Enterococcus

TAXONOMY Back to top

Early documentation on the microorganisms that are now included in the

genus Enterococcus is mainly related to the “streptococci of fecal origin” or “enterococci”

(see reference 31 for a brief historical overview). For a long time, they were considered a

major category within the genus Streptococcus, distinguished by their higher resistance to

chemical and physical agents and accommodating most of the serological group D

streptococci. After the introduction of molecular methods for studying these microorganisms,

however, the enterococci have undergone considerable changes in taxonomy, which started

with the splitting of the genusStreptococcus, and the recognition of Enterococcus as a

separate genus, in 1984 (87). Streptococcus faecalisand Streptococcus faecium were the

first species to be transferred to the new genus as Enterococcus faecalis(the type species)

and Enterococcus faecium, respectively. Subsequently, other earlier streptococcal species

and subspecies were transferred and received new denominations as species of the

genus Enterococcus (18). Since then, several new species have been described and proposed

for inclusion in the genus Enterococcus(30, 31).

The enterococci belong to the low-guanine-plus-cytosine-content (G+C <50 mol%) branch of

the phylumFirmicutes. Phylogenetic analysis based on the comparison of the 16S rRNA gene

sequences showed that members of the genus Enterococcus are more closely related to

those included in the genera Vagococcus, Tetragenococcus, and Carnobacterium than they

are to Streptococcus and Lactococcus, genera to which they have been phenotypically

associated (25, 31).

Current criteria for inclusion in the genus Enterococcus and for the description of new

enterococcal species encompass a polyphasic approach resulting from a combination of

different molecular techniques (frequently involving DNA-DNA reassociation experiments,

16S rRNA gene sequencing, and whole-cell protein profiling analysis) and phenotypic tests.

Partial or nearly entire sequencing of the 16S rDNA is nowadays considered a practical and

powerful tool in aiding the identification of enterococcal species, and it has been performed

for all recognized species of Enterococcus. Figure 1 shows the phylogenetic relationships

among the species ofEnterococcus based on the analysis of 16S rRNA gene sequences, which

are available from the GenBank database. Several other molecular methods, mostly nucleic

acid-based assays, have been used as additional tools to assess the phylogenetic

relationships among enterococcal species and to formulate the description of new species,

but their use is still limited.

DESCRIPTION OF THE GENUS Back to top

The members of the genus Enterococcus are gram-positive, catalase-negative cocci that

occur singly or are arranged in pairs or as short chains. Cells are sometimes coccobacillary

when Gram stains are prepared from growth on solid medium but tend to be ovoid and in

chains when grown in liquid media, such as thioglycolate broth. After growth on blood agar

media for 24 h, colonies are usually between 1 and 2 mm in diameter, although some

variants may appear smaller. Some (about one-third) cultures of E. faecalis may be β-

hemolytic on agar containing rabbit, horse, or human blood but nonhemolytic on agar

containing sheep blood. Some cultures of E. faecalis and Enterococcus durans may be β-

hemolytic regardless of the type of blood used. All other species are usually α-hemolytic or

nonhemolytic. Enterococci are facultative anaerobes with a homofermentative metabolism

that results in the production of L-(+)-lactic acid as the major end product of glucose

fermentation. Because of their ability to ferment a wide range of carbohydrates to lactic acid,

the enterococci are referred to as typical lactic acid bacteria. Gas is not produced. These

microorganisms are usually able to grow at temperatures ranging from 10 to 45°C, with

optimum growth at 35 to 37°C. The majority of the species grow in broth containing 6.5%

NaCl, and they hydrolyze esculin in the presence of bile salts (bile-esculin [BE] test). They

also hydrolyze leucine-β-naphthylamide by producing leucine aminopeptidase (LAP). Most

enterococci, apart from Enterococcus cecorum, Enterococcus columbae, Enterococcus

pallens, Enterococcus saccharolyticus, and some strains of the recently described

speciesEnterococcus canintestini, Enterococcus devriesei, Enterococcus

moraviensis, and Enterococcus termitis,hydrolyze L-pyrrolidonyl-β-naphthylamide (PYR) by

producing pyrrolidonyl arylamidase (pyrrolidonase). Results for both LAP and PYR testing,

especially for some of the more recently described species, may vary according to the

methodology, including the test format and media used to grow the bacterial cells. A few

species are motile (Enterococcus casseliflavus and Enterococcus gallinarum), and some are

pigmented (E. casseliflavus, Enterococcus gilvus, Enterococcus mundtii, E.

pallens, and Enterococcus sulfureus) (25, 31, 46). Methods used for detection of

enterococcal motility have to be selected carefully, as differences in motility due to the

composition of the medium have been demonstrated (108). Enterococci are not able to

synthesize porphyrins and therefore do not produce cytochrome enzymes (46). However,

cytochrome activity is sometimes expressed when strains of E. faecalis are grown on bloodcontaining

media, and a weak effervescence is observed in the catalase test. Positive

catalase testing has also been reported for strains of Enterococcus haemoperoxidus (94)

and Enterococcus silesiacus (95) when cultivated on blood-containing agar media. Most

enterococcal strains produce a cell wall-associated glycerol teichoic acid that is identified as

Lancefield’s serological group D antigen. The G+C content of the DNA ranges from 32 to 44

mol%. The genome size is in the range of approximately 2.0 to 3.5 Mb (5, 78, 89). Genome

sequencing of E. faecalis V583 (80), the first vancomycin-resistant clinical isolate in the

United States, has opened many lines of investigation to improve our understanding about

the genus Enterococcus.

The other genera of catalase-negative gram-positive cocci and the characteristics that

distinguish them from the enterococci are discussed in chapters 20 and 22. No phenotypic

criteria are available for clearly distinguishing the genus Enterococcus unequivocally from

other genera, since there are no particular characteristics that are common to all

enterococci. However, certain characteristics are usually found in the majority of the strains

belonging to the most frequently isolated enterococcal species. Presumptive identification of

a gram-positive, catalase-negative coccus as an Enterococcus can be accomplished by

demonstrating that the strain is positive for the BE, PYR, and LAP tests and grows in the

presence of 6.5% NaCl and at 45°C. Because strains of Lactococcus,

Leuconostoc, Pediococcus, and Vagococcus with phenotypic similarities have been isolated

from human infections (33, 98), the presumptive identification of enterococci based only on

BE reaction and growth in 6.5% NaCl broth can be erroneous. Demonstrating the presence of

group D antigen by serological reaction may be helpful in identification, although this antigen

is detected in only about 80% of the enterococcal strains. On the other hand, pediococci and

leuconostocs (33), as well as some vagococcal strains (98), can also react with anti-group D

serum. Reactivity with the AccuProbeEnterococcus genetic probe (GenProbe, Inc., San Diego,

CA) test can also be used to confirm an unknown strain as an Enterococcus. Strains of most

known species of Enterococcus react with this probe, except for the type strains

of Enterococcus aquimarinus, Enterococcus asini, Enterococcus canis, E. cecorum, E.

columbae, E. haemoperoxidus, E. moraviensis, E. pallens, E. saccharolyticus, E.

silesiacus, and E. termitis. However,Vagococcus strains may also react (98).

EPIDEMIOLOGY AND TRANSMISSION Back to top

Several intrinsic characteristics of the enterococci allow them to grow and survive in harsh

conditions and persist almost everywhere, colonizing several ecological niches. These

microorganisms are widespread in nature and can be found in soil, plants, water, food, and

animals, including mammals, birds, insects, and reptiles (25, 97). In humans, they are

predominantly inhabitants of the gastrointestinal tract and are less commonly found in other

sites, such as in the genitourinary tract, the oral cavity, and skin, especially in the perineal

area (25, 97). The prevalence of the different enterococcal species appears to vary according

to the host and is also influenced by age, diet, and other factors that may be related to

changes in physiologic conditions, such as underlying diseases and prior antimicrobial

therapy. Enterococci are considered the most abundant gram- positive cocci colonizing the

intestine, with E. faecalis being one of the most common bacteria isolated from this site

(25, 97). Other species, such as E. faecium, E. casseliflavus, E. durans, and E.

gallinarum,are also found in variable proportions in the gastrointestinal tract of humans.

Since the enterococci are opportunistic pathogens, the incidence of each species found in

human infections probably reflects the distribution of the different species of Enterococcus in

the human gastrointestinal tract. This site is believed to represent an important reservoir for

strains associated with disease; from this location they may migrate to cause infections and

can also disseminate to other hosts and to the environment. On the other hand, the

occurrence of high numbers of enterococci in the feces, as well as their ability to resist

different chemical and physical conditions and to survive in the environment, implies that the

enterococci can be used as indicators of fecal contamination and of the hygienic quality of

food, milk, and drinking water (36). The occurrence of enterococci as members of the

intestinal microbiota of humans (97) and the relationship between the presence of

enterococci in foods and human safety (36) have been extensively reviewed.

CLINICAL SIGNIFICANCE Back to top

The enterococci are commensal microorganisms that act as opportunistic agents, causing a

variety of infections in humans. Many of these infections have been suggested to arise from

translocation of the enterococcal cells from their major site of colonization in the

gastrointestinal tract. They most commonly infect the urinary tract, bloodstream,

endocardium, burn and surgical site wounds, abdomen, biliary tract, catheters, and other

implanted medical devices (62, 69, 71, 84). The ubiquitous presence of enterococci,

however, requires the use of caution in establishing the clinical significance of a particular

isolate. Unnecessary work and potentially misleading laboratory reports should be avoided

whenever possible, especially with respect to in vitro susceptibility testing decisions (see

“Antimicrobial Susceptibilities” below). Over the last decades, they have emerged from long

being considered virtually harmless bacteria to medically important multiple-antibioticresistant

nosocomial pathogens that contribute significantly to patient morbidity and

mortality, as well as healthcare costs. Changes in the dynamics of the host-commensal

relationship, such as those promoted by the use of broad-spectrum antibiotics, host injury,

or diminished host immunity, could allow these bacteria to gain access to extraintestinal host

sites and cause infection. Therefore, elderly patients with serious underlying diseases and

other severely ill immunocompromised patients who have been hospitalized for prolonged

periods, treated with invasive devices, and/or received broad-spectrum antimicrobial therapy

are at higher risk to acquire enterococcal infections (2, 62, 71).

The pathogenesis of enterococcal infections is still poorly understood. Although a debate

subsists over whether serious enterococcal infections arise from one’s own indigenous biota

or from exogenously acquired strains, epidemiological studies show the existence of clonal

relationships among outbreak isolates and support the notion that a subset of virulent

lineages with greater propensity to cause disease exist and are often responsible for

infections of epidemic proportions (57, 100, 105, 114). Several potential virulence factors

were identified in enterococcal isolates and have been suggested to play a role in the

pathogenesis of enterococcal infections; these include the surface adhesins Esp (enterococcal

surface protein) and aggregation substance, the secreted toxin cytolysin/hemolysin, the

secreted proteases gelatinase and serine protease, MSCRAMM Ace (adhesin to collagen of E.

faecalis), E. faecalis antigen A, enterococcal capsule, cell wall polysaccharides, and

extracellular superoxide (47, 83, 100). Nevertheless, none has been established as having a

major contribution to enterococcal virulence in humans. One mechanism by which the

enterococci can deviate from their commensal behavior is through the acquisition of new

traits that allow the bacterium to overcome host defenses and colonize new niches, as

suggested by the identification of the E. faecalispathogenicity island, which highlights genetic

differences between infection-derived and commensal strains (64, 88, 100). In this context,

acquired antimicrobial resistance is considered one of the many traits that virulent

enterococci possess compared with commensal isolates, as it may be fundamental to allow

members of this genus to survive for extended periods of time in the host or environment,

leading to their persistence and role as prominent nosocomial pathogens (47, 88, 100). In

addition, enterococci can transfer resistance determinants to other bacteria, for example,

staphylococci, which further increases the clinical importance of the enterococci. The ability

to form biofilms has recently been listed among the most prominent virulence properties of

these microorganisms, allowing colonization of inert and biological surfaces while protecting

against antimicrobial substances and mediating adhesion and invasion of host cells (2, 28).

Biofilm formation may be of particular importance in the development of endocarditis,

endodontic and urinary infections, and implant- as well as other medical device-associated

infections (2, 117).

The variety of infections associated with the enterococci has been thoroughly reviewed and

summarized (62,71). Although the spectrum of infections has remained relatively unchanged

since the extensive review by Murray (71), trends to increasing prevalence of these

organisms as nosocomial pathogens have been frequently observed. Enterococci have

become the second or third leading cause of nosocomial urinary tract infections (UTIs),

wound infections (mostly surgical, decubitus ulcers, and burn wounds), and bacteremia in

the United States (4, 53, 62, 71, 73, 74). UTIs are the most common of the enterococcal

infections: enterococci have been implicated in approximately 10% of all UTIs and in up to

approximately 16% of nosocomial UTIs. Enterococcal bacteremia is frequently associated

with metastatic abscesses in multiple organs and high mortality rates. Enterococci have also

been considered an important cause of endocarditis; they are estimated to account for about

20% of the cases of native valve bacterial endocarditis and for about 6 to 7% of prosthetic

valve endocarditis. Endocarditis remains among the most difficult-to-treat enterococcal

infections because of limitations on bactericidal antimicrobial therapy for enterococcal

infections, especially when caused by vancomycin-resistant enterococci (VRE). Intraabdominal

and pelvic infections are also commonly associated with enterococci. However,

cultures from patients with peritonitis, intra-abdominal or pelvic abscesses, biliary tract

infections, surgical site infections, and endomyometritis are frequently polymicrobial, and the

role of enterococci in these settings remains controversial. The significance of isolates from

some of these sites, then, should be carefully evaluated before clinical decisions are made.

There is also a growing concern about the role of the enterococci in endodontic and implantand

medical device-associated infections (2, 117). Infections of the respiratory tract or the

central nervous system, as well as otitis, sinusitis, septic arthritis, and endophthalmitis, may

occur but are rare.

E. faecalis is usually the enterococcal species most frequently isolated from human clinical

specimens, representing 80 to 90% of the isolates, followed by E. faecium, which is found in

5 to 10% of enterococcal infections (8, 32, 40, 70). However, the ratios of isolation of the

different enterococcal species can vary according to each setting and can be affected by a

number of aspects, including the increasing dissemination of outbreak-related strains, such

as vancomycin-resistant E. faecium (42, 49, 53, 105). A trend for a progressive decline in

the ratio of E. faecalis to E. faecium seems to be particularly evident among bloodstream

isolates (42, 49, 53). The other enterococcal species are identified less frequently, even

though clusters of infections associated with E. casseliflavus (75), E. gallinarum (19, 67, 76),

and E. raffinosus (55, 113) have been reported. Several of the other enterococcal species,

including E. avium, E. caccae, E. cecorum, E. dispar, E. durans, E. gallinarum, E. gilvus, “E.

hawaiiensis,” E. hirae, E. italicus, E. malodoratus, E. mundtii, E. pallens, E.

pseudoavium, and E. sanguinicola, have also been isolated from human sources.

Although the enterococci can cause human infections in the community and in the hospital,

these microorganisms began to be recognized with increasing frequency as common causes

of hospital-acquired infections in the late 1970s, paralleling the increasing resistance to most

currently used antimicrobial agents. A major impact on the incidence and epidemiology of

enterococcal infections was noted after the first reports on the occurrence and epidemic

increase of VRE in hospitals in the United States. By 1993, the rates of VRE had already

increased 34-fold in intensive care units of U.S. hospitals (12). The percentage of VRE

isolates reported by U.S. hospitals increased from 0.3% in 1989 to over 25% of all isolates in

1999 (73). Since then, the enterococci have usually been listed as the second or third most

frequent nosocomial pathogen isolated from intensive care unit patients in the United States,

depending on the type of infection (42, 74, 84). In the 2006–2007 report from the National

Healthcare Safety Network at the Centers for Disease Control and Prevention (CDC), the

enterococci were listed as the third most common antimicrobial-resistant pathogen asso

ciated with healthcare-associated infections, accounting for 12% of the them (42).

Considering both the Surveillance and Control of Pathogens of Epidemiological Importance

(SCOPE) and the Antimicrobial Resistance Surveillance Program (SENTRY) databases, about

2% of E. faecalis and 60% of E. faecium isolates recovered from the bloodstream were

resistant to vancomycin (4). The occurrence and spread of VRE have now reached a more

global dimension. According to the European Antimicrobial Resistance Surveillance System

(www.earss.rivm.nl) data, the prevalence of VRE in nosocomial enterococcal bacteremia is

already ranging from 5 to 30% in several European countries (112). VRE are also

encountered at different rates in several other parts of the world, including South America,

Asia, and Australia, illustrating the pandemic spread of hospital-associated VRE (114).

Hospitalized patients with gastrointestinal carriage of VRE appear to be the major reservoir

of the organism, and once colonized, the patients remain so for weeks or months. Thus, as

colonized patients leave the hospital, the possibility that transmission might occur in the

community cannot be discounted. VRE can be disseminated by direct patient-to-patient

contact or indirectly via transient carriage on healthcare workers’ hands; contaminated

medical instruments, such as glucose meters, blood pressure cuffs, electronic thermometers,

and electrocardiogram monitors and wires; and environmental surfaces, such as patient

gowns and linens, beds, bedside rails, overbed tables, floors, door knobs, and wash basins

(56, 118).

As a response to the rising rates of VRE colonization and infection in U.S. hospitals, the

CDC’s Hospital Infection Control Practices Advisory Committee established guidelines with

recommendations for preventing the spread of VRE (44). These include prudent vancomycin

use, implementation of surveillance procedures for early detection of VRE, and infection

control procedures to limit cross-contamination, such as isolation precautions, hand washing,

and education about transmission of VRE.

Although only a small percentage of colonized patients develop serious systemic enterococcal

infections, intestinal colonization with VRE has been clearly associated with subsequent VRE

infections. However, in certain specific clinical situations, such as liver transplant recipients,

patients on chronic hemodialysis, and oncology patients, particularly those with

hematological malignancies, VRE-colonized patients appear to be at increased risk for

developing serious enterococcal infections (2, 16, 118). This underscores the importance of

active surveillance in high-risk patient groups to prevent transmission and outbreaks.

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

The standard methods for collecting blood, urine, wound secretions, and other secretions or

swab specimens suspected of harboring enterococci are adequate (see chapter 16). No

special methods or procedures are usually necessary for transport and storage of clinical

specimens containing enterococci because these microorganisms are easily recovered and

are relatively resistant to environmental changes and adverse conditions. Transport can be

performed on almost any transport medium or on swabs that are kept dry. Like most clinical

samples, the material should be cultured as soon as possible.

Enterococcal strains can be stored indefinitely when lyophilized. In our experience, cultures

frozen at −70°C or less can be stored for several years as heavy cell suspensions made

directly in defibrinated sheep or rabbit blood or in a skim milk (10%) solution containing

glycerol (10%). These are the preferable methods for preservation of enterococcal strains.

Cultures can also be preserved for many years at -20°C in other cryopreservative media

commonly used for maintenance of bacteria. Most strains of enterococci can survive for

several months at 4°C on agar slants prepared with ordinary agar bases, such as brain heart

infusion agar and Trypticase soy agar. Certain of the less well known species, however, are

not as resistant to adverse conditions and may not survive long if more adequate

preservation procedures are not used.

DIRECT EXAMINATION Back to top

The direct microscopic examination of Gram-stained smears of normally sterile clinical

specimens, such as blood, may be useful for the diagnosis of enterococcal infections. Direct

examination of certain nonsterile specimens may also be informative but should not be

overemphasized. In any case, only a presumptive report of the “presence of gram-positive

cocci” should be made, as microscopy by itself cannot differentiate the enterococci from most

of the other gram-positive cocci. Culture and appropriate identification techniques should be

performed for confirmation.

As the occurrence of VRE continues to represent an important problem worldwide, hospitals

are encouraged to implement surveillance programs for VRE detection. In an attempt to

overcome the inherent limitations of the culture-based methods of detection (discussed in

“Isolation Procedures” below), conventional PCR and real-time PCR-based methods have

been developed and evaluated for direct detection of these microorganisms in clinical and

surveillance specimens (3, 29, 63, 79, 86, 90, 92, 116). Commercially available molecular

tests that have been approved by the U.S. Food and Drug Administration (FDA) for VRE

screening directly from rectal swabs include the BD GeneOhm VanR assay (BD GeneOhm,

San Diego, CA) (92) and the Xpert vanA assay (Cepheid, Sunnyvale, CA). The

LightCycler vanA/vanB detection assay (Roche Diagnostics, Basel, Switzerland) has also been

evaluated (29, 90, 116), but it has not been licensed for use in the United States yet.

Different investigators have developed assays independently (3, 86). All of these studies

have reported improved detection of VRE in rectal swabs and fecal specimens over

conventional culture techniques. It is important to note that most systems use different

primers for detection of the van genes and that sensitivities may vary (3, 63, 116).

Specificity is also a potential problem unless PCR controls for genus and/or species

identification are included, because van genes may be found in bacteria other than

enterococci (3, 63,116).

Assays for detection and identification of enterococci directly in blood samples have been

reported. An in-house real-time PCR assay for quantitative detection of E. faecalis DNA in

blood samples without prior cultivation has been proposed for the diagnosis of bacteremia

(82). The LightCycler SeptiFast Test (Roche Molecular Systems, Branchburg, NJ) is, to date,

the only multiplex real-time PCR assay licensed for use in the United States for the rapid

detection and identification of major pathogens involved in nosocomial bacteremia,

including E. faecalis and E. faecium, directly from whole blood (11). As the technology

evolves, these molecular methods may become widely available for the rapid and precise

detection of enterococci directly in clinical samples. However, further evaluation is needed to

determine the real impact of their use on laboratory diagnosis of invasive enterococcal

infections and on patient management.

ISOLATION PROCEDURES Back to top

The source of clinical specimens to be tested for the presence of enterococci influences the

type of medium needed for primary isolation. Clinical specimens from normally sterile body

sites can be plated directly onto a nonselective medium, such as Trypticase soy agar, brain

heart infusion agar, or other blood agar base containing 5% sheep, horse, or rabbit blood. In

general, strains of the most clinically relevant species grow well at 35 to 37°C and do not

require an atmosphere containing increased levels of CO2, although some strains grow better

in this atmosphere. Samples for blood culture can be inoculated into conventional blood

culture systems. For clinical specimens obtained from nonsterile sites, especially those

heavily contaminated with gram-negative bacteria, use of selective media is a good option

for primary isolation. Many different media have been devised for selective isolation of

enterococci, but none has been proven to be specific. Furthermore, not all enterococcal

species grow on these selective media. Most of these media contain sodium azide, bile salts,

and/or antibiotics as selective components and esculin or tetrazolium as indicator

substances. Some of these media are supplied under different designations by various

manufacturers. The diversity of media used for the isolation of enterococci from various

sources has been reviewed (26). Consideration must also be given to whether or not an

enrichment broth should be employed when rectal or fecal samples are tested. The use of a

broth enrichment step in the primary isolation delays identification but increases the

recovery rates of enterococci (23, 77). Enrichment procedures are applied mainly to detect

VRE present in low numbers (48).

The increasing incidence of vancomycin resistance among the enterococci has raised the

importance of selective isolation of VRE. Early identification of infection or colonization by

VRE is recommended to prevent the spread of these microorganisms (44, 110). Current

recommendations for hospital infection control include VRE fecal surveillance cultures, but

the optimal methods for obtaining these cultures are still unclear. Different selective agar

and/or broth media formulations and several procedures have been employed for the

isolation of VRE from sources containing normal biota, such as stool samples and rectal or

perianal swab specimens (21, 26, 45, 77, 79, 86). Most of them are variations of selective

media differing with regard to the antimicrobial agents or the antimicrobial concentrations

used. However, no consensus has been established for medium base, vancomycin

concentration, or method of use. Although there is not a single generally accepted screening

method for isolation at this point, the use of a selective enrichment broth to enhance the

recovery of VRE seems to be a highly effective procedure. Enterococcosel broth (a bileesculin

azide [BEA] medium supplied by Becton Dickinson Microbiology Systems) has been

used in a number of studies as the base medium supplemented with different concentrations

of vancomycin, with 6 μg/ml being the most common concentration. A commercially

available medium, labeled BEAV and containing 6 μg/ml vancomycin (distributed by Remel

Inc., Lenexa, KS), has also been evaluated (59).

Media containing chromogenic substrates have also been proposed for the isolation and

presumptive identification of enterococci. The first of these media, CHROMagar orientation

(from Becton Dickinson Microbiology Systems), was promoted as an isolation and

identification medium for enterococci from urine (66). Another chromogenic medium, CPS

ID3 (bioMerieux, Marcy l’Etoile, France), has been made available (14) to do the same.

Several studies have employed another new chromogenic agar medium, ChromID VRE

(bioMerieux), on which E. faecium colonies appear purple and E. faecalis colonies appear

blue or blue-green (21, 23, 41, 59, 63). Thus, the two most common species of VRE are

identified on this chromogenic primary isolation medium. Results obtained by using ChromID

VRE medium have been considered more rapid (41) and more specific than those obtained

with BEA or BEAV-based media (21).

Culture-based screening methods for VRE may be especially demanding and can take several

days to complete, besides having variable degrees of sensitivity, which affects the timely

implementation of infection control procedures. Therefore, some microbiology laboratories

have recently considered the introduction of molecular techniques to detect VRE to facilitate

the rapid and accurate identification of these organisms and to improve sensitivity for

detecting this pathogen. However, most of the current approaches still require bacterial

growth in culture prior to detection, requiring 24 h or more to complete. The application of

methods for a more rapid detection of VRE directly from clinical samples is still an area of

major interest (see “Direct Examination” above).

IDENTIFICATION OF ENTEROCOCCUS SPECIES Back to top

Identification by Conventional Physiological Testing

Once it is established that an unknown catalase-negative gram-positive coccus is

an Enterococcus or closely related genus (see “Description of the Genus” above), the tests

(see reference 33 and chapters 3, 17, and18) listed in Table 1 can be used to identify the

species. The data presented in Table 1 for the phenotypic characteristics are derived from

conventional testing (except for β-galactosidase) performed at the

CDCStreptococcus laboratory (see http://www.cdc.gov/ncidod/biotech/strep/strepdoc/

index.htm). Most of the information presented in Table 1 for the most frequent species is

related to the phenotypic characteristics of isolates obtained from humans. Isolates from

nonhuman sources, even those belonging to well-known species, may have different results

for some tests. Species that have not been recovered from humans to date are also included

in Table 1 due to the possibility that they will be isolated from human sources in the future.

Enterococcal species can be initially separated into five physiological groups of species based

on acid formation from mannitol and sorbose and hydrolysis of arginine (Table 1).

Identification of enterococcal species by conventional tests is not rapid and may require

incubation of the tests for up to 10 days. However, the majority of the isolates recovered

from human sources can be identified after 2 days of incubation.

Group I includes nine species see Table 1 for details). “E. hawaiiensis” is a denomination that

has been proposed for the new species previously designated Enterococcus sp. nov. CDC

PNS-E3 (10). E. avium and E. raffinosus are the most relevant species in this group,

considering the association with human clinical sources. Group II comprises eight species.

The majority of the isolates recovered from human sources belong to species included in this

group. Atypical strains that do not hydrolyze arginine or do not form acid from mannitol have

been documented. Lactococcus sp. is also listed in this group because the phenotypic

characteristics of some strains can lead to misidentification as an Enterococcus. If nonmotile

variants of E. casseliflavus and E. gallinarum are encountered, production of acid from

methyl-α-D-glucopyranoside can be used to help in the identification of these species. E.

sanguinicola is the denomination proposed for the species provisionally

designated Enterococcus sp. nov. CDC PNS-E2 (9, 10). Group III consists of six species.

Three of these species(E. durans, E. ratti, and E. villorum) have very similar phenotypic

profiles in the tests listed in Table 1. Reactions in litmus milk and hydrolysis of hippurate, in

addition to the reactions listed in Table 1, may also be used to help differentiate the species.

In litmus milk, E. durans forms acid and clot, E. villorum forms acid but no clot, and E.

ratti does not form acid or clot. E. durans hydrolyzes hippurate, while E. villorum does not. E.

ratti is variable in the hippurate hydrolysis test. The other members of this group are easily

identified by the reactions shown in Table 1. Uncommon mannitol-negative variant strains

of E. faecalis and E. faecium resemble species in this group. However, E. faecalis strains are

positive in the pyruvate test but not for acid formation from arabinose, raffinose, or sucrose,

and E. faecium variant strains form acid from arabinose. Group IV includes eight species. E.

caccae and E. cecorum are the only species in this group that have been isolated from

human sources to date. Group V comprises six species. Variant strains of E. casseliflavus, E.

gallinarum,and E. faecalis that fail to hydrolyze arginine resemble the microorganisms

included in this group. However, these variant strains have characteristics similar to the

strains that hydrolyze arginine and can be differentiated by the same phenotypic

tests. Vagococcus fluvialis is listed here because the phenotypic characteristics of this species

are very similar to those of the genus Enterococcus, and some strains may be identified as

enterococci (98). E. italicus corresponds to the new species previously

designated Enterococcussp. nov. CDC PNS-E1 (9, 10, 35). Among this group, only a single

strain of E. italicus has been isolated from human sources (9).

Identification by Commercial Systems

There are several commercially available miniaturized, manual, semiautomated, and

automated identification systems that may be an alternative to conventional testing for the

phenotypic identification of enterococcal species in routine diagnostic laboratories. Since

their introduction, these systems have been updated to improve their performance

characteristics and expand their identification capabilities as investigators have become more

aware of inaccuracies (22, 38, 50, 113). In general, these systems are reliable for the

identification of the most common species: E. faecalis and, to a lesser extent, E. faecium.

Precise identification of other species by most systems depends on additional testing,

although improvements have been observed with updated formats and databases.

Commercial systems available for the identification of enterococcal species include the API

20S and the API Rapid ID32 STREP systems (bioMerieux Vitek, Inc., Hazelwood, MO), the

Crystal Gram-Positive and the Crystal Rapid Gram-Positive identification systems (Becton

Dickinson Microbiology Systems, Sparks, MD), the Gram Positive Identification Card of the

Vitek system (bioMerieux), the Gram-Positive Identification panel of the MicroScan

Walk/Away system (Dade MicroScan, West Sacramento, CA), and the BD Phoenix Automated

Microbiology system (Becton Dickinson Microbiology Systems). Overall, a large proportion of

enterococcal isolates recovered from human sources can be accurately identified by most of

the commercial systems now available; however, the accuracy is dependent on the

distribution of species found in each specific setting. Difficulties in the identification of a

variety of enterococcal species, including E. faecalis and E. faecium, are still being reported

(7, 50, 81). Strict adherence to the instructions provided by the manufacturer, including the

base of the media used to grow strains for testing, is of paramount importance. Our own

unpublished experimental results indicate that differences in growth conditions can lead to

variation in the results of some tests, interfering with the accuracy of the identification.

Identification of an unusual enterococcal species by a commercial system should be

confirmed by a reference method before being reported.

Identification by Molecular Methods

Molecular methods, such as DNA-DNA hybridization and sequencing of the 16S rRNA genes,

have been used primarily for taxonomic purposes in reference or research laboratories. In

the past 2 decades, however, the application of molecular techniques for the rapid

identification of Enterococcus species has expanded dramatically for use in clinical

microbiology laboratories.

A variety of molecular procedures have been proposed for the identification of enterococcal

species, as previously reviewed and summarized (27, 31). Many of these molecular

procedures have been performed in only a few laboratories and have not been evaluated for

all of the species of Enterococcus. Most of them are potentially applicable to all enterococcal

species, and others are species specific. Several of these methods deserve consideration for

expanded testing and future improvements, as they represent promising adjunct tools for a

more rapid and precise identification of enterococcal species.

Among the molecular techniques proposed to identify the different enterococcal species,

sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell protein

(WCP) profiles and sequencing of the 16S rRNA genes have been more extensively evaluated

in reference laboratories. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis

of WCP profiles was shown to be a reliable tool for the differentiation and identification of

typical and atypical Enterococcus strains, since WCP profiles are species specific

(10, 31, 68, 99). Table 1, which depicts the phenotypic characteristics of

the Enterococcus species discussed in this chapter, is based on correlations between the WCP

profiles and the phenotypic tests, in conjunction with DNA-DNA reassociation experiments

and 16S DNA sequencing. Sequencing of the 16S rRNA gene is currently the most frequently

used among the nucleic acid-based methods for identification of enterococcal species. It has

been performed for all species of enterococci, and the sequences are available for

comparison purposes via public databanks of nucleotide sequences, such as GenBank.

Comparisons can be made by using one of several sequence-comparing software packages,

many of which are available for public access. Figure 1 depicts a dendrogram generated by

comparison of 16S rDNA sequences of the type strains of the species included in the

genus Enterococcus. However, clear-cut differentiation is not always obtained for all

enterococcal species, since some of them differ by only 2 or 3 bases over the approximately

1,500-base span of the 16S rRNA gene. Therefore, this procedure should not be used alone,

but together with phenotypic characterization and other alternative molecular methods, it

can be an important tool to establish enterococcal species identity.

PCR-based techniques have also been a focus of major interest for rapid and accurate

enterococcal identification. Schemes for amplification of the ddl (24, 50) or sodA (50) genes

have already been designed for several enterococcal species and constitute convenient

alternative approaches for rapid routine identification.

The use of molecular methods for the rapid identification of enterococci directly in routine

blood cultures has been investigated. The usefulness of the commercially available DNA

probe kit AccuProbe (Gen-Probe, Inc.) for the identification of enterococci directly in blood

cultures has been demonstrated (61), although this test has been validated by the

manufacturer only with the use of fresh growth from solid medium or from broth cultures. In

addition, fluorescence in situ hybridization (FISH) techniques have been developed and

evaluated for the identification of enterococci from positive blood culture bottles (34, 39).

One of these assays, the E. faecalis/other Enterococcus species PNA FISH (AdvanDx,

Woburn, MA), is a peptide nucleic acid FISH test that is FDA cleared (34). This assay has

been considered relatively easy to implement, leading to earlier identification

of Enterococcus species in comparison with conventional microbiological methods (34).

TYPING SYSTEMS Back to top

The increasing documentation of Enterococcus as a leading nosocomial pathogen frequently

resistant to several antimicrobial agents, as well as the evidence supporting the concept of

exogenous acquisition of enterococcal infections, has generated demand for strain typing and

epidemiological studies. Classic phenotypic methods used to investigate the diversity among

isolates of a given enterococcal species have frequently failed to adequately discriminate

among strains, and they have limited value in epidemiological studies. However, phenotypic

information in association with molecular data can constitute valuable information (27, 31).

The introduction of molecular techniques has substantially improved the ability to

discriminate enterococcal isolates and has provided critical insights into the epidemiology of

the enterococci. By using molecular typing approaches, it was possible to demonstrate the

exogenous acquisition of enterococcal strains by direct and indirect contact among patients,

breaking the traditional conception that enterococcal infections were endogenous in nature.

Intrahospital transmission and interhospital spread have been extensively documented for

antimicrobial-resistant enterococci, especially VRE (31, 37, 62, 107). In addition to

epidemiological investigations, some of the molecular typing techniques are now used to

trace the dissemination of enterococci in different environments and hosts and the evolution

of multidrug-resistant strains, greatly expanding our understanding of enterococcal

epidemiology, population structure, antimicrobial resistance, and virulence. Emergence and

global dispersion of certain epidemic enterococcal clonal complexes have been identified

(57, 64, 91, 105, 114, 115).

Several molecular methods have been proposed to type enterococcal isolates, as previously

reviewed (27,31). In addition to differences in complexity and costs, these methods vary in

their reproducibility and discriminatory power. Overall, there is not a single definitive typing

technique for enterococci, so a strong match among the results of different typing

techniques, particularly those based on different genomic polymorphisms, should be used as

indicative of high relatedness. Among these techniques, analysis of chromosomal DNA

restriction profiles by pulsed-field gel electrophoresis (PFGE) has been extensively evaluated

for epidemiological characterization of enterococcal outbreaks, showing improved

discrimination and allowing the identification of clonal complexes that predominate among

multidrug-resistant enterococci, mainly high-level resistance (HLR) to aminoglycosides and

VRE (31, 37, 40, 72). SmaI is the restriction enzyme most frequently used to digest

enterococcal DNA, although the usefulness of others, such as ApaI and SfiI, has also been

reported (31). PFGE is possibly the typing method most commonly used in clinical

microbiology settings, and it is considered by many investigators to be the gold standard for

the epidemiological analysis of enterococcal outbreaks. Several protocols for performing

PFGE typing of enterococcal strains have been published. However, the development of

standardized protocols for execution, interpretation, and nomenclature as a result of

collaborative studies is still needed in order to allow for interlaboratory data exchange and

comparisons. On the other hand, although PFGE is quite discriminatory, epidemiological

interpretation of PFGE profiles is not always clear-cut. The occurrence of genetic events can

be associated with substantial changes in the PFGE profiles, leading to problems in clonality

assessment (54). Due to the possibility of such inconsistencies in DNA banding patterns of

enterococci, PFGE is recommended mostly for the purpose of evaluating the genetic

relatedness and tracing the transmission of strains that are associated in time and location,

as usefulness for long-term epidemiological studies may be limited. The use of PFGE in

conjunction with at least one additional typing technique, or independent PFGE analysis using

different restriction enzymes, is highly recommended to help in clarifying epidemiological

interpretation. General principles proposed for the interpretation of molecular typing data

based on fragment differences are usually applied to interpret PFGE profiles obtained for

enterococcal strains (31, 101). Well-characterized control strains should be evaluated along

with unknown isolates. For that purpose, two reference strains, E. faecalisOG1RF (ATCC

47077) and E. faecium GE1 (ATCC 51558), have been proposed (101).

Two other robust molecular techniques became available more recently, named multilocus

sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA).

These techniques circumvent the difficulties in data exchange between different laboratories

by generating information that is suitable for the development of Web-based databases.

MLST is based on identifying alleles after sequencing of internal fragments of a number of

selected housekeeping genes, resulting in a numeric allelic profile. Each profile is assigned a

sequence type. Internet sites with the possibility for data exchange

(www.mlst.net andwww.pubMLST.org), which contain MLST schemes for E. faecium (43)

and E. faecalis (85), have been de veloped. Application of MLST has revealed the occurrence

of host-specific genogroups of E. faecium and allowed the recognition of a hospitaladapted

E. faecium subpopulation (initially named the C1 lineage) that seems to

predominate in several geographic areas (57, 64, 105, 107, 115). This hospital-adapted

lineage was later renamed clonal complex 17 (CC17) and classified as an example of the socalled

high-risk enterococcal complexes. Major clonal complexes have also been identified

among E. faecalis isolates (37, 64, 85) by using MLST. Two simultaneously published studies

described the development of MLVA typing schemes for E. faecalis(102) and E.

faecium (104). MLVA is based on variation in variable-number tandem repeat loci dispersed

over the enterococcal genome. For each variable-number tandem repeat locus, the number

of repeats is determined by PCR using primers based on the conserved flanking regions of

the tandem repeats. PCR products are separated on agarose gels, and the band size is

determined by the number of repeats. These numbers together result in an MLVA profile, and

each profile is assigned an MLVA type. An internet site has been developed

(www.mlva.umcutrecht.nl) to serve as a database and also for the submission of MLVA

profiles to assign MLVA types. Comparative studies indicate that both techniques can achieve

high degrees of discrimination between isolates and have comparable discriminatory power

(103) that appears to be similar to that of PFGE- based typing (37, 85, 104). In contrast to

the overt advantages (being reproducible, portable, highly discriminatory, and

unambiguous), MLST is time-consuming, expensive, and still limited to laboratories that have

facilities for both PCR and sequencing, while MLVA requires PCR and basic electrophoresis

facilities. Thus, MLVA may be a quicker and less expensive alternative to MLST for clinical

laboratory settings.

SEROLOGIC TESTS Back to top

Serologic tests for detecting antibody responses to different enterococcal antigens have been

proposed (15,93). However, their usefulness in the clinical laboratory setting for the

diagnosis of enterococcal infections has not been demonstrated.

ANTIMICROBIAL SUSCEPTIBILITIES Back to top

Resistance to several commonly used antimicrobial agents is a remarkable characteristic of

most enterococcal species. Moreover, most of the information about this is based on studies

with E. faecalis and E. faecium, the two species that are more frequently associated with

human infections. Antimicrobial resistance can be classified as either intrinsic or acquired.

Intrinsic resistance is related to inherent or natural chromosomally encoded characteristics

present in all or most of the enterococci. Furthermore, certain specific mechanisms of

intrinsic resistance to some antimicrobial agents are typically associated with a particular

enterococcal species or groups of species. In contrast, the occurrence of acquired resistance

is more variable, resulting from either mutations in existing DNA or acquisition of new

genetic determinants carried in plasmids or transposons (20,47, 51, 58, 71). Enterococcal

intrinsic resistance involves several antimicrobial agents, particularly two major groups: the

aminoglycosides and the β-lactams. Because of the poor activity of several antimicrobial

agents against enterococci due to intrinsic resistance, the recommended therapy for serious

infections (i.e., endocarditis, meningitis, and other systemic infections), especially in

immunocompromised patients, includes a combination of a cell wall-active agent, such as a

β-lactam (usually penicillin or ampicillin) or vancomycin, and an aminoglycoside (usually

gentamicin or streptomycin). These combinations overcome the intrinsic resistance exhibited

by the enterococci, and a synergistic bactericidal effect is generally achieved, since the

intracellular penetration of the aminoglycoside is facilitated by the cell wall-active agent.

In addition to the intrinsic resistance traits, enterococci have acquired different genetic

determinants that confer resistance to several classes of antimicrobial agents, including

chloramphenicol, tetracyclines, macrolides, lincosamides and streptogramins,

aminoglycosides, β-lactams, glycopeptides, quinolones, and even some of the more recently

available drugs, such as linezolid, daptomycin, and quinupristin- dalfopristin (51, 60, 118).

During the past several decades, the occurrence of acquired antimicrobial resistance among

enterococci, especially HLR to aminoglycosides and β-lactams and resistance to

glycopeptides (especially vancomycin), has been increasingly reported. Isolates that are

resistant to the cell wall-active agent or have HLR to aminoglycosides are resistant to the

synergistic effects of combination therapy and constitute a problem of peculiar importance.

Therefore, the detection of resistance to these groups of antimicrobial agents is critical in

order to predict the likelihood of synergy by using antimicrobial combinations as a

therapeutic strategy. Enterococcal isolates exhibiting HLR to aminoglycosides have been

described with increasing frequencies (40, 47, 51, 70, 71) and are now present in large

proportions in several geographic areas. Such strains frequently have MICs of >2,000 μg/ml

and cannot be detected by diffusion tests with conventional disks. Special tests using highcontent

gentamicin and streptomycin disks, as well as a single dilution method, were

developed to screen for this type of resistance (see references 17 and 31 and

chapters 68 and 70). Strains exhibiting HLR to penicillin and ampicillin due to altered

penicillin-binding proteins have also disseminated widely (47, 51, 71), while strains

producing β-lactamase have been rarely identified (40, 71). Results of susceptibility testing

with ampicillin may be used to predict susceptibility to amoxicillin, amoxicillin- clavulanate,

ampicillin-sulbactam, piperacillin, and piperacillin-tazobactam among non-β- lactamaseproducing

enterococci. Ampicillin susceptibility can also be used to predict imipenem

susceptibility, providing the species is E. faecalis (17, 111). However, enterococcal

susceptibility to penicillin cannot be predicted on the basis of ampicillin testing results. If

penicillin results are needed, testing of penicillin is required. On the other hand, enterococci

susceptible to penicillin are predictably susceptible to ampicillin and the other β-lactams

mentioned above. Penicillin and ampicillin resistance due to β- lactamase production is not

reliably detected by routine disk or dilution methods but is detected by using a nitrocefinbased

β-lactamase test (17, 31).

The emergence of vancomycin resistance as a therapeutic problem in enterococcal strains

was first documented in western Europe and in the United States (52, 58, 106). Thereafter,

the isolation of VRE has been continuously reported, showing epidemic proportions in diverse

geographic locations (13, 47, 51). VRE strains have been classified according to phenotypic

and genotypic features (Table 2). Seven types of glycopeptide resistance have already been

described among enterococci. Each type is associated with different genetic elements, some

of which, in turn, can be divided into subtypes. Three of them are the most common: the

VanA phenotype, encoded by the vanA gene, with inducible HLR to vancomycin as well as to

teicoplanin; the VanB phenotype, encoded by the vanB genes, with variable (moderate to

high) levels of inducible resistance to vancomycin only; and the VanC phenotype, encoded by

the vanC genes, conferring constitutive low-level resistance to vancomycin. VanA and VanB

are considered the most clinically relevant phenotypes and are usually associated with E.

faecium and E. faecalis isolates, while VanC resistance is an intrinsic characteristic of E.

gallinarum (vanC1 genotype) and E. casseliflavus (vanC2 to vanC4 genotypes)

(13,16, 20, 47, 109). The additional types of glycopeptide resistance encoded by

the vanD, vanE, vanG, and vanL(6, 20, 65) genes seem to occur rarely among enterococci.

Furthermore, the isolation of vancomycin-dependent (96) and of vancomycin-heteroresistant

(1) enterococcal strains from clinically significant infections, although sporadically reported,

may also represent additional serious threats for the treatment and control of enterococcal

infections.

While in vitro methods for detecting vancomycin resistance are discussed in detail in

references 17 and 31, as well as in chapters 67 and 68, some aspects regarding vanCcontaining

species (i.e., E. gallinarum and E. casseliflavus) need to be emphasized.

Resistance associated with vanC genotypes is not usually detected by disk diffusion, but

VanC strains frequently grow on vancomycin agar screen tests. Because of the low clinical

significance of the VanC resistance, the implications of susceptibility testing for patient

management may be unclear. However, the need to differentiate VanA or VanB strains from

VanC strains is quite evident for therapeutic, infection control, and surveillance reasons.

Because growth on vancomycin agar screening fails to help with this important distinction,

species identification is necessary. VanC resistance is yet to be described in E. faecalis or E.

faecium, so that growth on the vancomycin screening agar test by either of these species is

likely due to the presence of the vanA or vanB genes. Although rare, the occurrence of the

other kinds of vancomycin resistance may also be considered. Additionally, simultaneous

occurrence of the vanA gene has been increasingly reported for vanC-carrying species,

especially E. gallinarum, so that identification of a species that usually harbors only VanC

resistance does not completely rule out moderate to high levels of vancomycin resistance

(8, 62, 76). In this regard, determining vancomycin MICs is useful, as VanC resistance

frequently results in MICs <32 μg/ml, whereas VanA and VanB usually result in MICs >32

μg/ml. In such cases, determination of the genetic elements associated with vancomycin

resistance has important epidemiological and infection control implications. Also, resistance

to other agents such as ampicillin and aminoglycosides is uncommon among VanC isolates.

Because of limited alternatives, chloramphenicol, erythromycin, tetracycline (or doxycycline

or minocycline), and rifampin may be tested for VRE. Testing of quinupristin-dalfopristin,

linezolid, and daptomycin is recommended when reporting on vancomycin-resistant E.

faecium.

Molecular methods (see chapter 74) have been used to detect specific antimicrobial

resistance genes and have substantially contributed to the understanding of the spread of

acquired resistance among enterococci, especially resistance to vancomycin. However,

because of their high specificity, molecular methods do not detect antimicrobial resistance

due to mechanisms not targeted by the testing, including emerging resistance mechanisms.

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

The diversity and species specificity of acquired antimicrobial resistance traits among

enterococcal isolates created an additional need for accurate identification at the species

level and for in vitro evaluation of susceptibility to antimicrobial agents. The significance of a

particular enterococcal isolate is a major factor in determining when antimicrobial testing

should be done. Once the need to test a particular isolate has been established, selection of

the appropriate antimicrobial agents for testing must be considered on the basis of the site of

infection. Testing of antimicrobial agents to which enterococci are intrinsically resistant is

contraindicated. The drugs that should not be reported include aminoglycosides at standard

concentrations, cephalosporins, clindamycin, and trimethoprim-sulfamethoxazole: they may

appear active for enterococci in vitro but are not effective clinically, and isolates should not

be reported as susceptible. Updated guidelines (17) for the selection of antimicrobial agents

should be followed for routine testing and reporting. The in vitro methods for detecting

antimicrobial resistance in enterococcal isolates were reviewed and summarized (31) and are

also discussed in chapters 68 and 70.

As already mentioned, synergy testing should be done with any enterococcal isolate

implicated in infections for which combination therapy is indicated, e.g., from systemic

infections. Enterococci are also frequently encountered in polymicrobial infections associated

with the gastrointestinal tract or superficial wounds of hospitalized patients. Their pathogenic

significance in such settings is uncertain, but susceptibility testing is warranted when

predominant or heavy growth is observed (69). Testing of E. faecalis isolates from lower

urinary tract infections is optional, as these infections usually respond to therapy with

ampicillin or nitrofurantoin. However, many hospital infection control programs require

routine testing as part of surveillance programs for VRE. For those instances when testing a

urinary tract isolate is appropriate, ciprofloxacin, fosfomycin, levofloxacin, norfloxacin, and

tetracycline could be selected, in addition to nitrofurantoin and ampicillin (17, 47, 60, 62). In

cases of treatment failure, testing is always warranted.