Propionibacterium, Lactobacillus & Others

Phylum Actinobacteria

The genus Actinomyces, and the related clinically relevant

genera Actinobaculum, Mobiluncus, and Varibaculum,contain anaerobic and aerotolerant,

non-acid-fast, gram-positive organisms with variable morphology, ranging from

characteristic branching rods to coccobacilli. The genus Actinomyces includes a number of

species associated with disease, and the majority of species produce succinic acid from

glucose. Recently, a novelActinomyces species, A. massiliensis, has been described (146),

and the classification of the common oral species A. naeslundii has been clarified with the

proposal of two new species, A. johnsonii and A. oris (85). The

genus Actinobaculum includes A. massiliense, A. schaalii, and A. urinale (71, 76, 112), all of

which are associated with urinary tract and other infections, including septicemia and

osteomyelitis. The genusMobiluncus contains two species, M. curtisii and M. mulieris, which

are strictly anaerobic, curved bacilli with variable Gram reactions and corkscrew motility.

They resemble Actinomyces in that succinic acid is the major metabolic end product from

glucose. Varibaculum (80) is related to Mobiluncus on the basis of 16S rRNA gene sequence

analysis, as is Actinomyces neuii, which is distantly related to Actinomyces sensu stricto and

would appear to be worthy of proposal as a novel genus (90).

Propionibacterium species are anaerobic and aerotolerant, pleomorphic, gram-positive rods

that produce propionic acid from glucose. Five Propionibacterium species have been isolated

from human clinical infections: P. acnes, P. avidum, P. granulosum, P. propionicum, and the

recently described P. acidifaciens (44). A related species, Propionimicrobium

lymphophilum, formerly a member of Propionibacterium, has also been isolated from clinical

material, while Propioniferax innocua is a member of the normal skin microbiota.

Members of the genus Bifidobacterium and the closely related

genera Alloscardovia (92), Parascardovia, andScardovia (96) are strictly anaerobic or

occasionally microaerobic, gram-positive, pleomorphic rods, appearing as uniform to

branched or club shaped. Typically, bifidobacteria produce fructose-6-phosphate

phosphoketolase as well as acetic and lactic acids as major metabolic end products.

Bifidobacteria are aciduric and are nutritionally fastidious. There are currently

32 Bifidobacterium species. Of these, 11 species (B. adolescentis, B. angulatum, B. bifidum,

B. breve, B. catenulatum, B. dentium, B. gallicum, B. longum, B. pseudocatenulatum, B.

subtile, and B. scardovii) have been isolated from the human gut and oral cavity. B.

longum now includes the former species B. infantis and B. suis as subspecies (122, 153).

Numerous taxa have been misassigned to Lactobacillus in the past, including the so-called

“anaerobic lactobacilli,” which are now recognized to constitute two

genera, Atopobium and Olsenella, within the

familyCoriobacteriaceae (37, 41). Atopobium species produce lactic acid as the major

glucose metabolic end product.A. minutum and A. rimae were formerly Lactobacillus species,

and A. parvulum and A. fossor formerly belonged to the

genera Streptococcus and Eubacterium, respectively (37, 100). The genus Olsenella is

closely related toAtopobium and currently includes two species: O. uli (formerly Lactobacillus

uli) and O. profusa, both isolated from the human oral cavity.

The genus Eggerthella (191) includes the human pathogens E. lenta and E. sinensis, while

the former E. hongkongensis has been moved to the new genus Paraeggerthella (200). Other

members of theCoriobacteriaceae found in human infections include the

genera Collinsella (101), Slackia (191), andCryptobacterium (130), while the

genera Adlercreutzia and Gordonibacter have been isolated recently from feces and the

colon, respectively (121, 200).

Phylum Firmicutes

Lactobacillus, a large and heterogeneous genus, contains microaerobic, catalase-negative,

non-spore-forming, gram-positive rods, which produce lactic acid as their single or major

metabolic end product from glucose fermentation. The majority of Lactobacillus species are

found within the family Lactobacillaceae and orderLactobacillales. However, Catenibacterium

mitsuokai (99), isolated from human feces, forms a cluster with L. vitulinus and L.

catenaformis, two misclassified Lactobacillus species, within the

family Erysipelotrichaceae (117).

Organisms assigned to Eubacterium are defined by default; they do not produce propionic

acid as a major acid product, lactic acid as the sole major acid product, succinic and lactic

acids with small amounts of acetic or formic acids, or acetic and lactic (acetic > lactic) acids,

with or without formic acid, as the sole major acid products (125). It has been proposed

that Eubacterium sensu stricto should be restricted to E. limosum, E. callanderi, and E.

barkeri (194). Using this definition, the family Eubacteriaceae then includes the

generaEubacterium sensu stricto, Anaerofustis (with one species, A. stercorihominis, isolated

from human feces) (56), and Pseudoramibacter alactolyticus, a saccharolytic species found in

the oral cavity of humans (194). The remaining Eubacterium species would therefore require

reclassification. Clinically important Eubacteriumspecies are widely distributed among

the Firmicutes. E. biforme, E. cylindroides, and E. dolichum fall within the

family Erysipelotrichaceae mentioned above, together with the saccharolytic

species, Bulleidia extructa, isolated from the human mouth (43), and Holdemania

filiformis (195) and Solobacterium moorei (100), isolated from human feces. Turicibacter

sanguinis, isolated from a blood culture (11), also belongs to this family (117).

Eubacterium brachy, E. infirmum, E. minutum, E. nodatum, E. saphenum, and E.

sulci (formerly Fusobacterium sulci), together with Mogibacterium, a genus of five species

which are difficult to differentiate by phenotypic tests (131, 132), are a group of

asaccharolytic taxa isolated from the human mouth. On the basis of 16S rRNA phylogeny and

their phenotypic characteristics, novel genera should be created for the majority of these

species, with the exception of the pairs of species E. minutum and E. nodatum and E.

sulci and E. infirmum,which are closely related. Many Eubacterium species are closely related

to clostridia. Spore formation has long been the primary criterion for assignation of grampositive

anaerobic rods to Clostridium, but phylogenetic analysis indicates that the taxonomic

importance of this characteristic may have been overemphasized. Numerous phylogenetic

clusters contain both sporing and nonsporing representatives. Eubacterium budayi, E.

moniliforme, and E. nitritogenes are found in clostridial cluster I described by Collins et al.

(36), while E. siraeumbelongs to a group that includes Clostridium leptum and Anaerotruncus

colihominis (114). E. tenue and E. yuriibelong to cluster XI. E. tenue is related to Clostridium

ghonii and Clostridium sordellii; further work is required to determine whether these species

constitute a novel genus. Strains of Eubacterium plautii and Clostridium orbiscindens have

been shown to belong to the same taxon and renamed Flavonifractor plautii (26). Spore

formation is variable among strains of the species; the type and other strains do not produce

spores but have sporulation-specific genes. E. yurii is related to the genus Filifactor, which

includes Filifactor alocis (formerlyFusobacterium alocis) isolated from oral infections in

humans (95). E. contortum, E. eligens, E. hadrum, E. hallii, E. ramulus, E. rectale, E.

saburreum, and E. ventriosum belong to the family Lachnospiraceae. E. rectale and E.

ramulus are related to Roseburia intestinalis (45). This family also includes the recently

described formate-requiring

species Marvinbryantia (formerly Bryantella) formatexigens (45, 197, 198), isolated from

human feces without any disease association so far, and Oribacterium sinus (25), a highly

motile species isolated from pus of a human sinus. E. eligens and Lachnospira

pectinoschiza are close phylogenetic neighbors and are both motile rods whose growth in

broth culture is stimulated by the presence of fermentable carbohydrate. E. eligens should

therefore be transferred to the genus Lachnospira. Anaerostipes caccae (163) forms a loose

group with E. hallii and Coprococcus eutactus, all common species in human

feces. “Catabacter hongkongensis”is a deep-branching member of the order Clostridiales,

isolated from blood cultures (109), but as yet not validly published.

EPIDEMIOLOGY AND TRANSMISSION Back to top

The majority of the organisms described in this chapter are part of the commensal

microbiota associated with the mucocutaneous surfaces of the human and animal digestive

tract, being found in the mouth, small and large intestines, urogenital tract, and skin

(2, 8, 46, 140, 204). Microbial colonization of an individual occurs in a successive manner

during the first weeks and months of life. Actinomyces species are among the initial

colonizers of the mouth (158), whereas bifidobacteria and lactobacilli play an important role

in the development of the healthy gut and its associated immune defenses (20, 164). Where

members of this group cause infections, the host itself is the most likely source, although the

commensal microbiota of other humans can be responsible, for example, in the case of

infections resulting from human bites or clenched fist injuries from striking the face and

mouth (17).

CLINICAL SIGNIFICANCE Back to top

Non-spore-forming anaerobic gram-positive rods seldom cause infections alone but are

typically found in polymicrobial infections associated with mucosal surfaces (Table 2). Many

anaerobes involved in infections of the head and neck originate from the oral cavity, and

most vaginal and bladder pathogens are of fecal origin. In intra-abdominal infections due to

organ perforation, the predominant recoveries reflect the microbiota at the site of the

leakage (193). For surgical patients, anaerobes are a significant cause of morbidity and

mortality (48). Anaerobic bacteria can occasionally spread to adjacent tissues and even the

bloodstream, with serious consequences. For anaerobic bacteremias, the gastrointestinal

tract is the most common source, followed by abscesses, gynecologic infections, and wound

infections (155). The incidence and range of anaerobic gram-positive bacilli found in blood

cultures may be underestimated because many of them are slow growing and have fastidious

nutritional requirements. Anaerobic blood culture methods tend to be targeted

atClostridium species and Bacteroides fragilis, which grow readily and rapidly in commonly

used broth media.

Actinomyces and Related Bacteria

Actinomyces and related bacteria are associated with a wide range of infections, normally as

part of a polymicrobial consortium (Table 2) (32). Actinomycosis is a chronic, granulomatous

infection affecting the cervicofacial, thoracic, and abdominopelvic regions and is caused

primarily by Actinomyces species, particularlyA. israelii, A. gerencseriae, and A.

graevenitzii, and Propionibacterium propionicum (15, 74). Cervicofacial lesions normally arise

as a consequence of untreated dental caries or are associated with dental extractions or

trauma. These allow the causative organisms, which are part of the oral commensal biota, to

enter the tissues. Although actinomyces are regarded as the primary cause and form the

characteristic aggregates of branching bacilli seen macroscopically as sulfur granules, there

are always multiple species present, withAggregatibacter actinomycetemcomitans the most

typical, together with a variety of oral organisms, including viridans group streptococci,

anaerobic cocci, and gram-negative anaerobic bacilli (143). Thoracic actinomycosis most

commonly affects the lungs following aspiration of oral bacteria in saliva, while the majority

of pelvic infections are found in women using intrauterine contraceptive devices (177) and

abdominal infection normally arises following perforation of the bowel as a result of disease

or surgery (192).

Actinomyces make up a significant proportion of the microbiota in dental plaque in healthy

individuals but are also associated with a wide range of dental and oral infections, including

dental caries, endodontic infections, odontogenic abscesses, and dental implant-associated

infections (7, 13, 35, 77, 78, 128, 157). Recently, there has been increasing evidence

implicating Actinomyces species in infected osteoradionecrosis lesions, based on detection of

the organisms within lesions and histological evidence of sulfur granule formation (39, 82).

In a study in which anaerobic culture methods were used for sputum of adult cystic fibrosis

patients, Actinomycesspecies were frequently isolated (182).

The most frequently isolated Actinomyces species from clinical infections are A. turicensis, A.

radingae, and A. neuii and are found in a range of soft tissue infections including peri-anal,

groin, axillary, breast, and peri-aural abscesses (74). A number of species, especially A.

israelii and A. turicensis but also A. cardiffensis, A. gerencseriae, A. naeslundii, A.

odontolyticus, A. urogenitalis, and a novel, closely related genus and species,Varibaculum

cambriense, have been isolated from intrauterine device-associated infections in the female

genital tract (9, 50, 63, 75, 80). In addition, A. naeslundii and A. israelii have been isolated

from infectious hip prostheses (173, 201). A. turicensis and the Actinobaculum species A.

massiliense, A. schaalii, and A. urinale are particularly associated with genital and urinary

tract infections in both females and males (71, 76, 151). Pericarditis cases caused by A.

israelii and A. meyeri have been reported (17). Oral Actinomyces species can be detected in

the bloodstream following dental procedures (172), while A. funkei, A. massiliensis, A.

naeslundii, A. odontolyticus, A. turicensis, and Actinobaculum schaalii and Actinobaculum

urinale have been demonstrated to cause infections of the blood, particularly in individuals

with predisposing conditions (38, 54, 113, 138, 145,146).

Propionibacterium

Propionibacteria can be found in various systemic or disseminated opportunistic infections

(Table 2), such as endocarditis, central nervous system infections, osteomyelitis, osteitis,

and arthritis (18, 51, 94), and in about 20% of infected dog and cat bite wounds (179).

Because P. acnes is a common skin commensal, its isolation from blood is often discounted

as contamination of the blood during collection or as clinically insignificant (110,155).

However, P. acnes has been shown to be a significant cause of endocarditis, and one third of

those cases are complicated by intracardiac abscess formation (171). The pathogenic

potential of P. acnes should not be underestimated when there are predisposing factors

present, such as a foreign body, surgery or trauma, diabetes, or immunosuppression

(4, 72, 94, 134, 184, 187). Prosthetic joints are particularly susceptible to P. acnes infection

(115, 141), and biofilm formation appears to be a specific virulence factor associated with

invasive strains (88). P. acnes has also been isolated as part of the mixed bacterial

community found in the sputum of adult cystic fibrosis patients (182). P. propionicum is part

of the normal oral microbiota and causes oral and eye infections (14, 27, 167) as well as

actinomycosis, in which it displays a spectrum of pathogenicity similar to those of A.

israelii and A. gerencseriae (15). The recently described P. acidifaciens is particularly

associated with dental caries (44).

Lactobacillus

Despite the reputation of lactobacilli as beneficial organisms, they can be involved in serious

infections (Table 2), especially in immunocompromised individuals

(16, 19, 24, 53, 133, 154). The Lactobacillus species most frequently isolated from various

human infections are L. rhamnosus, L. casei, L. fermentum, L. gasseri, L. plantarum, L.

acidophilus, and L. ultunensis (23, 24, 154). Lactobacilli are particularly associated with

advanced dental caries (23, 28, 128), where they are considered a secondary colonizer

because of their preference for low-pH habitats, but probably play a role in exacerbating

existing lesions (5). The clinical infections most commonly caused by lactobacilli are

bacteremia and endocarditis, with an associated relatively high mortality rate (24), with the

mouth the primary route of entry to the bloodstream, either as a result of normal chewing

and brushing or following dental procedures (24). Detection of lactobacilli, alone or with

other microorganisms, in blood cultures of patients with underlying diseases may be clinically

significant. L. rhamnosus was the most frequent species detected in Lactobacillus bacteremia

(24, 53, 110, 154). Concern has been expressed that probiotic strains consumed in

foodstuffs may cause disease in some individuals. Although such reports are rare (175),

there have been reports of sepsis and endocarditis attributed to

probiotic Lactobacillus strains (106, 203). In some cases, these have been attributed to

inappropriate dosages and routes of administration, and it should be remembered that

organisms used as probiotics are defined at the strain level and that, although infections may

be caused by other strains within the same species, this does not imply that probiotic strains

are unsafe. Vancomycin-resistant lactobacilli have been implicated in dialysis-related

peritonitis after extended use of glycopeptides (102, 133). In contrast to

other Lactobacillus species, L. iners has been associated with an intermediate state of

bacterial vaginosis (190).

Eubacterium and Related Bacteria

The genus Eubacterium remains poorly defined, but species belonging to this genus and its

relatives in the phylum Firmicutes are commonly isolated from oral infections (Table 2),

particularly when nutrient-rich media and extended incubation times are used. For example,

careful isolation of tiny-colony-forming anaerobes from periodontal pockets in adult patients

with advanced periodontitis showed that “Eubacterium” species (mainly asaccharolytic)

dominated (185), suggesting a role in the etiology of chronic periodontitis. When molecular

identification methods were applied to a collection of Eubacterium-like strains from oral

infections,Mogibacterium timidum was one of the most frequently detected species

(42). Mogibacterium vescum, Bulleidia extructa, Filifactor alocis, and Pseudoramibacter

alactolyticus were also found among the isolates from severe (some of them requiring

treatment in intensive care units) odontogenic infections. Less frequently isolated were E.

sulci, E. saburreum, and E. yurii (42). Many species found in odontogenic infections are also

common in endodontic infections (59, 83, 129, 166, 168). F. alocis, E. nodatum, E.

saphenum, and M. timidum have been associated with periodontal diseases

(10, 40, 104, 105, 139), and “Eubacterium” species in general with failing dental implants

(176). Due to their presence in the oral cavity, various Eubacterium and related species are

among the anaerobic findings in human bite wound infections (178). Filifactor villosus, a

species of animal origin, has been isolated from infected cat bite wounds in humans (179). E.

nodatum has been found in infections of the female genital tract (86). E. tenue and E.

callanderi, an environmental anaerobe, have been detected in clinically significant

bacteremia (110, 180).

Eggerthella and Related Bacteria

Species of the genera Eggerthella and Paraeggerthella are recovered from a wide range of

human infections (Table 2). E. lenta (formerly Eubacterium lentum) is a well-recognized

pathogen particularly of intra- and peri-abdominal sites (16, 19, 111, 144). E. lenta, E.

sinensis, and P. hongkongensis have been found in blood in association with clinically

significant infections of relatively high mortality (110, 111). Cryptobacterium

curtumand Slackia exigua have been associated with chronic periodontitis (10, 104), and the

latter has also been associated with endodontic infections (83). An, as yet

unnamed, Eggerthella-like taxon is associated with bacterial vaginosis (61).

Atopobium

Several species of the genus Atopobium are isolated from various infections (Table 2).

Although A. vaginae is a prominent member of the commensal microbiota of the healthy

vagina (148, 202), it has been increasingly reported to be involved in infections of the

genital tract, especially bacterial vaginosis (22, 55, 64, 124, 190).A. minutum has been

isolated from various infections of the lower part of the body, and A. parvulum has been

isolated from respiratory specimens (136). Although A. parvulum and A. rimae have been

detected in the pockets formed as a result of periodontitis (136, 139), in a comprehensive

study of the microbiota of the subgingival region, these species were found to be associated

with oral health rather than disease (94). Among Eubacterium-like isolates from severe

odontogenic infections, A. rimae was the most frequently isolated (42).

Olsenella

Olsenella species show disease associations similar to those observed for lactobacilli in the

oral cavity and have been found in root caries (142), with O. profusa specifically detected in

dental caries lesions (128), and O. uli, in particular, in endodontic infections

(27, 129, 147, 165) and acute dental abscesses (165). Both species can also be found in

subgingival sites of periodontitis patients (41, 136). In addition, O. uli has been reported as

one of the causative organisms in clinically significant bacteremia (110).

Bifidobacterium and Related Bacteria

Culture-independent analyses have shown that although members of the

phyla Bacteroidetes and Firmicutesdominate the gut microbiota numerically, bifidobacteria

appear to be functionally of great importance to intestinal health (183). Because of this, they

are generally considered to be nonpathogenic but nevertheless are isolated from infections of

polymicrobial etiology (Table 2). Dental caries is the most common clinical entity in

which Bifidobacterium, mainly B. dentium, and the related species Parascardovia

denticolens, Scardovia inopinata, and the unnamed Scardovia species C1 may have a

pathogenic role (1, 7, 28, 119, 120). B. adolescentis, B. dentium, B. breve, and B.

longum are occasionally isolated from other infections, mainly in immunocompromised

individuals (16, 18, 19, 118). Although B. scardovii has been isolated from human clinical

samples, including blood, urine, and hip (91), its clinical relevance is not known. In addition,

a novel species related to Bifidobacterium, Alloscardovia omnicolens, has been detected in

infections at various body sites, including urine and the genitourinary tract, in particular, and

the oral cavity, tonsils, lung and aortic abscesses, abdominal wounds, and blood (118).

Mobiluncus

Although the etiology of bacterial vaginosis, the most common infection in the female genital

tract, remains unclear, the presence of vibrio-like Mobiluncus species in smears of vaginal

fluid has been widely used as one of the indicators of bacterial vaginosis (135).

Indeed, Mobiluncus curtisii is seldom present in the vaginas of healthy women but, instead,

is highly associated with bacterial vaginosis and its treatment failure due to persistence of

the organism (123, 161). The altered vaginal microbial ecology seen in bacterial vaginosis

can be a risk for adverse pregnancy outcome when ascending to the upper genital tract (89).

In addition to bacterial vaginosis, M. curtisii has been isolated occasionally from endometrial

smears and pus specimens of the female genital tract (6) and from blood (70, 152).

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

Many of the organisms described in this chapter are part of the human commensal

microbiota and cause disease as opportunistic pathogens. This makes specimen collection

difficult because at most sites of infection, the local commensal microbiota is close by. Thus,

appropriate and careful specimen collection is critical to avoid contamination of the specimen

with the commensal microbiota. Anaerobic transport techniques are also essential for the

successful recovery of clinically significant anaerobic bacteria (see chapter 16). Specimens

suitable for the isolation of non-spore-forming, gram-positive anaerobic rods present as

organisms of etiologic importance include aseptically collected peripheral blood, tissue biopsy

specimens, aspirates (e.g., cerebrospinal fluid, joint fluids, and pus), root canal exudates,

and subgingival plaque. Mucosal or cutaneous swabs are not recommended for the reasons

mentioned above. Instead of collecting periprosthetic tissue, sonication of the removed

implant followed by sonicate fluid culture has proven to be useful for microbiologic diagnosis

of prosthetic-joint infection (141, 181). A comprehensive description of different specimen

collection and transport methods for anaerobic bacteriology can be found elsewhere (98).

DIRECT EXAMINATION Back to top

Direct examination is of unequivocal value in the confirmation of a diagnosis of

actinomycosis. The macroscopic presence of “sulfur granules” in pus, which when crushed,

Gram stained, and viewed under the microscope reveal a mass of gram-positive branching

filaments, is characteristic of this disease. Similarly, in cervical smears of women with an

intrauterine contraceptive device, the presence of branching gram-positive organisms

suggests an infection with Actinomyces (58, 151). Gram stains of vaginal smears have been

considered more useful than culture for laboratory confirmation of bacterial vaginosis, and

the diagnostic criteria for this common infection have been based on the standardized

Nugent scoring system (135). The system relies on Gram stain characteristics of vaginal

smears, recognizing individual morphotypes or their combination. Although intercenter

reliability for gram-positive cocci was poor, the study documented moderate agreement for

large gram-positive rods (lactobacilli), small gram-variable and/or gram-negative

rods(Gardnerella vaginalis and Bacteroides/Prevotella), and good agreement for curved

gram-variable rods(Mobiluncus) (135). A simplified assessment of Gram-stained smears,

taking lactobacillary and mixed bacterial morphotypes into account, has been proposed (93).

It has been noted that the image area observed with microscopes requires standardization,

in particular, when interpreting the intermediate state of bacterial vaginosis (108).

In cases in which there is no typical microbiota associated with a particular infection, care

should be taken in determining appropriate empiric antimicrobial treatment on the basis of

the Gram stain. For example, branching/ pleomorphic rods can be tentatively identified as

facultatively anaerobic Actinomyces or strictly anaerobic E. nodatum (86), and the coccoid

cells of Actinomyces radicidentis are atypical for the genusActinomyces (35), while easily

decolorizing species (e.g., Eubacterium-like species) can yield a false gram-negative reaction

(42). The misinterpretation can lead to antimicrobial coverage targeted against facultative

organisms instead of anaerobes and/or gram-negative bacteria.

ISOLATION PROCEDURES Back to top

Specimens should be processed without delay using appropriate culture media, including

standard anaerobic blood agar enriched with hemin and vitamin K1 and a variety of selective

media based on the expected microbiota at the collection site, or in the case of bite wounds,

on the oral microbiota of the attacker (human or animal). Fresh or prereduced culture media,

including phenyl ethyl alcohol blood agar and/or colistin nalidixic acid blood agar, can be

useful for enhanced recovery rates of gram-positive organisms (29). The growth of many

asaccharolytic species on solid media is enhanced by the addition of 0.5% arginine (186). In

general, members of the aciduric genera Bifidobacterium and Lactobacillus can be selectively

cultured using agar media with an acidic pH, such as Rogosa or deMan-Rogosa-Sharpe agar.

However, some nutritionally fastidiousLactobacillus strains fail to grow on these agar media.

Lactobacilli isolated from dental caries were recovered equally well on nonselective bloodcontaining

media and on Rogosa agar, and it was concluded that acidic-pH medium is not

required for their detection (128). Notably, L. iners, one of the predominant lactobacilli in the

vagina, can grow only on blood agar and not on typical solid media used

for Lactobacillus (52).

Although some members of this group, particularly Actinomyces species, are facultative

anaerobes and can grow well on aerobically incubated culture media, anaerobic incubation is

recommended for optimal recovery. If anaerobic jars are used for incubation, anaerobic

growth should not be exposed to oxygen by opening the jar before 48 h of incubation, in

order to facilitate the detection of slow-growing, oxygen-sensitive organisms (29). The

availability of an anaerobic chamber may enable examination of the culture whenever

necessary. For reliable detection of slow-growing organisms, the incubation time should be

sufficient; for instance, an extended incubation period may be needed for some clinically

relevant Eubacterium-like species (42, 130, 131,180, 185). In heart tissue specimens from

endocarditis patients, grinding the tissue can improve the detection of anaerobic bacteria

(72, 94). A lytic anaerobic medium can increase the recovery rate of anaerobes and

facultative bacteria in automated blood culture systems (149).

IDENTIFICATION Back to top

Traditionally, the identification of bacterial isolates in clinical microbiology laboratories is

performed by phenotypic tests. For organisms inert in most conventional biochemical tests or

with unusual biochemical profiles, as is the case for many Eubacterium and related species,

or in cases where fastidious organisms require specific nutrients or temperatures,

identification strategies based on phenotypic characteristics can be challenging.

Presumptive Identification

The initial differentiation is based on aerotolerance (growth in air or in air plus 5% CO2),

colonial morphology, pigmentation, fluorescence under long-wave UV illumination (365 nm

wavelength), and presence of hemolysis. The colonial morphology can provide clues

regarding the organism involved; for instance, an easily recognizable “molar tooth”

appearance is typical for A. israelii but, notably, also for E. nodatum, although its colonies

are smaller (86). Other rapidly recognizable features are fluorescence and/or pigment

production: E. lenta shows orange or red fluorescence under UV light (126), and pink/red

pigmentation of colonies is typical for A. odontolyticus, but some other Actinomyces can also

produce pigment, especially on rabbit laked blood agar, with A. graevenitzii appearing as

nearly black, A. radicidentis as brown, and A. urogenitalis as reddish colonies (81, 159).

Gram stain morphology can contribute to a presumptive identification; it can show whether

organisms are gram-positive anaerobic rods, which can be very short (e.g., C. curtum and E.

lenta), long (e.g., manyLactobacillus spp.), pleomorphic (e.g., Bifidobacterium), branching

(e.g., many Actinomyces spp.), or curved and motile (e.g., Mobiluncus spp.); sometimes a

specific cell morphology can be seen, such as “flying birds” (e.g., P. alactolyticus). The

morphology may vary when cells are grown on different culture media. The

two Mobiluncusspecies can be tentatively separated based on the length of the curved,

motile cells: in contrast to the short, gram-variable cells of M. curtisii, M. mulieris reveals

clearly longer cells, which often appear as gram negative (162).

Although Actinomyces organisms have been traditionally described as branching rods, many

new species within the genus are nonbranching, and some have very short or even coccoid

cells (35, 188). Staining of cells can vary with different culture conditions. Certain grampositive

anaerobes, e.g., F. alocis and M. mulieris, routinely stain gram negative, whereas

older cultures (>3 days) of Actinobaculum and someEubacterium species and species of

related genera are gram variable (112, 130, 132). Decolorization of gram-positive organisms

may be due to exposure to oxygen or to damage from fixatives and reagents causing a

breakdown of the physical integrity of the cell wall; therefore, anaerobic working conditions

or, if not available, limited exposure time to oxygen between incubation and staining

improves the reliability of the Gram stain for anaerobic bacteria (97). For rapid confirmation

of the Gram reaction, a simple test based on dissolution of the gram-negative cell wall and

cytoplasmic membrane with a solution of 3% potassium hydroxide ((73) can be used: when

suspended in the solution, gram-negative cells display increased viscosity and stringing

within 30 s, whereas the absence of stringing, i.e., a negative reaction, suggests that the

isolate is gram positive. Routine screening of special-potency antibiotic susceptibility disk

patterns is valuable in confirming the accuracy of the Gram stain reaction (98): grampositive

species are generally resistant to colistin (10 μg) and susceptible to vancomycin (5

μg) and often to kanamycin (1 mg). However, the intrinsic resistance of

someLactobacillus species/strains, e.g., L. rhamnosus, to glycopeptides should be considered

(53, 102, 133), in addition to the intrinsic resistance of Bifidobacterium to aminoglycosides

(127). Holdemania filiformis has been reported to be resistant to vancomycin (43).

Additional rapid tests for initial grouping of non- spore-forming gram-positive anaerobes

include testing for production of catalase (H2O2 at a concentration of 15%) and indole,

nitrate reduction, and motility (29). If presumptive identification to the genus level has been

made correctly, this may give valuable information to clinicians in deciding the initial

treatment. However, differentiating members of the “normal flora” of human skin and

mucous membranes from pathogenic non-spore-forming gram-positive rods can be difficult.

Identification of nonsporing gram-positive rods to the species level should be performed

whenever they are present in pure cultures in clinical specimens or as the predominant

organism from normally sterile sites; otherwise, the potential pathogenicity of these lessoften

suspected species may remain undetected.

Biochemical Testing

For a more advanced phenotypic classification of anaerobic organisms and distinguishing of

individual species, sugar fermentation reactions, preferably using prereduced, anaerobically

sterilized carbohydrates, and enzyme profiles with individual diagnostic tablets, fluorogenic

substrate tests, or preformed enzyme kits must be determined. Insufficient growth or poor

reproducibility of reactions can cause difficulties in interpretation of results obtained with

biochemical tests; therefore, young cultures and heavy inoculum should be used (159). A

well-designed selection of key tests provides a tentative identification of various isolates to

the species level prior to confirming their identifications by more definitive methods. Table

3 presents some biochemical characteristics of Actinomyces and related organisms. Since the

description of many novel species, such as A. d entalis, A. hongkongensis, A. massiliensis, A.

nasicola, A. oricola, A. urinale, and “Actinobaculum massiliae,” is based on a single strain

(71, 76–79, 146, 199), discrepancies in test reactions may appear. In addition, the

clarification of the taxonomy of the A. naeslundii/Actinomyces viscosus group with the

proposal of the new species A. johnsonii and A. oris (85), while taxonomically valuable and

consistent with their ecology, has resulted in a group of species that cannot be differentiated

by phenotypic tests alone. Housekeeping gene sequence analysis is required but may be

beyond the scope of routine laboratories. Table 4 presents simple enzymatic reactions useful

in distinguishing propionibacteria encountered in human infections, and Table 5shows tests

for Atopobium and Olsenella species. Although the cultivation and identification

of Eubacterium-like species can be very laborious, not only because of their oxygen

sensitivity and slow growth but also due to their nonreactivity in conventional biochemical

testing, some simple reactions are helpful for grouping these organisms (Table 6).

In culture-based identification of anaerobic non-spore-forming gram-positive rods, the

determination of major volatile fatty acid end products of glucose metabolism, as detected

by gas chromatography, is useful for assigning isolates to genus level (Table 1).

Typically, Actinomyces strains produce succinic and lactic acids as their major metabolic end

products, but A. dentalis is reported not to produce succinic acid (75). TheActinomyceslike

Propionibacterium species, P. propionicum, is easily separated from Actinomyces based

on its production of propionic acid (62). For Lactobacillus spp., defining characteristics are

their ability to grow in acid media and ferment carbohydrates to produce lactic acid as the

major end product with or without small amounts of acetate, whereas Bifidobacterium spp.

produce acetic acid as a major product. A combination of phenotypic tests, specifically the

determination of metabolic end products by gas chromatography together with sugar

fermentation by prereduced, anaerobically sterilized carbohydrates and enzyme profiles

generated by a commercial identification kit (API Rapid ID 32A; bioMerieux, Marcy-l’Etoile,

France), have successfully been used to identify oral Eubacterium-like isolates to genus and

species level (42); for example, the lack of enzyme activity and formation of caproic acid or

phenylacetic acid distinguish P. alactolyticus or Mogibacterium spp., respectively, from other

related taxa. However, as already mentioned, phenotypic criteria are particularly unreliable

for identification of many Actinomyces species (81) and members of the L.

acidophilus complex and related species (189). In addition, gas chromatographic analysis of

cellular fatty acids (98) and examination of protein patterns by polyacrylamide gel

electrophoresis have been used taxonomically to distinguish among strains within a species

and among organisms within a genus or family.

Preformed enzyme and carbohydrate fermentation profiles can be obtained using

commercially available identification test kits, such as the API (bioMerieux), RapID (Remel,

Lenexa, KS), and BBL Crystal (Becton Dickinson Diagnostic Systems, Sparks, MD) systems,

according to manufacturers’ instructions. Although this approach is often hindered by

similarities in fermentation profiles of separate species within a genus, kits serve as a widely

used adjunct to anaerobe diagnostics in most hospital laboratories (65), since they are easy

to use and much faster than conventional anaerobic procedures. The main problem with

these kits are their incomplete or inaccurate databases (12, 156). The databases of the API

Coryne (bioMerieux) and RapID CB Plus (Remel) tests, designed for coryneform bacteria,

currently include some aerotolerant Actinomyces andPropionibacterium species as well.

However, the same test performed by different methodologies may give conflicting results;

this is particularly true for the commercial identification kits in which the tests are “poised,”

to give a definitive positive or negative reaction to aid interpretation. This can have the effect

of making the test insufficiently sensitive, giving false negatives compared to conventional

tests, or oversensitive, giving rise to false positives (156, 159). Isolates of A. vaginae have

been misidentified as Gemella morbillorum by the API Rapid ID 32A (bioMerieux) and RapID

ANA II (Remel) test kits (55, 64). In contrast, a clinically relevantBifidobacterium species, B.

scardovii, was readily separated from other bifidobacteria by using the Rapid ID 32A kit

(bioMerieux) (91). The carbohydrate fermentation test kit API 50 CH (bioMerieux), which is

specifically designed for lactobacilli, can be valuable in identification to the genus level but

fail at the species level (12). Despite the lack of reliability of species level identifications,

commercial test kits can be useful for the detection of positive reactions and identification of

many organisms from clinical sources to the genus level. Clinical microbiologists should be

aware of the possibility of erroneous identification and adjust the interpretation of their

results accordingly, in conjunction with cellular and colonial morphology and other

information available. Indeed, practical, discriminatory, and cost-effective methods are

needed for identification of fastidious gram-positive bacteria.

Identification by DNA Sequence Analysis

As discussed above, the use of conventional and biochemical tests for the identification of

this group carries a significant risk of misidentification. Far-more-precise identifications can

be obtained by 16S rRNA gene sequence analysis (103). DNA can be rapidly and reliably

purified from members of this group by using commercially available kits, such as GenElute

(Sigma-Aldrich), and the 16S rRNA gene can be amplified using “universal” primers that

amplify all members of the domain Bacteria (60, 107). Amplicons can be sequenced in-house

or submitted to commercial sequencing facilities. The 5′ region of the gene is the most

informative for identification purposes; the use of primer 519R for sequencing is

recommended (107). Sequences are identified by comparison with those held in the DNA

sequence databases, such as GenBank. BLAST interrogation (3) is useful, but care needs to

be taken because many sequences in the databases are mislabeled and some pairs or even

groups of species have virtually identical 16S rRNA gene sequences. Identification to genus

level is ensured, but at species level, some investigation of the phylogenetic status of the

genus should be made. This technique, while extremely powerful, should not be regarded as

an infallible “black box” method. A major advantage is that methods do not need to be

adapted for different groups of organisms; thus, there are no special considerations needed

when analyzing gram-positive non-spore-forming anaerobes, except that the DNA extraction

method needs to be suitable for lysing the rigid gram-positive cell wall.

A major impetus for the study of as yet uncultured bacteria has been the development of

16S rRNA/PCR amplification/cloning/sequencing methodology (196). The

phylum Firmicutes, in particular, has been found to harbor a number of lineages without

culturable representatives. For example, branches within the

familiesEubacteriaceae and Lachnospiraceae have been found in advanced carious lesions,

endodontic infections, and subgingival plaque in periodontitis (28, 129, 139). Similarly, the

distal esophagus, stomach, and colonic microbiotas include novel branches within

the Clostridiaceae, Erysipelotrichaceae, and Lachnospiraceae (8, 140,174). Cultureindependent

analysis of the microbiota in bacterial vaginosis identified a novel taxon related

toA. vaginae (190), and novel taxa belonging to both the Actinobacteria and Firmicutes were

found in a corneal ulcer (160).

SEROLOGIC TESTS Back to top

Serological tests are of little diagnostic value for this group of organisms, which are found

almost exclusively in polymicrobial infections. Furthermore, infections are frequently

opportunistic in nature, with the causative organism being a member of the commensal

microbiota, rendering serological tests difficult to interpret.

ANTIMICROBIAL SUSCEPTIBILITIES Back to top

In the clinical setting, empirical information is used for the initial diagnosis of infection and

choice of antimicrobial therapy, while awaiting culture and susceptibility test results. This is

particularly important for this group of organisms because many of them are slow growing,

and if they are isolated as part of a mixed infection, it may take some time to obtain pure

cultures for testing.

Published data regarding antimicrobial susceptibilities of nonsporing gram-positive anaerobes

can be difficult to interpret. Changes in bacterial taxonomy, e.g., among the species of the

former Eubacterium genus, and more precise classification of tested isolates may result in

antimicrobial resistance patterns different from those given in previously published surveys

(169). In general, penicillin and other β-lactams are active against gram-positive bacteria

together with parenteral carbapenems, such as doripenem, erta penem, imipenem, and

meropenem (21, 49, 66, 68, 87, 116, 127, 169, 170). Metronidazole has been considered a

drug of choice for treatment of anaerobic infections; however, the facultative anaerobes

among the genera Propionibacterium,Actinobaculum, Actinomyces, Bifidobacterium,

and Lactobacillus are intrinsically resistant, and resistant strains can also be found among

the strictly anaerobic genera Atopobium,

Eggerthella, Eubacterium, and Mobiluncus (6,30, 55, 87, 116). Failures or relapses are

common in the treatment of bacterial vaginosis, but whether metronidazole-resistant A.

vaginae or M. curtisii (6, 55, 64) play a role is not known. Occasional strains among various

genera of non-spore-forming gram-positive anaerobic rods show resistance to clindamycin

(21, 30, 47,69, 84, 137, 169). Although vancomycin and teicoplanin are considered active

against most gram-positive bacteria, species-related resistance to glycopeptides is frequent

among species of the genus Lactobacillus. Less than one-quarter of the isolates from 80

cases of Lactobacillus infections were reported as susceptible to vancomycin (24). Notably,

the vancomycin-resistant L. rhamnosus is the most common Lactobacillus species in clinical

specimens (53, 154). In contrast to vancomycin and teicoplanin, ramoplanin, a novel

glycolipodepsipeptide, showed good activity against lactobacilli (30, 57). A novel

glycopeptide, telavancin, has proved to be more active than vancomycin against lactobacilli,

except for L. casei, and demonstrated very good activity against the tested strains

of Propionibacterium, Actinomyces, Eggerthella, and Eubacterium (68). Tigecycline, a

glycylcycline antimicrobial agent, has been shown to be active against lactobacilli,

including L. casei, and Actinomyces species (67). Oxazolidinones represent a new class of

synthetic antimicrobial agents, having relatively good in vitro activities against gram-positive

cocci but also against anaerobes, and ranbezolid may have lower MICs than linezolid

(21, 49). Also telithromycin, a novel ketolide, and streptogramin antimicrobial agents, such

as pristinamycin and quinupristin-dalfopristin, have considerable activities against nonspore-

forming gram-positive rods (21, 69, 127). Fluoroquinolones have a broad spectrum of

antibacterial activity and good absorption from the gastrointestinal tract. Novel quinolones,

such as garenoxacin, gatifloxacin (topical application), and moxifloxacin, exhibit better

antianaerobic activity than the older quinolone compounds levofloxacin and ciprofloxacin

(21, 47, 84, 116), suggesting their potential in treating mixed organism infections.

The testing of anaerobic isolates for susceptibility to antimicrobials by clinical laboratories

remains problematic (see chapter 72). The Clinical and Laboratory Standards Institute

defines the agar dilution method as the gold standard but recommends it only for reference

laboratories (34). Specific guidance is available for lactobacilli (33). Broth microdilution is

recommended for clinical laboratories but is currently limited to fragilis groupBacteroides.

There are a number of reasons for the current lack of susceptibility testing for this group of

organisms. Firstly, anaerobic gram-positive bacilli are frequently isolated from polymicrobial

infections from which 10 or more species may be cultivated. The relevance of individual

susceptibility testing and its interpretation in this scenario are unclear. Secondly, as has been

described in this chapter, there are a large number of species that may be found in clinical

material, but each laboratory may encounter them relatively rarely. There are therefore

insufficient reference data on the susceptibility profile of each species, but even if

appropriate data were available, quality control procedures and strains would be required for

each species. A recent survey (65) revealed that the method most commonly used for

testing anaerobes was the Etest, which has been found to be useful and reliable (31, 150).

Etests should be performed on Brucella blood agar and are optimally read after 48 h, to allow

sufficient bacterial growth. Some slow-growing species may require longer incubation.

INTERPRETATION AND REPORTING OF RESULTS Back to top

Members of this group are commonly found as part of the normal microbiota at mucosal

surfaces. The primary considerations then in interpreting laboratory data are the site from

which the sample was collected, the method used for collection, and whether the sample was

likely to have been contaminated by the commensal biota. Culture plates from samples from

mucosal and cutaneous sites should be interpreted with reference to the normal commensal

biota expected for that site, and any recent or current antimicrobial therapy. It is important

that incubation times are sufficiently long to allow growth of the slow-growing members of

this group. Premature reporting of only the fastest growing species can be misleading since

growth rates in vivo and in vitro may be very different. Incubation should be continued for at

least 7 days before the final report is issued. The commensal microbiota is normally diverse.

The finding of a culture from a specimen dominated by a restricted number of organisms is

normally suggestive of infection, particularly if suspected clinically, although recent

administration of broad-spectrum antimicrobials may also reduce the diversity of the

commensal microbiota.

All isolations of members of this group from normally sterile sites, including blood, spinal

fluid, internal organs, and body cavities, are significant, and the organism should be

identified. Members of the group are generally of low-grade pathogenicity and do not

produce classical virulence factors, such as protein toxins. All isolates should be regarded as

equally important and reported with sensitivities to antimicrobials appropriate to the clinical

diagnosis and the site of the infection. Obtaining pure cultures of all of the organisms present

in a polymicrobial infection can be difficult and time consuming but should be attempted.

Collation of data regarding the identity and antimicrobial susceptibility profiles of isolates

causing confirmed infections will be invaluable in formulating recommendations for empirical

treatment, which are lacking at present, and in allowing associations between particular

species and diseases to be made.

One specific disease caused by gram-positive nonsporing anaerobes is actinomycosis,

typically affecting the cervico-facial region but which can occur at a range of body sites. Pus

collected from suspected lesions should be examined macro- and microscopically for the

presence of sulfur granules and, if present, should be reported as confirmation of a clinical

history consistent with actinomycosis. Culture of Actinomyces or related genera alone from a

site where the specimen is likely to have been contaminated with the commensal microbiota

should be interpreted with caution. This is particularly the case for the head and neck regions

sinceActinomyces is one of the predominant genera among the normal oral microbiota.