Aerococcus

TAXONOMY Back to top

The catalase-negative, gram-positive cocci included in this chapter form a taxonomically

diverse group of bacteria that are isolated infrequently as opportunistic agents of infection.

Most of these organisms resemble other more well-known clinical isolates (i.e., streptococci

and enterococci) and consequently may be mistaken for members of those genera. Although

probably misidentified or overlooked in clinical cultures in the past, these organisms may

represent emerging pathogens in immunocompromised patient populations. Table 1 lists the

organisms included here, along with some of their basic characteristics. The bacteria

discussed in this chapter are members of the phylum Firmicutes (low-G+C, gram-positive

bacteria). Helcococcus is the only genus in the group to reside in the class “Clostridia,” while

the remaining genera are classified in the class“Bacilli” (W. Ludwig, K.-H. Schleifer, and W.

B. Whitman, Bergey’s Taxonomic Outlines, vol. 3 [http://www.bergeys.org/outlines.html]).

The reader is referred to chapter 19 for information on Rothia mucilaginosa, another

infrequently isolated gram-positive coccus that may be catalase negative.

The genus Lactococcus is composed of organisms formerly classified as Lancefield group N

streptococci (122). The species Lactococcus lactis and Lactococcus garvieae have been

documented to be associated with human infections. Motile Lactococcus-like organisms with

Lancefield’s group N antigen (a teichoic acid antigen) are classified in the

genus Vagococcus (29, 141). The vagococci also resemble the enterococci, and Facklam and

Elliott (55) reported that Vagococcus fluvialis (the principal species described to occur in

human clinical specimens to date) isolates examined at the CDC gave positive reactions in a

commercially available nucleic acid probe test for enterococci.

The genera Abiotrophia and Granulicatella accommodate organisms previously known as

nutritionally variant or satelliting streptococci (37, 80). These bacteria were originally

thought to be nutritional mutants of viridans group streptococcal strains, most notably of the

species Streptococcus mitis. Bouvet and colleagues (15) suggested that this group of

organisms were really members of two novel streptococcal species given the

names Streptococcus defectivus and Streptococcus adjacens. A comparative analysis of 16S

rRNA sequences led Kawamura and coworkers to propose the creation of a new

genus, Abiotrophia, containing two species,Abiotrophia defectiva and Abiotrophia

adiacens, to accommodate these bacteria (80). A third species from human

sources, Abiotrophia elegans, was described in 1998 (119). Kanamoto et al. noted the

heterogeneity amongAbiotrophia strains and proposed a fourth species, Abiotrophia paraadiacens

(78). In 2000 Collins and Lawson proposed a new

genus, Granulicatella, with Granulicatella adiacens and Granulicatella elegans representing

strains formerly called A. adiacens and A. elegans. A. defectiva remains as the

sole Abiotrophia species (37).

Among the intrinsically vancomycin-resistant catalase-negative, gram-positive cocci, a

number of Leuconostocspecies have been noted in human infection (Leuconostoc

mesenteroides, Leuconostoc lactis, Leuconostoc pseudomesenteroides, and Leuconostoc

citreum [50]). In 1993, the former Leuconostoc paramesenteroides and related species were

placed into a novel genus, Weissella (41). Pediococcus acidilactici and Pediococcus

pentosaceus are the most common clinical isolates of pediococci (11). The vancomycinsusceptible

species formerly named Pediococcus halophilus was reclassified in the

genus Tetragenococcus (42). The organism formerly called Enterococcus solitarius has also

been transferred to the Tetragenococcus genus asTetragenococcus solitarius (52). Little is

known about the role of the tetragenococci in human infection.

The organism we now know as Gemella morbillorum was described in 1917 by Tunnicliff

(134) as an isolate from the blood of patients with measles. G. morbillorum was originally

named Diplococcus rubeolae and was also called Diplococcus morbillorum,

Peptostreptococcus morbillorum, and Streptococcus morbillorum until a proposal to include it

in the genus Gemella as Gemella morbillorum was made in 1988 (83). A second

species,Gemella haemolysans, was originally classified as a Neisseria species, due to its

gram-variable or even gram-negative nature and its cellular morphology (diplococci with

flattened adjacent sides). Collins and coworkers described two additional Gemella species

isolated from human sources, Gemella bergeri (originally namedGemella bergeriae [34])

and Gemella sanguinis (35). The genus Dolosigranulum shows phenotypic similarities

toGemella, although it is not phylogenetically closely related to Gemella strains (2, 87).

Aerococcus urinae, described in 1992, is negative for pyrrolidonyl arylamidase production

(PYR) and positive for leucine aminopeptidase production (LAP), showing opposite reactions

of Aerococcus viridans in these important identification tests (1). In spite of these phenotypic

differences, molecular taxonomic studies suggest that A. urinae should remain in

the Aerococcus genus. Organisms currently included in the A. urinae species are fairly

heterogeneous and can probably be subdivided into at least two subspecies (24). Aerococcus

christensenii,isolated from the human genitourinary tract, was described by Collins and

coworkers in 1999 (36) and was joined by the species Aerococcus sanguinicola (originally

named Aerococcus sanguicola [56, 94]) and Aerococcus urinaehominis (93) in 2001.

Globicatella, Facklamia, Ignavigranum, and Dolosicoccus are related genera that are isolated

infrequently from clinical specimens. Globicatella sanguinis, initially named Globicatella

sanguis, was described in 1992 (28).Facklamia currently contains four species isolated from

human sources: Facklamia hominis (32), Facklamia sourekii (33), Facklamia ignava (38),

and Facklamia languida (92). The genus Ignavigranum, currently consisting of a single

species, Ignavigranum ruoffiae, was described by Collins and coworkers (39), along with the

genusDolosicoccus and its single species, Dolosicoccus paucivorans (40).

The genus Helcococcus, originally composed of the single species Helcococcus kunzii (30),

came to include a new species isolated from humans, Helcococcus sueciensis, in 2004 (31).

A third human species, “Helcococcus pyogenes,” has been proposed, but to date it has not

received official taxonomic standing (107, 108).Helcococcus ovis, isolated from infections in

animals, displays satelliting growth, unlike the human Helcococcusspecies (86).

DESCRIPTION OF THE GENERA Back to top

The organisms included in this chapter form gram-positive coccoid cells, but G.

haemolysans may appear gram variable or gram negative due to the ease with which its cells

are decolorized. Cell shape and arrangement can be used to divide these organisms into two

broad groups: those with a “streptococcal-like” Gram stain (coccobacilli in pairs and chains)

or those with a “staphylococcal-like” Gram stain (more spherical cocci in pairs, tetrads,

clusters, or irregular groups). Abiotrophia and Granulicatella isolates (formerly the

nutritionally variant streptococci) form coccobacilli arranged in pairs and chains, but these

organisms may also appear pleomorphic, especially when grown under suboptimal nutritional

conditions (22). Dividing these diverse bacteria into two groups based on cellular shape and

arrangement serves only as an aid in identification; no relatedness of organisms is implied

by this grouping. With the exception of the infrequently isolated vagococci, these bacteria

are all nonmotile.

Most of the genera described here are catalase-negative facultative anaerobes, but A.

viridans is classified as a microaerophile that grows poorly, if at all, under anaerobic

conditions. Some strains of Aerococcus may exhibit weakly positive catalase reactions due to

nonheme catalase activity. None of the genera are beta-hemolytic on routinely employed

blood agars, but strains of G. haemolysans, G. bergeri, and G. sanguinis have been

described as beta-hemolytic on agars supplemented with horse blood (34, 35, 114).

EPIDEMIOLOGY AND TRANSMISSION Back to top

The organisms discussed in this chapter are opportunistic pathogens. Some of the genera

have been characterized as constituents of the normal microbiota of the human oral cavity or

upper respiratory tract(Gemella, Abiotrophia, and Granulicatella) and

skin (Helcococcus). Lactococci, pediococci, and leuconostocs can be isolated from foods and

vegetation (60, 61) and may also be found as part of the normal microbiota of the

alimentary tract. Aerococci are environmental isolates that can also be found on human skin.

Although they have been isolated from human clinical cultures, the natural habitats of many

of the organisms mentioned here are not well characterized.

The bacteria examined here seem to be of low virulence and are usually pathogenic only in

immunocompromised hosts. Infection often occurs in previously damaged tissues (e.g., heart

valves) or may be nosocomial and associated with prolonged hospitalization, antibiotic

treatment, invasive procedures, and the presence of foreign bodies.

CLINICAL SIGNIFICANCE Back to top

The bacteria described in this chapter may be present as contaminants in clinical cultures,

but they are also isolated infrequently as opportunistic pathogens. Blood, cerebrospinal fluid,

urine, and wound specimens are likely to yield significant isolates of these bacteria. Details

on reported infections due to each of the genera follow.

Lactococcus

Due to their phenotypic similarities with streptococci and enterococci, clinical isolates of

lactococci have probably been misidentified in the past, accounting at least in part for the

paucity of reports concerning the clinical role of these bacteria. Elliott and coworkers (49)

studied the phenotypic characteristics of a number of lactococcal strains isolated from blood,

urinary tract infections, and an eye wound culture. Lactococci have been associated with

prosthetic valve endocarditis (49, 59). Other reports have documented cases of lactococcal

native valve endocarditis (58, 101, 111, 140, 148), septicemia in an immunosuppressed

patient (103), osteomyelitis (74), peritonitis (65), and liver abscess (8, 66). Lactococcus

garvieae is a known pathogen of aquacultured fish, and human infections have been linked

to fish consumption (143).

Vagococcus

To date, only a handful of Vagococcus isolates from human sources have been reported in

the literature. Teixeira and coworkers (133) described strains isolated from blood, peritoneal

fluid, and a wound. Al-Ahmad and colleagues reported isolation of Vagococcus fluvialis from

an infected root canal system (3). Vagococci are motile organisms that, like lactococci,

elaborate Lancefield’s group N antigen (55). Difficulties encountered in identifying vagococci

may partially account for their infrequent recognition in clinical cultures.

Abiotrophia and Granulicatella

Organisms in the genera Abiotrophia and Granulicatella (formally known as nutritionally

variant streptococci) are normal residents of the oral cavity and are recognized as agents of

endocarditis involving both native and prosthetic valves (6, 21, 71, 75). These organisms

have also been isolated from other types of infection, including ophthalmic infections

(104, 106), central nervous system infections (19, 149), peritonitis in patients undergoing

continuous ambulatory peritoneal dialysis (9), musculoskeletal infection (144), septic

arthritis (132), and a breast implant-associated infection (45).

Leuconostoc, Pediococcus, and Weissella

The vancomycin-resistant genera Leuconostoc and Pediococcus were first recognized in

clinical specimens in the mid-1980s. Handwerger and colleagues (69) observed that host

defense impairment, invasive procedures breaching the integument, gastrointestinal

symptoms, and prior antibiotic treatment were common features among adult patients

with Leuconostoc infection. They also noted a predisposition to Leuconostoc bacteremia

among neonates, suggesting that infants may become colonized during delivery by

leuconostocs inhabiting the maternal genital tract. Leuconostocs have been isolated from

blood, cerebrospinal fluid, peritoneal dialysate fluid, and wounds. Case reports have

implicated leuconostocs as agents of infection in osteomyelitis (147), ventriculitis (47), brain

abscess (4), and postsurgical endophthalmitis (85).

Pediococcus strains have been isolated from bacteremia and cases of sepsis and hepatic

abscess in compromised patients (11, 12, 63, 102, 128). Barros and coworkers (11) noted

that Pediococcus acidilactici was isolated from clinical specimens more frequently

than Pediococcus pentosaceus and was also more commonly isolated from cases of

bacteremia. Barton and coworkers noted the role of Pediococcus in bacteremia in infants with

gastrointestinal malformations requiring surgical correction (12).

Weissella confusa, formerly classified as Lactobacillus confusus, has been reported

infrequently as an agent of bacteremia and endocarditis (126).

Gemella

G. haemolysans has been isolated from cases of endocarditis (82), meningitis (7), brain

abscess (96), and ocular infection (77, 113, 117) and a total knee arthroplasty (48). G.

morbillorum has been implicated in cases of endocarditis (5, 62), empyema and lung abscess

(136), septic shock (139), brain abscess (130), osteomyelitis (138), septic arthritis (118),

and peritonitis (90). The clinical significance of G. bergeri and G. sanguinis is not well

described, but strains of these species have been isolated from blood cultures, and they may

also be causative agents of endocarditis (34, 35, 100).

Dolosigranulum

Dolosigranulum, a genus phenotypically similar, but not closely related, to Gemella (2), has

been documented to occur in blood, eye, and respiratory specimens (87). The single species

of the genus, Dolosigranulum pigrum, has been associated with nosocomial pneumonia and

septicemia (95), synovitis (68), and acute cholecystitis accompanied by acute pancreatitis

(99).

Aerococcus

A. viridans has been noted as a contaminant in clinical cultures and infrequently as a

clinically significant isolate from cases of endocarditis and bacteremia and a case of

spondylodiscitis (43, 81, 105, 109). Four additionalAerococcus species isolated from humans

have been described since the early 1990s. A. urinae (1, 64) has been implicated as a

urinary tract pathogen in patients predisposed to infection (23, 127) and as an agent of

endocarditis (79, 84), lymphadenitis (121), and peritonitis (27). A. sanguinicola has been

isolated from blood and urine specimens (56, 94) and cases of urosepsis and endocarditis

(73). Little is currently known about the clinical significance of A. christensenii (isolated from

vaginal specimens [36]) and A. urinaehominis (isolated from urine [93]).

Globicatella

G. sanguinis, isolated from human clinical specimens, has been implicated in cases of

bacteremia, urinary tract infection, and meningitis (28, 91, 124). A second species in the

genus, Globicatella sulfidifaciens, has been isolated from purulent infections in domestic

mammals (137).

Facklamia

The Facklamia genus is closely related to, but phenotypically and phylogenetically distinct

from, Globicatella(32). Strains of the four Facklamia species isolated from humans have been

recovered from blood, wound, and genitourinary sites (32, 33, 38, 92) and a case of

chorioamnionitis (70).

Ignavigranum

A limited number of isolates of I. ruoffiae, the sole species of Ignavigranum, have been

described to date. Sites of isolation include a wound and an ear abscess (39).

Dolosicoccus

The single species of the genus Dolosicoccus, D. paucivorans, has been isolated from blood

cultures (40, 54).

Helcococcus

Helcococcus kunzii can be isolated from intact skin of the lower extremities (67) as well as

from mixed cultures of wounds, notably foot infections (30, 97). In such scenarios the clinical

significance of this organism is difficult to interpret, since it may be present merely as a

colonizer of the wound site. The ability of H. kunzii to function as an opportunist is, however,

suggested by its isolation as the sole or predominant organism from an infected sebaceous

cyst (110), a breast abscess (20), a postsurgical foot abscess (116), and cases of bacteremia

and empyema in intravenous drug users (146). Two additional species isolated from

humans, H. sueciensis and “H. pyogenes,” are based on single isolates from a wound and a

prosthetic joint infection, respectively (31, 107, 108).

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

No special requirements for collection and transport of specimens for isolation of the

organisms discussed in this chapter have been described. Routine procedures for collection,

transport, and storage of specimens for aerobic culture allow for the isolation of these

bacteria, since the majority are facultative anaerobes or microaerophiles. With the exception

of some Aerococcus strains that require an aerobic atmosphere for good growth, these

organisms should also be recovered from specimens that have been collected and

transported under anaerobic conditions (see chapter 16).

DIRECT EXAMINATION Back to top

The organisms described in this chapter can be visualized in direct Gram stains of clinical

material but have no outstanding morphological characteristics that distinguish them from

commonly isolated gram-positive cocci (streptococci and staphylococci).

Although Abiotrophia and Granulicatella isolates may appear pleomorphic in direct Gram

stains, they form gram-positive cocci in pairs and chains when grown on nutritionally

adequate media. Direct detection of these genera by antigenic methods has not been

described, but some authors have employed amplification of 16S rRNA genes for direct

detection in clinical specimens (71).

ISOLATION PROCEDURES Back to top

Generally, there are no special requirements for isolation of the group of bacteria discussed

here; general recommendations for the culture of blood, body fluids, and other specimens

should be followed (see chapter 16). These organisms are likely to be isolated on rich,

nonselective media (e.g., blood or chocolate agar and thioglycolate broth) since they are

nutritionally fastidious. If selective isolation of the vancomycin-resistant

genera Leuconostoc and Pediococcus is desired, Thayer-Martin medium may be used to

inhibit normal microbiota or other contaminating microorganisms (120). Some of the genera

(e.g., Helcococcus) grow slowly, forming tiny colonies that may not be visible unless

extended incubation (48 to 72 h) is employed. The recovery of many of the genera included

in this chapter may be enhanced by CO2 enrichment of the incubation atmosphere.

Members of the genera Abiotrophia and Granulicatella usually grow on chocolate agar, on

brucella agar with 5% horse blood, and in thioglycolate broth, but not on Trypticase soy agar

with 5% sheep blood. These organisms can be cultured on nonsupportive media that have

been appropriately supplemented (see “Procedures for Phenotypic

Differentiation, Abiotrophia and Granulicatella,” below).

IDENTIFICATION Back to top

Procedures for Phenotypic Differentiation

While molecular characterization may be required for accurate species-level identification of

the aerobic, catalase-negative, gram-positive cocci encountered infrequently in clinical

laboratories, phenotypic methods can be helpful in characterization of these bacteria to the

genus level. Gram stain morphology has been employed as a major decision point in the

identification protocols in Fig. 1 and 2 and Table 1, with two general categories: morphology

resembling that of streptococci, meaning cocci or coccobacilli in pairs and chains versus

staphylococcal morphology, consisting of coccoid cells arranged in pairs, clusters, tetrads, or

irregular groups. Broth-grown cells (thioglycolate broth is suitable) should be used for

making accurate morphological determinations. Note that Gemella and Facklamia strains

may display either type of cellular morphology, depending on the species. Figures

1 and 2 display phenotypic tests used to differentiate the genera of bacteria discussed in this

chapter. Descriptions of tests for catalase, PYR, LAP, beta-glucuronidase, and hippurate

hydrolysis, as well as bile esculin agar and lactobacillus MRS (deMan, Rogosa, Sharpe) broth

media, can be found in chapter 17 and reference 55. Additional phenotypic tests are

described below in the discussion of identification criteria for each genus.

Lactococcus and Vagococcus

The members of the genera Lactococcus and Vagococcus are usually PYR and LAP positive,

grow in the presence of 6.5% NaCl, and can be confused with enterococci or streptococci.

For the salt tolerance test, heart infusion broth supplemented with 6.0% NaCl (producing a

final NaCl concentration of 6.5%), with or without the acid-base indicator bromcresol purple,

is inoculated with two or three colonies and incubated at 35°C for up to 72 h. Turbidity with

or without a color change from purple to yellow indicates growth (55, 57). Facklam and

colleagues (55, 57) recommended growth temperature tests for distinguishing lactococci

from streptococci and enterococci. Consult Fig. 1 for growth temperature characteristics of

each of the genera. For growth temperature tests, broths (heart infusion broth containing

1% glucose and bromcresol purple indicator) are inoculated with a single colony or drop of

broth culture of the test strain and incubated at 35°C for up to 7 days. A water bath is

recommended for incubation of cultures at 45°C. Turbidity with or without a change in the

broth’s indicator to yellow indicates a positive test. The motile vagococci can be distinguished

from lactococci with modified motility test medium, stab-inoculated and incubated at 30°C

for up to 48 h, according to the method of Facklam and Elliott (55). Further information on

the phenotypic traits of Lactococcusand Vagococcus isolates may be found in

references 49, 51, 122, and 133.

Abiotrophia and Granulicatella

A test for satelliting behavior is important for identification of these two genera. The strain to

be examined is streaked for confluent growth on a medium that does not support growth or

supports only weak growth (e.g., sheep blood agar). A single cross streak of Staphylococcus

aureus (ATCC 25923 or another suitable strain) is applied to the inoculated area. After

incubation at 35°C in an atmosphere containing elevated CO2, strains

ofAbiotrophia or Granulicatella grow only in the vicinity of the staphylococcal growth. Some

strains ofIgnavigranum may also show satelliting behavior (39). Alternatively, media can be

supplemented with pyridoxal. An aqueous stock solution of filter-sterilized 0.01% pyridoxal

hydrochloride (which can be stored frozen) should be added to media to achieve a final

concentration of 0.001%. Pyridoxal disks (Remel, Lenexa, KS) may also be used in the

satelliting test.

Detailed phenotypic information for the PYR- and LAPpositive

Abiotrophia and Granulicatella species can be found in references 13, 16, 22,

and 37. Davis and Peel (44) reported that the API 20 Strep system (bioMerieux, Durham,

NC) was superior to the Rapid ID32 Strep system (bioMerieux) for identification of these

organisms.

Leuconostoc, Pediococcus, and Weissella

Members of the PYR-negative, vancomycin-resistant genera Leuconostoc,

Pediococcus, and Weissella produce small, alpha-hemolytic or nonhemolytic colonies on

blood agar. Vancomycin resistance can be tested by streaking several colonies over half of a

Trypticase soy agar with 5% sheep blood plate. After placing a 30-μg vancomycin disk in the

center of the inoculated area, the plate is incubated overnight in a CO2-enriched atmosphere

at 35°C. Any zone of inhibition indicates susceptibility, while resistant strains exhibit no

inhibition zone (55, 57). In addition to differing cellular morphologies (Table 1), these

vancomycin-resistant genera, along with vancomycin-resistant strains of lactobacilli that

form short coccoid cells, can be differentiated by tests for gas production from glucose and

arginine hydrolysis. Leuconostocs produce gas and are always arginine negative. Lactobacilli

are variable in both tests, but a positive arginine test for a gas-producing strain would rule

out identity of the organism as a leuconostoc. Pediococci are gas production negative and

show variable reactions in the arginine test, although P. acidilactici and Pediococcus

pentosaceus, the two species commonly found in clinical material, are arginine

positive. Weissella strains may be misidentified as leuconostocs or lactobacilli. These

organisms produce gas from glucose. The few clinical isolates reported in the literature have

been described as positive for hydrolysis of arginine (41, 126).

MRS broth (BD Diagnostic Systems, Franklin Lakes, NJ; Hardy Diagnostics, Santa Maria, CA;

see chapter 17), sealed with melted petrolatum and incubated for up to 7 days at 35°C, is

used to test for gas production, indicated by displacement of the petrolatum plug (55, 57).

The arginine hydrolysis test can be performed with Moeller’s decarboxylase broth containing

arginine (55). Lancefield group D antigen can be detected in pediococci (57).

References 10, 11, 50, 55, 57, and 115 should be consulted for further information on

identification of Leuconostoc and Pediococcus to the species level.

Gemella

On sheep blood agar media, members of the Gemella genus (usually PYR positive) form

small colonies that are similar in appearance to those of viridans group streptococci. Slow

growth of some Gemella strains may lead to confusion of these organisms

with Abiotrophia or Granulicatella (formerly called nutritionally variant streptococci). A test

for satelliting behavior separates these two groups of bacteria (57). Cells of G.

haemolysans are easily decolorized and resemble those of neisserias, since they occur in

pairs with the adjacent sides flattened. G. haemolysans prefers an aerobic growth

atmosphere. The esculin hydrolysis test for differentiation of G. haemolysans and Rothia

mucilaginosa in Fig. 2 is performed with esculin agar slants (heart infusion agar containing

0.1% esculin and 0.5% ferric citrate) that are inoculated and incubated at 35°C for up to 7

days. Partial or complete blackening of the agar indicates a positive reaction (55). G.

morbillorumcells are gram positive and arranged in pairs and short chains; individual cells in

a given pair may be of unequal sizes. Only a small number of strains of G. bergeri and G.

sanguinis have been reported on to date. Information on phenotypic characteristics of

these Gemella species can be found in references 34 and 35.

Aerococcus

The PYR-positive, LAP-negative member of the genus, A. viridans, is characterized by

displaying weak or no growth when incubated in an anaerobic atmosphere (53). This trait

can be tested by incubating duplicate blood agar plate cultures of the organism in question in

anaerobic and aerobic atmospheres and comparing growth after 24 to 48 h. A. viridans forms

alpha-hemolytic colonies that could be confused with those of either viridans group

streptococci or enterococci. A. sanguinicola is positive in the PYR and LAP tests, while A.

urinaehominis is negative in both. The PYR-negative, LAP-positive species, A. urinae and A.

christensenii, are differentiated by production of beta-glucuronidase (A. christensenii is

negative and A. urinae is positive). A. urinae forms small (0.5 mm in diameter after 24 h of

incubation) alpha-hemolytic, convex, shiny, transparent colonies on blood agar media.

Additional information on the identifying characteristics of A. urinae can be found in

reference 23, and a second biotype (esculin hydrolysis positive) of this species is described in

reference 24. Additional information on phenotypic traits of the species A. christensenii, A.

sanguinicola, and A. urinaehominiscan be found in Table 1, Fig. 2, and references 36, 56, 93,

and 94.

Dolosigranulum

D. pigrum, the sole species of Dolosigranulum described to date, displays positive PYR and

LAP reactions and was initially described as phenotypically similar, though not closely

related, to members of the genus Gemella(2). D. pigrum is distinguished

from Gemella species by its abilities to hydrolyze arginine and to grow in the presence of

6.5% NaCl.

Globicatella and Related Genera (Facklamia,

Dolosicoccus, and Ignavigranum)

Globicatella and the related genera Facklamia, Dolosicoccus, and Ignavigranum are all PYR

positive. Facklamiaand Ignavigranum are also LAP positive and salt tolerant. Globicatella is

LAP negative and salt tolerant, whileDolosicoccus is LAP negative and salt

intolerant. Dolosicoccus strains are also hippurate hydrolysis negative, which further

distinguishes them from strains of Facklamia and Globicatella (hippurate hydrolysis positive).

Strains of Facklamia hominis and Ignavigranum may produce urease. Ignavigranum strains

may exhibit satelliting behavior. Further details of phenotypic traits of these organisms can

be found in references 32, 33,38–40, 89, and 92.

Helcococcus

Colonial morphology (tiny gray, usually slightly alpha-hemolytic colonies), good growth

under anaerobic conditions, and stimulation of growth by addition of 1% horse serum or

0.1% Tween 80 to the medium differentiate H. kunzii from aerococci (30). Isolates of H.

kunzii are PYR positive, and most produce an API 20 Strep (bioMerieux) profile of 4100413.

Additional Helcococcus species isolated from humans (H. sueciensis and the proposed “H.

pyogenes”) are negative in the PYR test. Detailed phenotypic data on these organisms can be

found in references 30, 31, 107, and 108.

Commercially Available Kits and Automated Methods Based on

Phenotypic Traits

There have been no comprehensive evaluations of the ability of commercially available

products to identify the diverse and infrequently isolated bacteria described in this chapter.

Phenotypic variation among isolates classified in the same species, the relative metabolic

inactivity of some organisms, and a relatively small number of strains available for inclusion

in databases have challenged the capabilities of these products for accurate identification.

Manual methods for performance of some of the basic differentiation tests (e.g., PYR and

LAP) are available (e.g., BactiCard Strep [Remel]). Commercially available identification kits

or systems offering a more comprehensive array of phenotypic tests are improving in their

ability to identify many of the organisms discussed in this chapter (14, 56, 89, 142, 145).

These products include manual methods (e.g., API 20 Strep [bioMerieux] and RapID Strep

[Remel]) and automated systems (e.g., Vitek 2 [bioMerieux], MicroScan [Siemens

Healthcare Diagnostics, Inc., Deerfield, IL], and Phoenix [BD Diagnostics, Sparks, MD]). In

the absence of an accurate genus or species level identification, these systems will at least

provide additional phenotypic information that can be used to augment results of the basic

tests mentioned above.

Molecular Methods

16S rRNA gene sequencing-based identification methods appear to be more accurate than

phenotypic methods, either manual or automated, for identifying many of the infrequently

isolated aerobic catalase-negative, gram-positive cocci (14, 145). Bosshard and colleagues

(14) observed that this method produced more species or genus level identifications than a

commercially available phenotypic method (API 20 Strep), and identifications based on

phenotypic traits often disagreed with those determined by 16S rRNA gene

sequencing. Abiotrophia, Aerococcus, and Gemella strains were included in their study. Woo

and coworkers (145) examined strains of Abiotrophia, Granulicatella,

Gemella, and Helcococcus in their evaluation of a commercially available rRNA gene

sequence-based identification system (MicroSeq 500 [Perkin-Elmer Applied Biosystems

Division, Foster City, CA]). They noted disagreement in identifications obtained with

commercially available phenotypic test systems (API 20 Strep and Vitek) and the

commercially available sequence-based identification system compared with conventional

16S rRNA gene sequencing. The authors stressed the importance of adequate databases for

accurate rRNA gene sequence-based identification. The use of alternative sequencing targets

for identification of the organisms discussed in this chapter has not been extensively

investigated, but Drancourt and colleagues demonstrated the utility of rpoB gene sequencing

for identification of strains of Abiotrophia, Granulicatella, and Gemella (46).

TYPING SYSTEMS Back to top

Little information exists on typing methods for the genera of infrequently isolated grampositive

cocci included in this chapter. Typing is not routinely used for characterizing these

organisms.

SEROLOGIC TESTS Back to top

Serologic response to the organisms described in this chapter has not been extensively

investigated. No clinically useful tests have been described.

ANTIMICROBIAL SUSCEPTIBILITIES Back to top

Antimicrobial susceptibility studies on the organisms mentioned in this chapter have

generally employed dilution testing methods. Little or no data exist on the utility of disk

diffusion or the correlation of Etest results with those of broth or agar dilution methods.

Standardized dilution methods and interpretive criteria for observed MICs have been

described for only four of the genera (Abiotrophia, Granulicatella,

Leuconostoc, andPediococcus [reference 25 and chapter 71]). The lack of standardized

methods and interpretive criteria and the relatively small collections of isolates for some of

the genera discussed in this chapter make it difficult to accurately assess antimicrobial

susceptibility patterns. With the exception of Leuconostoc, Pediococcus, andWeissella, all of

the genera display susceptibility to vancomycin. While many of the genera are susceptible to

beta-lactams and other antimicrobials, observed strain variations suggest that MICs of

antimicrobials used for treatment should be determined for individual isolates. When

susceptibility testing is requested for isolates for which no guidelines exist, dilution methods

may be used to generate MICs which can be reported without interpretation. Since many of

the bacteria dealt with here are fairly fastidious, investigators have often employed bloodsupplemented

Mueller-Hinton media and, if necessary for good growth, incubation in a CO2-

enriched atmosphere for susceptibility testing. Pyridoxal hydrochloride (final concentration of

0.001%) should also be added to blood-supplemented media for testing strains

of Abiotrophia and Granulicatella (25, 26). Details of published susceptibility testing studies

for each of the genera appear below.

Information on the in vitro antimicrobial susceptibility of Lactococcus lactis and Lactococcus

garvieae strains isolated from humans suggests that L. garvieae isolates are less susceptible

to penicillin and cephalothin than are strains of L. lactis. The uniform resistance of L.

garvieae versus the uniform susceptibility to clindamycin of the L. lactis strains examined by

Elliott and Facklam (51) led them to propose a test for clindamycin susceptibility as an aid in

differentiation of these two species. In clinical practice, cases of lactococcal endocarditis have

been successfully treated either with penicillin alone or with penicillin and gentamicin

(101,111).

Teixeira and colleagues observed that a collection of Vagococcus isolates were all susceptible

to ampicillin, cefotaxime, and trimethoprim-sulfamethoxazole. All strains were resistant to

clindamycin, lomefloxacin, and ofloxacin. Variable results were observed with other

antimicrobial agents (133).

The vancomycin-resistant genera Leuconostoc and Pediococcus are considered penicillin

susceptible when MICs are interpreted using criteria adapted from those

for Enterococcus spp. (25, 131). They are usually susceptible to chloramphenicol,

tetracyclines, and aminoglycosides. Carbapenem and cephalosporin resistance has been

noted in some strains of Leuconostoc (25). Huang and colleagues (72) noted MIC ranges of

0.5 to 8 μg/ml for linezolid and 0.06 to 2 μg/ml for daptomycin in 68 strains

of Leuconostoc tested and ranges of 1 to 4 μg/ml for linezolid and 0.06 to 0.5 μg/ml for

daptomycin in 13 Pediococcus isolates.

Abiotrophia and Granulicatella isolates display a range of penicillin MICs, with authors

reporting reduced penicillin susceptibility in 33 to 65% of isolates (76, 98, 135).

Susceptibility to aminoglycosides is also variable, but no cases of high-level resistance have

been reported. A synergistic effect between beta-lactam agents and aminoglycosides has

been demonstrated for isolates of Abiotrophia, and combination therapy with penicillin and

gentamicin is the currently recommended treatment for endocarditis caused

by Abiotrophia andGranulicatella. High relapse rates have been reported, even with

appropriate therapy (76). Tuohy and colleagues (135) examined a collection of 27 G.

adiacens and 12 A. defectiva strains, noting susceptibility of all isolates to clindamycin,

rifampin, levofloxacin, ofloxacin, and quinupristin-dalfopristin. These authors noted that the

susceptibilities of G. adiacens and A. defective, respectively, to other agents tested were as

follows: penicillin, 55 and 8%; amoxicillin, 81 and 92%; ceftriaxone, 63 and 83%; and

meropenem, 96 and 100% (135). Zheng and coworkers reported high rates of beta-lactam

and macrolide resistance in a collection of pediatricAbiotrophia and Granulicatella isolates

(150). A daptomycin MIC range of ≤0.125 to 2 μg/ml was observed for 10 strains of this

group of bacteria (112).

A. viridans and G. haemolysans appear to be susceptible to penicillin and display a low level

of resistance to aminoglycosides (17, 18). Resistance to tetracycline and macrolides has

been described to occur in Gemellaisolates (151), as well as a synergistic effect for penicillin

and gentamicin (18). Piper and colleagues (112) noted daptomycin MICs of ≤0.125 μg/ml for

four strains of G. morbillorum. Buu-Hoi and colleagues (17) noted that while A.

viridans seems to be naturally susceptible to macrolides, tetracyclines, and chloramphenicol,

resistance to these agents has been observed. A. urinae has been described as susceptible to

penicillin, amoxicillin, piperacillin, cefepime, rifampin, and nitrofurantoin but resistant to

sulfonamides and netilmicin. Isolates displayed variable susceptibilities to trimethoprim and

co-trimoxazole (23, 123, 129). A. sanguinicolaisolates display susceptibility to penicillin,

amoxicillin, cefotaxime, cefuroxime, erythromycin, chloramphenicol, quinupristin-dalfopristin,

rifampin, linezolid, and tetracycline (56).

Clinical isolates of D. pigrum studied by LaClaire and Facklam (87) were all susceptible to

penicillin, amoxicillin, cefotaxime, cefuroxime, clindamycin, levofloxacin, meropenem,

quinupristin-dalfopristin, rifampin, and tetracycline. Variable susceptibility to erythromycin

was noted, and 1 of the 27 strains examined was resistant to trimethoprimsulfamethoxazole.

The small number of Helcococcus isolates examined displayed

susceptibility to penicillin and clindamycin, and most strains were resistant to erythromycin

(20, 110). Woo and coworkers described an H. kunzii strain with ermA-mediated

erythromycin and clindamycin resistance (146). Strains ofFacklamia exhibit variable MICs for

a variety of antibiotics (88). A study of 27 strains of G. sanguinis reported susceptibility of all

isolates to amoxicillin but various levels of resistance to other antimicrobials tested (125).

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

Efforts to identify the gram-positive cocci included in this chapter should be made only when

isolates are considered to be clinically significant (i.e., isolated repeatedly, in pure culture, or

from normally sterile sites), since these organisms may also appear in clinical cultures as

contaminants or constituents of the normal microbiota. Communication with clinicians should

guide the microbiology laboratory in evaluating the significance of these infrequently isolated

organisms. The phenotypic tests mentioned in Table 1 and Fig. 1and 2 facilitate presumptive

identification of the infrequently isolated catalase-negative, gram-positive cocci. More

extensive phenotypic testing using commercially available identification systems and

molecular methods should be employed for definitive identification. Currently there are

susceptibility testing guidelines for only four of the genera mentioned in this chapter. The

MICs generated with dilution methods can be reported without interpretation when

susceptibility testing is requested for significant isolates for which no guidelines exist.

Abiotrophia, Granulicatella, and Gemella species are well-documented agents of endocarditis.

The satelliting behavior of Abiotrophia and Granulicatella and the positive PYR reactions of all

three genera are useful for distinguishing them from viridans group streptococci. CLSI

guidelines should be employed for susceptibility testing and interpretation of results

for Abiotrophia and Granulicatella. The vancomycin-resistant generaLeuconostoc,

Pediococcus, and Weissella are infrequent clinical isolates, but they have been described as

agents of bacteremia and central nervous system and other infections in compromised hosts.

Phenotypic testing for vancomycin resistance (see “Identification” above) is important for

identifying these genera and also helps guide antimicrobial therapy. Guidelines for

antimicrobial susceptibility testing and interpretation of results are available

for Leuconostoc and Pediococcus (25). Among the aerococci, A. urinae is a well-documented

urinary tract pathogen and should be reported when isolated in significant amounts as the

predominant organism in urine cultures. Phenotypic tests mentioned in this chapter

presumptively identify A. urinae, which has been described as susceptible to beta-lactam

agents and nitrofurantoin (23, 123, 129).