

Human isolates of S. indologenes have been very rare; they have been isolated from ocular

sources (140) and a blood culture in a case of endocarditis (75).

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

Collection of specimens should follow the guidelines described in chapter 16; the low viability

of many species makes the use of transport media, e.g., eSwab (Copan Diagnostics Inc.,

Murrieta, CA), whenever indicated, mandatory. Cultures of most bacteria described in this

chapter can be stored at room temperature for 1 to 2 weeks. However, for some other very

fastidious bacteria, e.g., C. canimorsus, subcultures must be performed frequently. For

keeping strains in a culture collection, the isolates should be frozen in a cryoprotective

solution, e.g., skim milk, at -70°C.

DIRECT EXAMINATION Back to top

Actinobacillus spp. are medium-sized, gram-negative rod-shaped or coccoid bacteria with a

tendency to bipolar staining. Their arrangement is single, in pairs, and, rarely, in short

chains.

Aggregatibacter spp. are coccoid or rod-shaped gram- negative bacteria, occasionally

exhibiting filamentous forms. A PCR for A. actinomycetemcomitans has been developed (50).

Capnocytophaga spp. are mainly fusiform, medium-to-long cells with tapered ends (Fig. 1a).

PCR has also been devised for their detection (65). Leptotrichia buccalis is a long, fusiform,

gram-negative rod but with one tapered and one square end (Fig. 1b). Differentiation is

supported by biochemical reactions or by analysis of fatty acids (9).

ISOLATION PROCEDURES Back to top

For culture of members of this group, the use of blood or chocolate agar and, wherever

normal flora and specific bacteria are suspected, of selective media is mandatory. HACEK

members do not need more than 5 days of incubation in modern blood culture systems

(115).

Actinobacillus spp. require enriched media but not necessarily hemin for growth, and growth

is improved by a 5 to 10% CO2 atmosphere.

Some A. aphrophilus (formerly H. paraphrophilus) strains and A. segnis require V factor;

none requires hemin. All of them grow better in a CO2 atmosphere. Selective media have

employed bacitracin and vancomycin (133).

Primary isolation of Capnocytophaga spp. requires 5 to 10% CO2 and enriched media; the

composition of the blood agar base influences the ability to grow (38, 146). For detection in

mixed cultures, selective media containing bacitracin, polymyxin B, vancomycin, and

trimethoprim have been used (26), as have Thayer-Martin and Martin-Lewis agars (116).

Inhibition by sodium polyanethol sulfonate in blood culture media has been experimentally

verified (122). C. canimorsus has most often been isolated from blood. However, it may not

be detected by commonly used automated blood culture systems; therefore, clinicians should

inform the laboratory about risk factors for C. canimorsus infection so that subcultures on

enriched media can be performed in a blind manner (146).

Cardiobacterium spp. mostly require 5 to 10% CO2 and increased humidity for initial growth

on blood agar.

C. violaceum grows on routine media, even on most enteric ones, at 30 to 35°C, its optimal

temperature.

For Dysgonomonas spp., a selective medium containing cefoperazone, vancomycin, and

amphotericin B has been used for stool cultures (58).

With a few exceptions, E. corrodens strains require hemin for growth unless 5 to 10% CO2 is

present (55). Detection is improved by a selective medium containing clindamycin (126).

Recovery of K. kingae from body fluids and pus can be difficult because these specimens

seem to be inhibitory. For isolation from mixed cultures, media containing clindamycin or

vancomycin as well as Thayer-Martin agar have been recommended (143). The use of

various blood culture media has significantly improved the detection rate (142).

In contrast to some Haemophilus spp., Pasteurella spp. are hemin and CO2 independent and

will, therefore, grow on media without blood. Selective media containing vancomycin,

clindamycin, and/or amikacin have been employed (6). “Related” species may even grow on

enteric media.

S. muelleri grows well on blood agar but not on enteric agars.

S. moniliformis is best isolated from blood, joint fluid, or abscess material. For culture, media

enriched with sheep, horse, or rabbit blood (15% seems to be optimal), serum, or ascitic

fluid and a 5 to 10% CO2atmosphere at 37°C are required. Sodium polyanethol sulfonate in

blood culture media is inhibitory (40).

S. indologenes grows slowly on blood agar (35).

IDENTIFICATION Back to top

Phenotypic identification of fastidious gram-negative rods presents several challenges. Triple

sugar iron or Kligler’s agar may not support the growth of fastidious genera (e.g., Eikenella).

Media should be rich in peptones (e.g., c ystine Trypticase agar); serum (except rabbit

serum) should not be used because it may split maltose. The inoculum should be large (cell

paste or agar blocks). Unsupplemented media used to check acid formation from

carbohydrates may yield false-negative reactions. Gas formation from carbohydrates is scant

or absent in most species. Indole may have to be extracted with xylene. Correct

identification to the species level often requires multiple substrates that may not be available

to routine laboratories (60, 89) and may not even be provided by automated systems (135).

In view of the phenotypic closeness of the species, molecular methods (e.g., 16S rRNA gene

or rpoB gene sequencing) seem optimal for the identification to the species level

of Actinobacillus, Aggregatibacter, C apnocytophaga, and Pasteurella spp. (44, 52, 83, 89).

They have mostly replaced the analysis of cellular fatty acids, which was able to separate

only some species from others (140).

Colonies of Actinobacillus spp. are approximately 2 mm in diameter after 24 h of growth at

37°C, smooth or rough, viscous, and often adherent to the agar. Smooth colonies are dome

shaped and have a bluish hue when viewed by transmitted light. Biochemical reactions are

listed in Table 1.

A. actinomycetemcomitans colonies initially show a central dot and a slightly irregular edge

and, on further incubation, develop a star-like configuration resembling “crossed cigars” and

pit the agar. After several subcultures, this rough morphology may give way to smooth and

opaque, nonpitting colonies, reflecting loss of fimbriae. In liquid media, the bacterium forms

granules which adhere to the sides and to the bottom of the tube. Colonies of

other Aggregatibacter spp. are granular or smooth, grayish white to yellowish, and opaque;

without CO2, there is pleomorphism, with small and large colonies. For species identification,

a battery of tests (requirements for V- and X-factors, biochemical tests, colonial morphology

[Table 1]) is necessary to avoid confusion with Haemophilus spp. Aggregatibacter spp. have

negative reactions for production of indole, ornithine decarboxylase, and urease and are not

dependent on X-factor; Haemophilus spp. are at least positive for one of those three

reactions or cannot synthesize heme components from delta-amino-levulinic acid. Automated

systems may present difficulties in identifying to the species level (53, 89, 135).

Colonies of Capnocytophaga spp. on blood agar are very small after 24 h at 37°C and reach

2 to 4 mm in diameter after 2 to 4 days; they are convex or flat and often slightly yellow

when scraped off agar, show regular or spreading edges, and adhere to the agar surface.

Phenotypic differentiation of species in the oxidase-negative group may be inconclusive due

to the similarity of many biochemical reactions (Table 2) and the lack of suitable substrates

even in automated systems (25, 43, 135). This has frequently given rise to identification as

“Capnocytophaga sp.” 16S rRNA gene sequencing is at present the most adequate diagnostic

tool (25).

Colonies of C. violaceum measure 1 to 2 mm in diameter after 24 h of growth, are round and

smooth, have an almond-like smell, and may be beta-hemolytic. Most strains produce a

violet pigment called violacein, which is soluble in ethanol but not in water. Identification is

easy if this pigment is produced, although the positive oxidase reaction will be detected only

by a modified technique (127). Biochemical reactions are listed in Table 3. This species may

even grow on most enteric media. Nonpigmented strains may be confused

with Aeromonasspp. but are lysine, maltose, and mannitol negative. Principal fatty acids do

not differentiate between these two genera.

Colonies of Dysgonomonas spp. are entire, measure 1 to 2 mm in diameter after 24 h of

growth, have a strawberry-like odor, and do not spread or adhere. The species show few

biochemical differences (Table 2). Aerobically growing isolates of Leptotrichia buccalis may

be confused with Dysgonomonas; microscopic examination of morphology, different cellular

fatty acid profiles, and determination of the production of lactic acid from glucose

in Leptotrichia (Dysgonomonas produces propionic and succinic acids) are of help if 16S rRNA

gene sequencing is not available (9).

Colonies of Eikenella corrodens are 1 to 2 mm in diameter after 48 h of growth, show clear

centers that are often surrounded by spreading growth, may pit the agar, and assume a

slightly yellow hue after several days. In liquid media, granules are produced. Typical

isolates fail to form acid from carbohydrates in nonsupplemented media and are ornithine

decarboxylase and nitratase positive; lysine decarboxylase activity is variable (Table 3). E.

corrodens grows poorly or not at all on triple sugar iron or Kligler’s agar.

Kingella colonies on blood agar in 5 to 10% CO2 (which enhances growth) are 1 to 2 mm in

diameter after 48 h of growth. One type is smooth with a central papilla, and the other

spreads and pits the medium. As the only species, K. kingae shows a small but distinct zone

of hemolysis on blood agar. Colonies have a short viability and have to be subcultured

frequently. Biochemical test results are listed in Table 3. In addition to microscopic and

colonial morphology, the tests serve to separate kingellae from rod-shaped members of the

genusNeisseria (Table 4), with which they may be confused in automated systems (135).

Since K. denitrificans may grow on Thayer-Martin or Martin-Lewis agar (143), it may be

misidentified as Neisseria gonorrhoeae unless the catalase reaction, negative for Kingella, is

performed.

Colonies of Simonsiella spp. are 1 to 2 mm in diameter after 24 h, may show gliding motility,

and produce a pale yellow pigment. S. muelleri is beta-hemolytic. An optimal medium for

recognition is BSTSY agar, which contains bovine serum, glucose, tryptic soy broth, and

yeast extract (85). Biochemical tests are listed in Table 3.

S. moniliformis may show eubacterial and L-phase colonies in the same culture. The former

are 1 to 3 mm in diameter after 48 to 72 h of growth on blood agar and are round and

smooth. L-phase colonies grow better on clear media, yielding the “fried egg” appearance,

with irregular outlines and coarse lipid globules. In liquid media, growth occurs mainly in the

form of “puff balls” at the bottom of the tube. The organism dies quickly unless subcultured

(40). It is biochemically inert; glucose is acidified weakly and in a delayed fashion (Table 2).

Fatty acid analysis can confirm the diagnosis (Table 2), as can 16S rRNA gene sequencing.

Colonies of S. indologenes may spread or pit the agar surface of the blood agar. Biochemical

tests are listed inTable 3.

TYPING SYSTEMS AND SEROLOGIC TESTS Back to top

On the basis of surface polysaccharides, five serotypes of A. actinomycetemcomitans can be

distinguished, of which a, b, and c are most common (commercial antisera are not

available). Serotype b is associated with periodontitis, endocarditis, and penicillin resistance;

serotype c is associated with periodontal health and extraoral infections (109).

Typing of Capnocytophaga spp. has been done by multilocus enzyme electrophoresis or by

restriction fragment length polymorphism analysis (43). Typing of C. violaceum has

employed recA PCR-restriction fragment length polymorphism analysis (120). For E.

corrodens, typing has been done by arbitrarily primed PCR (48) and by restriction

endonuclease analysis (19), which have demonstrated the unstable clonality of E.

corrodens in the oral cavity. For typing of Pasteurella spp., PCR profiling, restriction

endonuclease analysis, ribotyping, and pulsed-field gel electrophoresis have been employed

(22, 73).

Detection of antibodies directed against any of the bacteria discussed in this chapter has

been tried on a small scale only and does not seem to offer much value.

ANTIMICROBIAL SUSCEPTIBILITIES Back to top

Approved guidelines for broth microdilution susceptibility testing of the HACEK group and for

antimicrobial dilution and disk susceptibility testing of Pasteurella spp. have recently been

published by the CLSI (28) (seechapter 71). Beta-lactamase production among members of

the HACEK group is well documented, and beta-lactamase-producing isolates are ampicillin

resistant; some isolates may be resistant to ampicillin due to mechanisms other than betalactamase

production (28).

Susceptibility studies of Actinobacillus spp. are extant for a few isolates of the humanpathogenic

species that are susceptible to many antimicrobials, including penicillin (47, 78).

Aggregatibacter spp. are susceptible to cephalosporins, tetracyclines, and aminoglycosides

(84, 110). Resistance to ampicillin is not uncommon (29, 144), but amoxicillin combined with

a beta-lactamase inhibitor has been effective (84).

Capnocytophaga spp. are usually susceptible to broad-spectrum cephalosporins,

carbapenems, lincosamides, macrolides, tetracyclines, and fluoroquinolones but are resistant

to colistin and aminoglycosides (4). Multidrug-resistant isolates have occasionally been

encountered (138).

C. hominis and C. valvarum are susceptible to many antimicrobials, including penicillin

(61, 84, 94). Beta- lactamase production is rare, and its effect can be neutralized by

clavulanic acid (28, 97).

C. violaceum is resistant to many antimicrobials (beta-lactams and colistin) but is mostly

susceptible to imipenem, fluoroquinolones, gentamicin, tetracyclines, and co-trimoxazole

(1, 93).

D. capnocytophagoides strains are susceptible to tetracyclines, clindamycin, macrolides, and

co-trimoxazole, whereas they are resistant to cephalosporins, aminoglycosides, and

fluoroquinolones and variably susceptible to other beta-lactam antibiotics and to imipenem

(58, 95).

E. corrodens is generally susceptible to penicillin, ceph-alosporins, carbapenems,

doxycycline, azithromycin, and fluoroquinolones but is often resistant to narrow-spectrum

cephalosporins, macrolides, and clindamycin (84, 98, 134). Beta-lactamase-positive strains

have been reported, but the enzyme was inhibited by beta-lactamase inhibitors (28, 88).

Kingella spp. are generally susceptible to beta-lactam antibiotics, macrolides, tetracyclines,

co-trimoxazole, and quinolones (84, 142). Beta-lactamase-positive isolates have been

reported to be susceptible to combinations with beta-lactam inhibitors (99, 131).

Pasteurella spp. are generally susceptible to penicillin, broad-spectrum cephalosporins,

tetracyclines, quinolones, and co-trimoxazole but often resistant to oral narrow-spectrum

cephalosporins, macrolides, and amikacin; other a minoglycosides are only moderately active

(27, 56). Rare penicillin-resistant isolates ofPasteurella spp. have been encountered, but

their effect can be neutralized by clavulanic acid (28, 102). For isolates of Pasteurella spp.

from bite wounds, routine testing is usually not necessary; multiple organisms are often

present in these specimens. Empirical therapy directed toward these organisms is generally

effective forP. multocida as well (28). A few beta-lactamase-positive isolates of

(P.) bettyae (12) have also been reported; they were susceptible to the combination of

penicillin and clavulanic acid.

One strain of S. muelleri was susceptible to beta-lactam antibiotics, tetracycline, and

gentamicin (141).

S. moniliformis is susceptible to penicillin and tetracyclines, the mainstays of treatment, and

to cephalosporins, carbapenems, aztreonam, clindamycin, erythromycin, and tetracycline; it

shows intermediate susceptibility to aminoglycosides and fluoroquinolones and is resistant to

colistin and co-trimoxazole (37, 40).

The susceptibility of S. indologenes resembles that of C. hominis (75).

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

Some bacteria of the group are colonizers of the human or animal oral cavity; therefore, the

evaluation of their isolation may be difficult. All should be identified to the species level if

isolated as pure cultures from normally sterile body sites. Interpretation as infectious agents

and results of susceptibility testing should be clearly reported to the physician.

With specimens normally colonized with aerobic and anaerobic bacteria, as well as with

specimens from wounds, e.g., bite wounds, the significance of the bacteria discussed in this

chapter depends on their predominance and the absence of other potentially pathogenic

bacteria. If these conditions are met, identification to the species level is needed for

adequate interpretation and reporting as infectious agents and for susceptibility testing. If

none of these conditions is present, a repeat culture and close cooperation between the

microbiology laboratory and the physician are necessary for interpretation, for identification

to the species or genus level, and for susceptibility testing.