EPIDEMIOLOGY AND TRANSMISSION
Staphylococcus and Related Genera
The major habitats of most staphylococcal species
are the skin and mucous membranes of
mammals and birds. S. aureus subsp. aureus
is considered to be the most important human
pathogen among staphylococci, followed by S.
epidermidis, S. haemolyticus, and S.
saprophyticus subsp. saprophyticus. S. auricularis, S. capitissubsp. capitis
and urealyticus, S.
caprae, S.cohnii subsp. cohnii and urealyticus, S.
hominis subsp. hominis andnovobiosepticus, S. lugdunensis, S. pasteuri,
S. pettenkoferi, S.
saccharolyticus, S. schleiferi subsp. schleiferi, S. simiae, S. simulans, S.
warneri, and S.
xylosus are also encountered in human specimens (164).
These species are found mainly as
part of the resident microbiota. For references
concerning species description, see “List of
Prokaryotic Names with Standing in Nomenclature” (http://www.bacterio.cict.fr/).
S. aureus and Other Coagulase-Positive Species
As determined in longitudinal studies, three types
of S. aureus nasal carriers have been
historically distinguished: persistent carriers
(10 to 35%, carrying one strain over time),
intermittent carriers (20 to 75%, carrying
different strains), and noncarriers (5 to 50%)
(203). Since intermittent carriers and noncarriers
share similar S. aureus nasal elimination
kinetics, it was recently proposed that there are
only two types of nasal carriers: persistent
carriers and others (189).
From the vestibulum nasi, S. aureus can be transferred to skin
and other body areas. Health care workers have a
high S. aureus nasal carriage rate (50% to
90%) as do patients with insulin-dependent
diabetes mellitus, patients receiving long-term
hemodialysis, and users of illegal intravenous
drugs (37). The vaginal carriage rate in adult
premenopausal women is about 10% (119).
The intertriginous skin folds, the axillae, and the
perineum are also found to be regularly colonized.
Of particular interest, nasal colonization
plays a crucial role as a source of invasive
infections (107, 195, 202).
The population of S. aureus presents a
highly clonal structure dominated by approximately a
dozen major clonal complexes comprising hundreds
of clonal lineages or sequence types
(58). The S. aureus populations are also
divided into four distinct groups based
on agr (accessory gene regulator system)
allelic variation (135).
Within health care facilities, S. aureus strains
are transmitted from patient to patient
primarily via hand carriage of medical personnel.
This is of utmost importance for the
transmission of methicillin-resistant S. aureus
(MRSA) strains (MRSA strains are further
discussed in “A ntimicrobial Susceptibilities”
below). The role of the external environment is
less important, except for certain areas such as
intensive care units and burn units.
Colonized or infected health care workers may act
as a reservoir (2). MRSA strains
circulating in livestock and found in meat
production differ from companion animal strains.
For companion animals, MRSA acquisition is
primarily a humanosis, in contrast to the newly
emerging livestock-associated MRSA and
methicillin-susceptibleS. aureus (MSSA) strains
such as ST398, presenting a genuine zoonotic risk
(130).
Staphylococcus aureus subsp. anaerobius has been recovered from
subcutaneous abscesses
of sheep (45). So far, it has not been
isolated from human clinical specimens. S.
intermedius, the recently delineated species S. pseudintermedius, and
two clusters of S.
delphini are closely related coagulase-positive species commonly recovered
from carnivores,
other mammals (e.g., horses and cats), and birds.
Since it is very difficult to differentiate
among these species, it is very likely that
previously reported S. intermedius isolates with
variant biotypes might have included S.
delphini and, in particular, S.
pseudintermedius (163). S. pseudintermediusmight be the
predominant coagulasepositive
Staphylococcus species recovered from normal and infected canine skin (48).
Further coagulase-positive animal-adapted
staphylococci are S.
schleiferi subsp. coagulans, S. felis, and S. lutrae.
Coagulase-Negative Staphylococci
In humans, S. epidermidis is the most
frequently recovered staphylococcal species colonizing
the body surface, where it is particularly
prevalent on moist areas such as the axillae,
inguinal and perineal area, anterior nares, and
toe webs. S. auricularis is part of the healthy
human external auditory canal microbiota
colonizing exclusively this region; S. capitis is
found surrounding the sebaceous glands on the
forehead and scalp following puberty; S.
haemolyticus and S. hominis are preferentially isolated from axillae and
pubic areas high in
apocrine glands; and S. saprophyticus subsp.
saprophyticus is frequently colonizing the
rectum and the genitourinary tract of young women
(106, 159, 164). S. lugdunensis is
frequently found on the lower extremities and on
the groin (28). However, these species may
be found occasionally on other body sites.
S. sciuri and S. xylosus are commensals of the skin and the mucous
membranes of many
animals and, occasionally, of humans. Both species
are also found in food; S.
xylosus represents one of the major starter cultures used for meat
fermentation. S.
sciuri subsp. carnaticus is recovered mainly from bovine hosts,
and subsp. rodentium is
found mainly in rodents. S. kloosii, S. equorum
subsp. equorum, and S. gallinarum are found
on several mammals and food products. S.
chromogenes, S. hyicus, and S. lentus are
common residents of cloven-hoofed animals and, in
addition, may be isolated from their food
products. S. vitulinus (syn. S.
pulvereri) is found preferentially on horses and whales. S.
arlettae is found on mammals and birds; S. nepalensis has been
isolated from Himalayan
goats; S. muscae is described on flies. S.
carnosus subsp. carnosus and utilis, S. condimenti,
S. equorum subsp. linens, S. fleurettii, S. piscifermentans, and S.
succinus subsp. casei have
been associated with fermented food and dairy
products;
and S. succinus subsp. succinus was
isolated from an amber fragment. However, the
complete and/or true natural habitat for many of
these species is still unclear.
Macrococcus, Salinicoccus,
Jeotgalicoccus, and Nosocomiicoccus
The Macrococcus genus comprises four
hoofed-animal-adapted species including M.
caseolyticus, first described as S. caseolyticus (102).
This species is found on food such as
sausages and meat products. The
halotolerant/halophilic Jeotgalicoccus and Salinicoccus
species are recovered from fermented
seafood and salted fish or found in saline and
desert soil or salt mines. Thus far, the only
reported recovery ofNosocomiicoccus ampullae has
been isolation from surfaces of bottles of
saline solution used in wound cleansing (4).
Micrococcaceae and Dermacoccaceae
The skin of humans and other mammals is the
primary habitat for
most Micrococcaceae and Dermacoccaceaeisolated
from clinical specimens. Cutaneous
populations of micrococci are carried by most
people (ca. 96%) with M. luteus as the most
frequent species followed by K. varians (104).
R. mucilaginosa is probably a normal
inhabitant of the mouth and upper respiratory
tract. Animal and dairy products may be
considered secondary sources of micrococci. Many
of the recently discovered members of
the Micrococcaceae and Dermacoccaceae are
associated with different environmental
habitats.
Alloiococcus
Recently, a high incidence of A. otitis in
the outer ear canal of healthy persons was
demonstrated, suggesting that alloiococci are part
of the normal bacterial microbiota (182).
CLINICAL SIGNIFICANCE Back to top
Staphylococcus
Many staphylococcal species are classical
opportunists colonizing skin and mucous
membranes but may become pathogenic in a species-
and strain-dependent manner
following breaks in the cutaneous epithelial
barrier through trauma or medical interventions.
The recovery of a staphylococcal isolate always
requires assessment of clinical significance to
determine whether it is a contaminant, colonizer,
or pathogen.
S. aureus is the clinically most important species, capable of causing a
wide range of human
and animal diseases. S. aureus possesses an
extensive, often redundant and overlapping
arsenal of virulence factors, such as adhesins,
enzymes, and toxins, and has various
strategies to evade the host immune response. In
addition, the pathogen has become
resistant to many of the therapeutic agents
available. National Nosocomial Infection
Surveillance and National Healthcare Safety
Network data indicate that S. aureus is the most
common cause of nosocomial pneumonia and skin and
soft tissue infections. S. aureus is
second only to CoNS as a cause of primary
bacteremia in hospitals (84, 205).
Disease entities caused by S. aureus can be
broadly divided into toxin-mediated diseases
and suppurative infections comprising skin and
soft tissue infections (SSTIs), organic and
systemic infections, and foreign-body-related
infections (FBRIs). The spectrum of SSTIs
ranges from superficial (impetigo, folliculitis,
furuncles/carbuncles, hydradenitis suppurativa,
pyoderma, and wound infections) to deep entities
(abscesses, mastitis, cellulitis, and
pyomyositis) to life-threatening necrotizing
fasciitis and myositis. SSTIs are the most
frequent infections associated with
community-acquired MRSA (CA-MRSA) with a single clone
(pulsed-field gel electrophoresis [PFGE] type USA
300; multilocus sequence typing [MLST]
type ST-8) being most prevalent (99).
Infection of deep sites may involve any body
compartments and organ systems resulting in
empyemas, osteomyelitis, arthritis,
endocarditis, pneumonia, otitis media, sinusitis,
mastoiditis, and parotitis. Any localizedS.
aureus infection can become invasive and lead to bacteremia. Systemic
infections comprise
primary and secondary bacteremia, meningitis, and
endocarditis. Bacteremia may be
complicated by metastatic foci (e.g., vertebral
osteomyelitis). Congenital or acquired defects
in host defense and the presence of foreign bodies
may predispose patients to serious
infections.
Besides an acute aggressive course, S. aureus may
also cause chronic, persistent, and
relapsing infections often due to a phenotypic
subpopulation designated small-colony
variants (SCVs) (147). SCVs of S. aureus and
other staphylococcal species (e.g., S.
epidermidis and S. lugdunensis) have been isolated from patients with
chronic osteomyelitis,
abscesses, and FBRIs as well as from cystic
fibrosis patients with chronic airway infection
(96, 167, 196).
Classical toxin-mediated diseases due to S.
aureus include the staphylococcal toxic shock
syndrome (TSS), staphylococcal food poisoning, and
staphylococcal scalded skin syndrome
(SSSS). TSS is associated with colonization by or
infection with an isolate of S. aureus that is
positive for TSS toxin-1 (TSST-1) or, less
frequently, for other members of the
staphylococcal pyrogenic toxin superantigen
(PTSAg) family (primarily staphylococcal
enterotoxin B or C). TSS is diagnosed on clinical
grounds characterized by high fever, rapidonset
hypotension, a diffuse erythematous rash that
becomes desquamating 1 to 2 weeks
after onset, and involvement of three or more
organ systems. After its initial description in
children, it was associated with menstruating
women who were using highly absorbent
tampons. While the incidence of menstrual TSS
decreased due to changes of the tampons’
absorbency and chemical composition, the frequency
of nonmenstrual TSS entities has
remained constant (78). Although commonly no
source of infection is confirmed,
nonmenstrual TSS is usually associated with focal
postoperative wound or soft tissue
infections.
Staphylococcal food poisoning is caused by
consumption of food contaminated with one or
more preformed, relatively heat-stable
enterotoxins. Nausea, vomiting, abdominal cramps,
and diarrhea occur 2 to 6 hours after food
ingestion. Symptoms usually subside 8 to 12
hours later.
SSSS is a type of bullous exfoliative dermatitis
caused by exfoliative (epidermolytic) toxins
(ETA and ETB) (112). The syndrome is
typically found in neonates and young children. In
addition to severe exfoliation affecting up to 90%
or more of the entire body surface
(“Ritter’s disease”), a localized form (pemphigus
neonatorum) with a few blisters is known.
Diagnosis is made on the basis of clinical
features, including Nikolsky’s sign, in which the skin
wrinkles on gentle pressure.
The other coagulase-positive or -variable
staphylococci are members of the skin microbiota
of various animal species and occasionally cause
infections in their hosts. The members of
the S. intermedius/pseudintermedius/delphini cluster
are the most common etiologic agents
of the canine pyoderma.S. hyicus is
predominantly associated with the exudative epidermitis
(greasy pig syndrome) in pigs, S. schleiferisubsp.coagulans
is found in dogs suffering from
external otitis, and S. aureus subsp. anaerobius
is the etiological agent of abscess disease, a
specific lymphadenitis of sheep and goats. In
humans, S.
intermedius/S. pseudintermedius appears to be occasionally responsible
for canine-inflicted
wound infections, FBRIs, food poisoning, and
invasive infections in immunocompromised
patients (19, 180).
Only a few reports are known for human infections due to S.
schleiferi subsp. coagulans and S. hyicus.
Since 1980, CoNS have been increasingly recognized
as nosocomial pathogens, especially S.
epidermidis. CoNS cause nosocomial infections in patients with predisposing
factors such as
immunodeficiency and/or indwelling or implanted
foreign polymer bodies
(84, 156, 194, 198). CoNS are less often implicated as the cause of
infections of natural
tissue. CoNS are the most common cause of
nosocomial bloodstream infection typically
associated with central and peripheral
intravascular catheters (205). Most important in the
pathogenesis of FBRIs is the ability of CoNS to
colonize the surface of the device by the
formation of a thick, multilayered biofilm (142).
S. epidermidis is the predominant cause of
infections associated with prosthetic vascular
grafts, prosthetic orthopedic devices, and
cerebrospinal fluid shunts. CoNS are frequently
isolated causative agents of prosthetic-valve
endocarditis; rarely they are involved in
infections of (previously damaged) native valves
(ca. 5%) (113). A right-sided native
valve endocarditis is observed in intravenous drug
abusers. Virtually any other surgically inserted
materials and devices may become infected
by CoNS. They account for 45 to 75% of all
late-onset bloodstream infections in preterm and
low-birth-weight neonates in neonatal intensive
care units (43).
In addition to prosthetic-valve endocarditis,
unusually fulminant cases of native-valve
endocarditis may be caused by S. lugdunensis, characterized
by an aggressive clinical course
with high mortality (8).
Thus, patients with S. lugdunensis bacteremia should be carefully
examined for signs of endocarditis. Besides other
invasive infections, this organism is also a
common pathogen involved in FBRIs (167).
S. lugdunensis infections resemble those caused
by S. aureus rather than those caused by
other CoNS.
Based on special urotropic and ecologic
characteristics, S.
saprophyticus subsp. saprophyticus is a well-documented causative agent
of acute,
commonly recurrent, urinary tract infections in
young, otherwise healthy, sexually active
women and, less frequently, in young men or boys.
This pathogen is the second most
common (after Escherichia coli) cause of
uncomplicated cystitis among young women. While
colony counts of ≥100,000 CFU/ml in two or more
cultures of midstream urine usually
indicate significant bacteriuria, lower counts may
be significant in the symptomatic patient.
Infections due to the recently described
subspecies S. saprophyticus subsp. bovis have not
been reported.
Since human infections due to Macrococcus,
Jeotgalicoccus,
Nosocomiicoccus, and Salinicoccus have not been described,
these genera are not discussed
further in this chapter.
Micrococcaceae and Dermacoccaceae
While “micrococci” are generally acknowledged as
harmless saprophytes, they can also act
as opportunistic pathogens. Micrococcus,
Kocuria, and Kytococcus species have been found
to cause infections such as endocarditis,
pneumonia, and sepsis or FBRIs predominantly in
immunocompromised patients (3,
25, 166). Recovery of the more recently described
micrococcal species associated primarily with the
environment must be assessed for clinical
significance as reported for K. rhizophila (23).
R. mucilaginosa has been implicated in cases of bacteremia, endocarditis,
endophthalmitis,
intravascular catheter-related and central nervous
system infections, pneumonia, peritonitis,
septicemia, and cervical necrotizing fasciitis (75,
76, 116).
Since human infections due to Acaricomes,
Citricoccus, Luteipulveratus, Nesterenkonia,
Renibacterium, Sinomonas, Yimella, and Zhihengliuella have not been reported,
these genera
are not discussed further in this chapter.
Alloiococcus
A. otitis has been associated with infections of the middle ear (114).
While
immunostimulatory capacity suggests that A.
otitis has pathogenic potential (80), other
studies revealed that A. otitis may be a
commensal rather than a cause of otitis media (182).
COLLECTION, TRANSPORT, AND STORAGE Back to top
The general principles of collection, transport,
and storage of specimens as given in chapters
9 and
chapter 16 of this Manual are applicable to the
microorganisms listed in this chapter.
No special methods or precautions are usually
required for these organisms because they are
easily obtained from clinical material of most
infection sites and are relatively resistant to
drying and to moderate temperature changes. While
some staphylococcal species may
require anaerobic conditions or CO2
supplementation for satisfactory growth, they survive
transport and limited storage in air.
DIRECT EXAMINATION Back to top
The direct microscopic examination of normally
sterile fluids such as cerebrospinal fluid and
joint aspirates may be helpful. Direct examination
of nonsterile fluids may also be useful, if
the presence of inflammatory cells versus
epithelial cells is taken into consideration. As the
result of direct microscopic examination, only a
presumptive report of “gram-positive cocci
resembling staphylococci” should be made. Cells of
microorganisms discussed here are
gram-positive, nonmotile, non-spore-forming cocci
that are arranged mostly in pairs and
tetrads but occur also singly, in irregular
(grape-like) clusters or in short chains (three or
four cells). However, within the Micrococcaceae
and Dermacoccaceae, some species exhibit
rod-shaped cells and have been shown to be motile.
Rapid PCR-based approaches have been introduced
for detection of MRSA directly from
surveillance swabs. The multiple-locus approach,
detecting the mecA gene and additionally
an S. aureus-specific target (see “Species
Identification by Nucleic Acid-Based and
Spectroscopic Approaches” below), may be influenced
by the coexistence of MSSA and MRCoNS
in the patient’s physiological microbiota and thus
may lead to false-positive MRSA
findings (21). Nevertheless, the
fundamental advantage of this approach is the direct
detection of the methicillin resistance-encoding mecA
gene. Tests applying this principle,
such as hyplex Staphylo Resist (plus) (BAG
Health Care, Lich, Germany) and StaphPlex Panel
(Qiagen), are commercially available.
The alternative single-locus amplification
strategy overcomes the MSSA/MR-CoNS
coexistence drawback by using oligonucleotide
primers binding on the staphylococcal
cassette chromosome (SCCmec) right
extremity and on the neighboring orfX region of the S.
aureus chromosome, amplifying both a taxonomic marker and a resistance
marker in one
step (89). This principle is the
basis for several rapid test systems (e.g., BD GeneOhm MRSA
assay; BD, Franklin Lakes, NJ; GenoType MRSA
Direct and Geno-Quick MRSA; Hain
Lifescience; Xpert MRSA; Cepheid, Sunnyvale, CA;
and LightCycler MRSA Advanced Test;
Roche, Basel, Switzerland). However, the use of
the surrogate marker SCCmec region
instead of mecA as target may lead to
false-positive or false-negative results, e.g., due to
the exchange of the mecA gene by other
genes, partial excision of the cassette, and
variability of the cassette primer binding sites (53).
Overall, the amplification-based rapid
MRSA screening assays are characterized by very
good negative predictive values
(approximately 97 to 99%) and are hampered by
moderate positive predictive values
(approximately 65 to 95%). To date, MRSA cultures
remain essential for confirming the
molecular results, for typing purposes, and for
determination of the complete susceptibility
profile.
ISOLATION PROCEDURES Back to top
Considering the widespread distribution of
staphylococci and “micrococci” on the skin and
mucous membranes, careful procedures should be
used to isolate organisms from the
presumed focus of infection without collecting
surrounding microbiota. The basic procedures
for culture and isolation described in chapter 16 of this Manual should be followed.
The primary culture plate used for the isolation
of staphylococci from clinical specimens is
Columbia blood agar containing 5% defibrinated
sheep blood (see also chapter 17).
Abundant growth of most staphylococcal species
occurs within 18 to 24 h. The simultaneous
use of an enrichment broth (e.g., dextrose broth)
streaked after 24 and 48 h on Columbia
blood agar may enhance the recovery rate of S.
aureus and other staphylococci.
The use of selective agars for S. aureus such
as mannitol salt agar, egg yolk-tellurite
pyruvate containing Baird-Parker medium, Columbia
colistin-nalidixic acid agar, lipase-saltmannitol
agar (Remel, Lenexa, KS), and phenylethyl alcohol
agar may be appropriate for
specimens from heavily contaminated sources such
as feces. It is mandatory to confirm
putative S. aureus isolates recovered on
these media.
Novel selective agars using chromogenic enzyme
substrates specifically for S. aureus, such
as CHROMagar Staph aureus (CHROMagar, Paris,
France), BBL CHROMagar Staph aureus
(BD Diagnostics, Sparks, MD), and S. aureus ID
(bioMerieux, La Balme Les Grottes, France),
have been launched on the market with
chromogen-dependent coloration of the colonies. In
particular for screening purposes, the chromogenic
agars have been proven to be suitably
sensitive and specific, allowing a presumptive but
not final identification (141). Chromogenic
agars designed for MRSA detection are discussed in
“Antimicrobial Susceptibilities” below.
The diagnosis of catheter-related bloodstream
infections by CoNS and other organisms
remains a major challenge. One of the most
frequently studied diagnostic techniques is
represented by the semiquantitative roll-plate
catheter culture. Here, the distal segment of
the central venous catheter is cut and rolled
across the surface of a Columbia blood agar
plate at least four times followed by overnight
incubation. A colony count of 15 CFU/ml or
more may indicate catheter colonization (118).
Examination of paired quantitative blood
cultures drawn simultaneously from the catheter
and a peripheral vein enhanced by the
analysis of differential time to positivity
represents an example of an approach that does not
require catheter removal (148)
(see also “Evaluation, Interpretation, and Reporting of
Results” below).
Cultivation of “micrococci” and R. mucilaginosa
should be performed as described for
staphylococci on Columbia blood agar at 35°C to
37°C under aerobic conditions. However,
abundant growth of Micrococcaceae andDermacoccaceae
needs consistent incubation times
of 36 to 48 h.
Because of the slow growth rate, it is difficult
to isolate A. otitis by conventional nonselective
culture methods (29). Blood agar plates with
6% NaCl were shown to be useful.
IDENTIFICATION Back to top
The basic criteria distinguishing
catalase-positive gram-positive cocci and their relatives
among themselves and from other microbial taxa are
given in “Description of the Genera”
above and in Table 1. Misidentification is
likely to occur if automated test system results are
accepted without critical review by skilled lab
personnel. In specialized settings, species can
be identified by chemotaxonomic procedures and
molecular methods.
Staphylococcus and Related Genera
Staphylococcus species can be identified phenotypically on the basis of a variety
of
conventional characteristics (Tables 1 to 3). The most clinically significant species in
human
and veterinary medicine can be identified on the
basis of several key characteristics (Table
3).
The application of the extensive scheme originally published by Kloos and
Schleifer in
1975 (105) has been mostly replaced by the use of
commercial identification systems.
Colony Morphology
Most staphylococcal colonies are 1 to 3 mm in
diameter within 24 h and 3 to 8 mm in
diameter after 72 h of incubation in air at 34 to
37°C. Exceptions are S.
aureus subsp. anaerobius, S. saccharolyticus, S. auricularis, S.
equorum, S. vitulinus, and S.
lentus, which grow more slowly and usually require 24 to 36 h for detectable
colony
development. Further incubation of agar plates for
a period of up to 48 to 72 hours
(optimally followed by 2-day incubation at room
temperature) enhances morphologic
differences.
On routine blood agar, the typical S. aureus colony
is pigmented (cream yellow to orange),
smooth, entire, slightly raised, and hemolytic (Fig. 1). Mucoid colonies due to highly
encapsulated strains are rarely encountered. A
number of isolates of S. aureus as well as
some CoNS species (e.g., S. haemolyticus and
S. lugdunensis) may have a hazy or distinct
zone of beta-hemolysis around the colonies ranging
from weak to strong. SCVs of S.
aureus or other staphylococcal species are characterized by pinpoint
colonies (1/10 the size
of the wild type), mostly nonpigmented and nonhemolytic
after 24 to 72 hours of incubation
(Fig.
1) (147,192).
They are often mixed with colonies displaying the normal phenotype,
thus giving the appearance of a mixed culture.
Upon subculture they may remain stable or
revert to the wild type. Depending on their
auxotrophy, normal growth may be restored if
the isolate is grown in the presence of hemin,
menadione, or thymidine and/or
CO2 supplementation (147).
Coagulase Production
A widely used criterion for the identification of S.
aureus in the clinical laboratory is the
clotting of plasma proven by two different tests:
(i) detection of the extracellular free
coagulase by the tube test due to staphylococcal
coagulase that converts fibrinogen to fibrin
and (ii) detection of the cell wall bound “coagulase”
(i.e., the clumping factor) by the slide
agglutination test (see below).
The tube coagulase test is performed by
transferring a large, well-isolated colony from a
noninhibitory agar into 0.5 ml of reconstituted
rabbit plasma. It is crucial to incubate the
tube at 37°C for 4 h and to observe the tube for
clot formation by slowly tilting the tube 90°
from the vertical. Any degree of clotting
represents a positive test. Aflocculent or fibrous
precipitate is not a true clot and should be
recorded as negative. If no clot is formed by 4 h,
the tube should be read again after 18 h of
incubation. False-negative results may occur for
some strains producing staphylokinase, which may
lyse the clot after formation (usually after
prolonged incubation). Inaccurate results may
occur if nonsterile plasma is used or the
colony tested is not pure.
Particularly in veterinary microbiological
laboratories, the other coagulase-positive or -
variable species (Table 1) must not be disregarded.
The detection of free coagulase in
staphylococci obtained from human specimens is
usually equated with the species
identification of S. aureus. However, for
animal-inflicted wounds, additional testing should be
performed to provide identification beyond
“coagulase-positive staphylococci.”
Agglutination Assays
The classical slide agglutination test detects
bacterial aggregation of S. aureus and other
clumping factor-positive staphylococcal species in
the presence of rabbit plasma through the
action of clumping factor A, a cell wall-associated
adhesin for fibrinogen. The detection of
this factor expressed by non-aureus species
(Table 1) may require the application of human
plasma. While this test is very quick to perform
(less than 1 minute), several limitations
concerning sensitivity and specificity (e.g.,
masking of the factor by capsular polysaccharides
and autoagglutination by picking colonies from
media with high salt concentrations) have to
be noted.
To overcome the disadvantages of the slide
coagulase test (low sensitivity) and of the tube
coagulase test (long incubation time), rapid latex
and hemagglutination assays allowing
presumptive identification of S. aureus have
been developed. Besides detecting protein A
and clumping factor A, recent third-generation
assays include monoclonal antibodies
recognizing the clinically most prevalent capsular
polysaccharide serotypes 5 and 8 or other
structures (e.g., Pastorex Staph-Plus [Bio-Rad
Laboratories, Hercules, CA]; Slidex Staph Plus
[bioMerieux, Marcy l’Etoile, France]; and
Staphaurex Plus and Staphytect Plus [Oxoid,
Cambridge, United Kingdom]). The higher
sensitivity (>98 to 100%) of the third-generation
tests has reduced their specificity (72 to 99%).
False-positive reactions occur with some
CoNS strains (S. haemolyticus and S.
hominis)possessing type 8 capsular polysaccharide or
owning a cell wall hemagglutinin (S.
saprophyticus). When an isolate is suspected to be S.
aureus, negative slide tests should be confirmed by the tube coagulase
test.
Identification of Species by Susceptibility Tests
Novobiocin resistance is intrinsic to S.
saprophyticus and other CoNS species (Table 2) but is
uncommon in the other clinically important CoNS
species of the “S. epidermidis group.” A
disk diffusion test for estimating novobiocin
susceptibility can be performed using a 5-μg
novobiocin disk on Mueller-Hinton agar or tryptic
soy sheep blood agar. With an inoculum
suspension equivalent in turbidity to a 0.5
McFarland opacity standard and incubation at 35
to 37°C overnight or up to 24 h, novobiocin resistance
is indicated by an inhibition zone
diameter of ≤16 mm with any of these media.
Polymyxin B resistance is usually observed for
isolates of S. aureus, S. epidermidis, S.
hyicus, S. chromogenes,and to a lesser extent, for some strains of S.
lugdunensis. A disk
diffusion test may be performed using a 300-U
polymyxin B disk on tryptic soy sheep blood
agar with the same test conditions as those
described for novobiocin. Polymyxin B resistance
is indicated by an inhibition zone diameter of
<10 mm.
Identification of Species by Biochemical
Procedures
For speed, standardization, cost reduction, and
convenience, the classical tests for
fermentation, oxidation, degradation, and
hydrolysis of various substrates (details are
available in chapter 17) have been incorporated
into commercial manual and automated
biochemical test systems (see below and chapter 3). They are often complemented by
simultaneously performed antimicrobial
susceptibility testing, an advanced expert system,
and an interface to the laboratory informatics
software. Results with conventional tests
(Tables
1 and 3)
may be slightly different from those obtained with rapid biochemical test
systems due to the use of other, more sensitive
indicators. Commercial identification
systems identify the staphylococci (and some other
aerobic gram-positive cocci) of clinical
importance with an accuracy of 70 to >90%. For
some systems, reliability depends on
additional testing as suggested by the
manufacturer. Uncommon strains or phenotypic
variants (e.g., SCVs) may have altered patterns of
biochemical reactions requiring molecular
testing for identification.
The API Staph (bioMerieux) strip represents an
overnight method for manual identification of
primarily staphylococci for health care and
product safety applications. Necessitating the
same incubation time and also fashioned in the
strip format, the ID32 Staph (bioMerieux)
may be read manually as well as automatically by
the bioMerieux ATB system. The
databases of both systems comprise more than 20
staphylococcal species with clinical
significance, some “micrococcal” species, and R.
mucilaginosa. A rapid version allowing 2-h
identification of S. aureus, S. epidermidis, and
S. saprophyticus is provided by the same
manufacturer (Rapidec Staph). The Vitek 2 (bioMerieux)
system is a fully automated
platform that performs bacterial identification
and antibiotic susceptibility testing. The VITEK
2 gram-positive identification card encompasses a
total of more than 100 species, including
26 staphylococcal species, a small spectrum of
“micrococci,” and R.
mucilaginosa;identification of CoNS usually requires 10 h.
The MicroScan product Pos ID family (Siemens
Health-care Diagnostics, Deerfield, IL)
includes “Conventional” (overnight identification
time), “Rapid” (2.5-h identification time),
and “Synergies plus” (2- to 2.5-h identification
time, with key antimicrobial results in as little
as 4.5 hours) panels in a microtiter format.
Identification of 19 clinical staphylococcal
species, some “micrococcal” species, and R.
mucilaginosa is available with either manual or
automated processing on the autoSCAN-4 and
WalkAway systems.
The BD BBL Crystal identification systems’ Rapid
Gram-Positive ID kit (BD Diagnostic
Systems, Sparks, MD) is a three-row panel that may
be read manually or with the BBL
Crystal AutoReader requiring a 4-hour incubation
period. Besides 14 staphylococcal species,
the database encompasses a total of 88 taxa
including M. caseolyticus, R. mucilaginosa, and
several “micrococci.” The BBL Crystal Gram-Positive
ID system represents the overnight
incubation (18-h) version with an extended taxa
profile of 121 gram-positive organisms. A
further enhanced taxa profile (covering about 200
taxa) is covered by the automated
nephelometry-based BD Phoenix Automated
Microbiology System using one combination
panel with the identification substrates on one
side and the antimicrobial agents on the other
side of the panel. For staphylococci, about 10 to
15.5 hours are required for complete results
(identification and antimicrobial susceptibility
testing) (56).
The gram-positive aerobic bacteria database (339
taxa) of the Biolog Systems family (Biolog,
Hayward, CA) comprises 34 staphylococcal and 4
macrococcal (sub-)species and many
members of the Micrococcaceae andDermacoccaceae
families not found in other commercial
phenotype-based systems. The system’s redox
chemistry based on the utilization of a wide
variety of carbon sources is used to generate a
“metabolic fingerprint” providing results in 4
hours or less and is available with different
automation levels.
The Sherlock Microbial Identification System
(MIDI, Newark, DE) represents an identification
system that automates microbial identification by
combining cellular fatty acid analysis with
computerized high-resolution gas chromatography.
In addition to a multitude of other
species, 30 staphylococcal species, M.
caseolyticus,and several “ micrococci” are listed in the
fatty acid-based database. A new sample
preparation method (Instant FAME) allows for rapid
identification from pure cultures in less than 15
minutes.
Most systems are fairly successful in
differentiating S. aureus, S. epidermidis, and S.
saprophyticus, while the accurate identification of less common species is more
variable
(152). Systems may fail in distinguishing commonly
encountered staphylococcal species, in
particular if phenotypic variants or isolates
recovered from livestock and food are tested
(18, 210). A verification of the identification result by
a second, independent approach is
recommended for isolates with presumptive
identification as S. aureus, particularly for
oxacillin-resistant strains. Additional tests
should be performed on clinically significant
isolates with questionable identification results
that impact patient management.
Species Identification by Nucleic Acid-Based and
Spectroscopic
Approaches
Extraction of staphylococcal nucleic acids may be
challenging due to the gram-positive
nature requiring special conditions for lysis of
the cell wall. For this purpose, lysostaphin,
lysozyme, proteinase K, and achromopeptidase have
been described.
Besides PCR fingerprinting techniques based on DNA
sequence polymorphisms, speciesspecific
variable regions of universal genes or genes
unique for S. aureus or other
staphylococcal species may serve as targets for
identification and differentiation of
staphylococcal isolates. Assays based on the
specific amplification of fragments of the
universal 16S and 23S rRNA genes and of their
spacer sequences have been published
(18,41, 55, 92, 124, 173, 178). The 16S rRNA gene is also used as target for
the in situ
detection and identification of S. aureus and
S. epidermidis (111). Other universal DNA
targets shown to be useful for identification of
staphylococci include the elongation factor
gene (tuf), the gyrase gene (gyrA), the
manganese-dependent superoxide dismutase
gene (sodA), the glyceraldehyde-3-phosphate
dehydrogenase-encoding gene (gap),and a
60-kDa heat shock protein (HSP60/GroE) (73,
74, 120, 144, 211).
Sequencing of selected universal phylogenetic
marker genes represents the ultimate
approach to identify known and not yet described
staphylococcal species. For 16S rRNA gene
sequencing, the regions between bp 70 to 300, 420
to 500, 1,000 to 1050, and 1,250
to1,290 (corresponding to nucleotides of the Escherichia
coli16S rRNA gene) are useful to
determine sequence differences among the
staphylococcal species. For differentiation of
staphylococcal subspecies, sequencing of partial rpoB
gene sequences seems to be superior
to partial 16S rRNA gene sequencing (123).
The recognized limitations of currently available
public sequence databases apply to sequencing of
staphylococcal isolates (18).
The most popular and well-studied specific target
for S. aureus identification is the nuc gene
that encodes thermostable nuclease (thermonuclease
or TNase) (31, 204). PCR methods
targeting the nuc gene are highly specific
for S. aureus. A specific PCR for the S.
intermedius nuc gene has also been described (24). Further specific
targets used for
identification of S. aureus include the
genes encoding clumping
factor (clfA), coagulase(coa), manganese-dependent
superoxide dismutase (sodM, absent in
CoNS), the factors essential for the expression of
methicillin resistance (femA and B), and for
MRSA only, the staphylococcal insertion element 431(108,
122, 188, 191, 211).
Misidentification using the fem factors may
occur due to fem-negative S. aureusstrains and
CoNS with a gene structurally related to femA.
For molecular identification of S. aureus and
some other staphylococcal species isolated from
culture, several commercial tests (e.g., GenoType
Staphylococcus [Hain Lifescience, Nehren,
Germany]; StaphPlex Panel [Qiagen, Germantown,
MD]; AccuProbe [Gen-Probe, San Diego,
CA]; and S. aureus Evigene [AdvanDx,
Woburn, MA]) are available. Some assays also detect
resistance genes (see below) and/or toxin genes.
The GeneXpert (Cepheid) and BD
GeneOhm (BD Diagnostics) instruments offer assays
to detect MSSA and MRSA directly from
positive blood cultures. The RiboPrinter microbial
characterization system (DuPont Qualicon,
Wilmington, DE) utilizes ribotype pattern analysis
for the differentiation of staphylococcal
species with patterns in the database.
The S. aureus PNA FISH (bioMerieux) is a
qualitative nucleic acid hybridization assay
targeting rRNA sequences based on peptide nucleic
acid (PNA) fluorescence in situ
hybridization (FISH). The assay is intended for
rapid identification of S. aureus in a smear
prepared from a positive blood culture. The S.
aureus/CNS PNA FISH is expanded to identify
simultaneously several CoNS species that commonly
cause bacteremia (83). Diagnostic DNA
oligonucleotide microarrays that identify the
genus Staphylococcus, clinically important
staphylococcal species, other pathogens, drug
resistance genes, and toxin genes have been
designed and tested on clinical isolates (66,
129).
Alternative high-throughput approaches involving
mass spectral analysis of surfaceassociated
proteins of intact staphylococcal cells are
represented by the matrix-assisted laser
desorption ionization–time of flight and related
methods (32, 150, 206). Here, the quality of
the database and the standardization of variable
parameters are crucial to achieve
reproducible results. Nondestructive techniques
such as Fourier transform infrared (FT-IR)
and Raman spectroscopy are currently being
developed as alternative methods for the rapid
identification of staphylococci (6).
This approach also allows discrimination between the SCV
and the normal S. aureus phenotype (20).
Diagnosis of Toxin-Mediated Staphylococcal
Syndromes
The diagnosis of TSS and SSSS is based on clinical
signs supplemented by serologic tests
and the detection of the toxin production by
staphylococcal isolates (S. aureus, rarely other
species). While the skin manifestations are mostly
culture negative, isolates are usually
recovered from the suspected site of infection.
Blood cultures are positive in fewer than 5%
of cases of staphylococcal TSS. In patients with
TSS, protective antibodies against causative
PTSAgs are absent or present at very low levels.
However, serocon-version after onset of the
disease and during convalescence may be observed.
The same phenomenon holds true for
exfoliatin antibodies in patients with SSSS.
Currently available immunoassays for antibody
detection are for research use only. Beyond the
recognition of the characteristic rapid onset
and the clinical signs, staphylococcal food
poisoning is difficult to verify because the
incriminated food source may not contain
cultivable staphylococcal cells and requires
detection of staphylococcal enterotoxin.
Traditional immunological procedures may be used
to measure the toxin in culture
supernatants of isolated strains, in contaminated
food extracts, or in patient specimens. Kits
for the detection of strains producing TSST-1
(TSTRPLA [Oxoid, Cambridge, United
Kingdom]; TST-RPLA “Seiken” [Denka Seiken, Tokyo,
Japan]; and TSST-1 Evigene
[AdvanDx, Woburn, MA]), staphylococcal
enterotoxins (Rida Screen set A, B, C, D, E [RBiopharm,
Darmstadt, Germany]; SET-RPLA “Seiken” [Denka
Seiken]; SET-RPLA Kit toxin
detection kit [Oxoid]), and ETA/ ETB (EXT-RPLA
“Seiken” [Denka Seiken]) are offered. A
quantitative real-time immuno-PCR was recently
described for the detection of small
amounts of enterotoxins (61).
An adaptation of a flow cytometry-assisted multiplex
immunoassay (Bio-Plex system; Bio-Rad, Hercules,
CA) for the detection of ETA and ETB has
been recently reported (94).
Since phenotypic methods may be hampered by low sensitivity
and specificity (cross-reactivity between PTSAgs)
and in vitro expression of PTSAgs might be
negatively influenced by various factors,
detection of PTSAg/ET-encoding sequences by
nucleic acid-based methods has come into favor (17,22,
128, 209).
Due to renewed interest in Panton-Valentine
leukocidin (PVL), PCR procedures targeting
leukocidal (synergohymenotropic) pore-forming
toxins produced by S. aureus were
established (115). The detection of
PVL-encoding genes is included in the GenoType
Staphylococcus and MRSA test systems (Hain
Lifescience) and offered by the PVL EVIGENE
kit (AdvanDx, Woburn, MA).
Micrococcaceae and Dermacoccaceae
Micrococci and staphylococci might be easily
confused with one another on the basis of
similar cellular morphologies, Gram stain
appearance, and positive catalase activities. The
exact species affiliation of “micrococci” may be
frequently misjudged. The frequent
pigmentation of micrococcal colonies with high
convex profile leads to their presumptive
identification as members of the Micrococcaceae
and Dermacoccaceae families. Colonies
of M. luteus, K. varians, and the
kytococcal species are characterized by yellowish tints. K.
kristinaeand D. nishinomiyaensis appear orange-like, and K. rosea
shows pink to red
colonies. Some species (e.g., M. lylae) or
strains of the usually pigmented species are nonpigmented.
On routine blood agar, R. mucilaginosacolonies
are mucoid or sticky, transparent
to white, and nonhemolytic and in the majority of
cases adhere to the agar (differentiation
from streptococci). This organism may be
distinguished from other similar organisms by its
weak catalase reaction and its inability to grow
in the presence of 5% NaCl.
In the clinical laboratory, “micrococcal” species
can be preliminarily distinguished from
staphylococci by their resistance to furazolidone
(100 μg/disk; resistance is indicated by a
≤9-mm zone diameter) and lysostaphin (200 μg/disk;
resistance, no zone) and susceptibility
to bacitracin (0.04 U/disk; susceptibility, ≥10-mm
zone diameter) in contrast to members of
the Staphylococcus genus, which show
inverse susceptibility patterns (zone diameters:
furazolidone, ≥15mm; lysostaphin, 10 to 16 mm;
bacitracin, no zone) (Table 1). Details are
available in previous editions of this Manual.
In contrast to most staphylococci, micrococci
are positive by the modified oxidase test (59).
Regarded as a reference method to
distinguish “micrococcal” species from
staphylococci, the fermentation of glucose in a
manner similar to the oxidation-fermentation test
for nonfermenters requires a specific
oxidation-fermentation medium and prolonged
incubation. In contrast to staphylococci,
“micrococci” are characterized by the lack of acid
production from glucose under anaerobic
conditions.
Key features for differentiation of species reported
to occur in human specimens are given
in Table 4. Data concerning the
applicability of manual and automated identification systems
for members of Micrococcaceae andDermacoccaceae
are given in the
respective Staphylococcus section; however,
their use is limited to a small spectrum of the
clinical “micrococcal” species. In cases of doubt
and extraordinary clinical relevance, the use
of sequencing-based approaches is recommended for definite species
recognition.
Alloiococcus
After
48 h of incubation at 37°C, alloiococci form small alpha-hemolytic colonies on
blood
agar.
Colonies formed on brain heart infusion agar with 5% rabbit blood are small,
moist,
and
slightly yellow at 72 h, and the blood is partially hemolyzed. Growth occurs in
the
presence
of 6.5% NaCl and on bile esculin agar. No growth occurs at 10°C or 45°C. A.
otitis
can be distinguished from similar organisms by its positive
catalase and negative
oxidase
activities, its obligate aerobic nature, and its inability to produce acid from
glucose or
other
carbohydrates (Table 1). Arginine dihydrolase is not produced. Pyrrolidonyl
arylamidase,
leucine aminopeptidase, and P-galactose are produced. Starch and esculin are
not
hydrolyzed; hippurate is mostly hydrolyzed (29). A.
otitis is included in the database of
the
API Strep gallery (bioMerieux).
As
the bacterium is quite inert biochemically, molecular methods are often
necessary to
confirm
the identification. For the molecular verification of suspected colonies, an A.
otitisspecific
PCR
assay has been described (1). A PCR assay for direct
detection of this
microorganism
in clinical specimens has been reported as part of a multiplex approach
targeting
pathogens that cause otitis media with effusion (82).
TYPING SYSTEMS Back to top
Traditional
phenotyping techniques such as phage typing, capsule serotyping, antibiotic
susceptibility
pattern analysis, and other biotyping methods have been replaced by molecular
band-based
and sequence-based typing methods (179).
The reference method for defining
the
core genetic population structure of S. aureus is MLST. In a
standardized manner, the
allelic
polymorphism of seven housekeeping genes is indexed. A web-based database is
available
(http://saureus.mlst.net/). MLST has limited
discriminatory power and low
throughput
capacity in the context of MRSA outbreak investigation and surveillance. In the
case
of common ancestry but assumed distinct epidemiological origin of MRSA
isolates,
subtyping
of SCCmec may provide additional information (155).
For
local MRSA outbreak investigation as well as for long-term MRSA surveillance,
SmaI
macrorestriction
pattern analysis by PFGE represents a highly discriminatory “gold standard”
tool
with detailed performance and interpretation guidelines (133, 183).
However, PFGE is
technically
demanding with low throughput, limited portability, problems with inter-center
reproducibility,
and different national nomenclatures. The emerging livestock-associated
ST398
isolates are mostly nontypeable by standard PFGE protocols (88). Attribution
of PFGE
clusters
to genetic lineages may be problematic (179).
The
number of polymorphisms and the sequence of tandem repeat elements of the
hypervariable
X region of the S. aureus protein A (spa) gene are the basis of a
single-locus
sequence
typing approach that has become one of the primary genotyping methods for
MRSA
surveillance (65, 170). Beside total reproducibility, other advantages include low
costs,
high
throughput, a standardized nomenclature, and complete portability of data
transferable
into
an international database (http://spaserver.ridom.de/)
curated by SeqNet.org
(http://www.seqnet.org/) (81).
For particular genetic lineages, misclassification may occur,
necessitating
the use of additional tests for reliable inference.
Other
molecular typing techniques used for typing of staphylococci include several
other
band-based
molecular fingerprinting approaches, ribotyping, and more recently,
multiplelocus
variable-number
tandem-repeat analysis and microarray-based approaches
(110, 160, 185).
Modern approaches based on phenotype structures applied for
staphylococci
include the matrix-assisted laser desorption ionization– time of flight/mass
spectrometry
and whole-cell fingerprinting techniques, such as the FT-IR spectroscopy (7).
While
many typing systems have been developed and evaluated for S. aureus, fewer
applications
are available for CoNS. These include antibiotic resistance analysis, phage
typing,
slime production, and some modern genotyping procedures (PFGE and ribotyping)
(69, 190).
For
“micrococcal” species, phage typing and PFGE approaches applying phage sets and
restriction
enzymes, respectively, different from those used for staphylococci (132)
have
been
described. A restriction fragment length polymorphism approach has been described
for Alloiococcus
(29).
SEROLOGIC TESTS Back to top
Because
serological testing for antistaphylococcal antibodies lacks specificity and
predictive
accuracy,
it plays no role for the diagnosis of most staphylococcal diseases. The one
exception
is the determination of protective antibodies in the case of toxin-mediated
syndromes
such as TSS and SSSS (see above). For the other microorganisms discussed in
this
chapter, detection of antibodies is not clinically useful.
ANTIMICROBIAL SUSCEPTIBILITIES Back
to top
Staphylococcus
and Related Genera
Genetic Basis and Prevalence of Antimicrobial
Susceptibilities
Methicillin
resistance mediated by the mecA gene has the greatest impact on patient
management
by excluding all traditional β-lactam antibiotics from the antibiotic
armamentarium.
Since the early 1980s, the prevalence of health care-associated MRSA (HAMRSA)
has
increased in many regions. In some areas of the United States, the prevalence
of
MRSA
is >50% (50, 101). In Europe, with the low-prevalence exception of The Netherlands
and
the Scandinavian countries, approximately 20% or more of S. aureus isolates
are
methicillin
resistant (177). In Asia, Australia, and Africa, high MRSA rates (approximately
20
to
80%) have also been noted (158, 169, 212).
The worldwide burden of infections caused
by
MRSA is increasing due to the advent of CA-MRSA in the past decade and, most
recently,
by
livestock-associated S. aureus infections (85, 109). A
number of studies and reviews
describing
antimicrobial and biocide susceptibilities of clinically important
staphylococcal
species
have been published (51, 64, 87). Networks have also been established that provide
online
or published resistance data for staphylococci: the National Nosocomial
Infections
Surveillance
System (http://www.cdc.gov/ncidod/dhqp/), the European
Antimicrobial
Resistance
Surveillance System (http://www.rivm.nl/earss/), and the International
Nosocomial
Infection Control Consortium (http://www.inicc.org/).
The mecA
gene is acquired by S. aureus and other staphylococcal species on a
foreign,
mobile
DNA element (SCCmec) and encodes an additional penicillin-binding
protein (PBP),
PBP2a.
Seven major variants of SCCmec, types I to VII, and many subtypes have
been
recognized
(27, 44, 90, 91, 136). SCCmec types I, II, III, and VI are predominantly
associated
with HA-MRSA (47).
Multidrug
resistance is regularly observed in HAMRSA and usually includes resistance to
aminoglycosides,
fosfomycin, fusidic acid, glycopeptides, ketolides, lincosamides, macrolides,
quinolones,
rifampin, tetracyclines, and trimethoprim- sulfamethoxazole (51, 62, 87).
There
are
rare reports of S. aureus and CoNS isolates resistant to new agents such
as linezolid,
daptomycin,
and tigecycline (54, 143, 171).
In
contrast to HA-MRSA, CA-MRSA strains are more susceptible to non-β-lactam
antibiotic
classes
and harbor different SCCmec (mostly types IVa, V, and VII) (13, 27, 44, 91).
CAMRSA
isolates
are more likely to carry PVL, a pore-forming toxin with potent cytolytic and
inflammatory
activities (72). Whereas in Europe an array of diverse CA-MRSA clones (mainly
the
“European” ST-80, spa type t044) have been reported, the PFGE type
USA300 (ST-
8, spa
types t008 and t024) predominates in the United States followed by USA400
(47, 101).
Populations at increased risk for CA-MRSA infections include children in day
care
centers,
athletes, military recruits, jailed inmates, and men who have sex with men
(52, 97, 138).
CA-MRSA causes superficial skin and soft tissue infections and can also be
associated
with necrotizing fasciitis and myositis, necrotizing pneumonia, and other
severe
entities
(115). Infections by PVL-positive S. aureus in young, otherwise
healthy children
following
a respiratory viral infection (most frequently influenza) can be a devastating
disease
(72). However, some of the distinctions between HA-MRSA and CA-MRSA
strains
may
disappear since CA-MRSA is now becoming endemic in hospitals and acquiring
additional
resistances
(52, 162, 168).
Since
1996, vancomycin-intermediate S. aureus (VISA) isolates (MIC of 4 to 8
μg/ml) (39)
and
their putative precursors, termed heterogeneous VISA (hVISA) strains, have been
identified
first in Japan followed by detection in the United States and other regions
(33, 86, 154).
Reduced susceptibility is thought to be caused by cell wall alterations
resulting
in
reorganization and thickening (30, 42).
In 2002, the first of multiple vancomycinresistant
S.
aureus (VRSA) strains containing the vanA gene were reported in
the United
States
(34). S. aureus isolates currently defined as
vancomycin-resistant exhibit MICs of ≥16
μg/ml
(39). Since most VISAs are also resistant to teicoplanin, the acronym
GISA
(glycopeptide-intermediate
S. aureus) is preferred by some authors. A few clinical isolates
that
are resistant to teicoplanin but fully susceptible to vancomycin have been
reported (57).
More
than 90% of all of nosocomial CoNS (mainly S. epidermidis and S.
haemolyticus) are
resistant
to penicillin due to β-lactamase production, and approximately 60 to 80% are
resistant
to methicillin and other agents (51,93, 121).
Rare acquisition of mecA-encoded
methicillin-resistance
in S. intermedius/S. pseudintermedius has been reported (15).
Determination of Antimicrobial Susceptibilities
Antimicrobial
susceptibility testing of staphylococci may be performed conventionally by
Clinical
and Laboratory Standards Institute (CLSI) reference methods (38) or
commercial
systems
as described in chapter 67 to 70 of this Manual. Direct detection of MRSA is
discussed
in “Direct Examination” above. For SCVs, no approved method has been developed
to
determine the susceptibility (146, 192).
Detection
of MRSA represents the most important task in determining the antimicrobial
susceptibilities
of staphylococci. To distinguish MRSA from MSSA, traditional methods may
have
reduced sensitivity and specificity due to heteroresistance and borderline
oxacillinresistant
S.
aureus characterized by β-lactamase hyperproduction. In the case of
heterogeneous
PBP2a expression, only a small fraction of the bacterial cell population
(10−8
to 10−4) expresses the resistance phenotype under in vitro test conditions. The
resistant
subpopulation may be overlooked because it usually grows more slowly than the
susceptible
population. The successful detection of heteroresistant strains is favored by
cooler
incubation temperatures (30 to 35°C), the presence of NaCl (2 to 4%), and
prolonged
incubation
time (up to 48 h). Cefoxitin (30 μg) disk diffusion tests correlate better with
the
presence
of mecA than do the oxacillin disks used previously. The cefoxitin disk
diffusion
method
may be used for testing S. aureus and S. lugdunensis (resistance
is indicated by a
zone
diameter of ≤21 mm) as well as other CoNS (resistance, ≤24 mm) for mecA-mediated
resistance
(60, 172). For broth microdilution testing, oxacillin or cefoxitin may be
used to
detect
mecA- mediated resistance in S. aureusand S. lugdunensis (resistance:
cefoxitin, MIC
≥ 8
μg/ml; oxacillin, MIC ≥ 4 μg/ml). For other CoNS isolates, the presence of mecA
is
predicted
by applying lower oxacillin MIC breakpoints (resistance: oxacillin, MIC ≥ 0.5
μg/ml).
Cefoxitin has also improved the detection of MRSA by automated susceptibility
testing
systems (157). The slow growth rate of SCVs prevents the use of disk diffusion
and
automated
methods to determine the susceptibility of these strains (147).
An
alternative method for detection of methicillin resistance is the use of
anti-PBP2a
monoclonal
antibodies available as a latex agglutination assay that may be performed on
isolated
colonies from a pure culture (MRSA Screen; Denka Seiken, Tokyo, Japan) (134).
If
the
latex test is used for SCVs, then the number of colonies must be increased
100-fold
(100).
For
heterogeneous methicillin-resistant staphylococci, strains displaying growth-impaired
phenotype
(SCVs) or borderline oxacillin-resistant S. aureus strains, the
methicillin
resistance
of cultivated staphylococcal isolates may be determined by detection of
the mecA
gene (131). For the genetic verification of MRSA isolates, the occurrence
of both
the mecA
gene and a species-specific marker (see above) has to be proven. The use of
pure
colony
material is a vital premise for this approach.
For
MRSA screening to detect colonization (preferentially nasal, also pharyngeal),
selective
media
(e.g., mannitol salt agar) supplemented with oxacillin are widely used.
Inclusion of a
broth
enrichment step prior to plating enhances sensitivity but delays results. Media
containing
chromogenic enzyme substrates (e.g., MRSA ID [bioMerieux]; BBL CHROMagar
MRSA
[BD]; and Brilliance MRSA agar [Oxoid]) have better specificity; however,
confirmation
of
MRSA with a coagulase test is recommended for some products (36, 49, 140).
Detection
of VISA is unreliable and probably underreported by routine susceptibility testing
methods,
including automated methods (9). According to current CLSI
recommendations, S.
aureus
isolates with vancomycin MICs of 4 to 8 μg/ml are classified as
VISA (39). These
vancomycin
MIC breakpoints were lowered for S. aureus (the intermediate category
remained
defined as 8 to 16 μg/ml for CoNS) in order to better detect hVISA strains that
are
potentially
associated with vancomycin clinical failure (35, 184).
Detection of hVISA requires
a
“modified population analysis profile–area under the curve” method or a
macroEtest
method
(201, 207). Recently, FT-IR spectroscopy has been successfully applied for
rapid and
accurate
identification of VISA and hVISA among isolates of MRSA (5).
VRSA strains (MIC,
≥16
μg/ml) are reliably detected by the broth microdilution reference method, most
FDAcleared
automated
systems, or a brain heart infusion vancomycin (6 μg/ml) agar screen
plate
(39). In 2009, the European Union Committee on Antimicrobial
Susceptibility Testing
reduced
the glycopeptide MIC breakpoints (resistance: vancomycin, >2 μg/ml for S.
aureus
and CoNS; teicoplanin, >2 μg/ml for S. aureus and >4
μg/ml for CoNS) to avoid
reporting
VISA isolates as “intermediate”
(http://www.srga.org/eucastwt/MICTAB/MICglycopeptides_v2.html).
Additional
information on the determination of antimicrobial susceptibilities is contained
in chapters
67 to 70 of this Manual. There are no CLSI methods for
susceptibility testing of
“micrococci”
and alloiococci.
Treatment
For
additional and annually updated information regarding treatment, the reader
should
consult
the current edition of The Sanford Guide to Antimicrobial Therapy (71) or
other
guidelines.
Effective treatment of focal infections such as empyema and abscesses requires
incision
and drainage. Penicillin G is the most effective compound for the treatment of
the
uncommon
penicillin-susceptible S. aureus strain. In general,
penicillin-resistant, oxacillinsusceptible
staphylococcal
strains should be treated with penicillinase-stable penicillins, β-
lactam/β-lactamase
inhibitor combinations, and cephalosporins (151).
For patients with
penicillin
allergy or chronic renal failure, clindamycin or vancomycin may be an option in
the
case
of MSSA. However, the use of vancomycin, known to be poorly bactericidal
against
staphylococci,
is not recommended for severe infections due to MSSA as it is inferior to β-
lactams
in terms of mortality and bacteriological outcome.
Strains
that are oxacillin- or cefoxitin-resistant (MRSA) should be considered
resistant to all
β-lactams
including penicillins, carbapenems, and cephalosporins (except for the new
“fifthgeneration”
cephalosporins
[ceftobiprole and ceftaroline] with anti-MRSA activity) (77).
Vancomycin
and the newer agents such as linezolid, daptomycin, dalbavancin, and
tigecycline
are suitable options for empiric therapy of MRSA infections (note the different
spectra
and the approved indications of these compounds) (63, 161, 208).
Occasionally,
trimethoprim-sulfamethoxazole
(SXT) may be helpful, but it should be used with caution
(145).
In particular in terms of “collateral damage,” quinolones should be avoided for
the
therapy
of staphylococcal infections (139).
While
there are only a few, partly uncorroborated studies and case reports available
supporting
combination therapy for treatment of severe staphylococcal infections,
aminoglycosides,
rifampin, fosfomycin, co-trimoxazole, and fusidic acid in combination with
glycopeptides
and β-lactams have been recommended (70). A
careful risk-benefit
assessment
concerning drug-drug interactions and side effects should be performed. Since
resistance
towards rifampin, fusidic acid, and fosfomycin develops rapidly, these
compounds
must
not be administered alone.
Since
the majority of clinically recovered CoNS strains are methicillin resistant, most
infections
by CoNS require treatment with vancomycin or, where appropriate, the new
agents
described above. Replacement of these by β-lactamase-resistant penicillins is
advisable
for methicillin-susceptible isolates. When used simultaneously, antibiotics
with cell
wall
activity (β-lactams and vancomycin) combined with rifampin were shown to act
synergistically;
however, this combination is not recommended for catheter-related
bloodstream
infections (125). FBRIs remain a therapeutic challenge and frequently require
removal
of the device (125, 198).
Prospective
studies on the most appropriate treatment for patients infected with
staphylococcal
SCVs are unavailable. A reduced susceptibility to aminoglycosides can be
expected
(16). Resistance to trimethoprim-sulfamethoxazole is observed in
thymidineauxotrophic
SCVs
(96). Due to the fact that SCVs may persist intracellularly, a
combined
treatment
regimen of rifampin (intracellular activity) with either β-lactam antibiotics
or
vancomycin
(for methicillin-resistant SCVs) may be effective.
Micrococcaceae
and Dermacoccaceae
Systematic
data on susceptibilities of the two
families
Micrococcaceae and Dermacoccaceae are rare, and the real species
affiliation in
older
reports is often unclear. Members of the genera Micrococcus and Kocuria
appear to be
susceptible
to β-lactams, macrolides, tetracycline, linezolid, rifampin, and the
glycopeptides;
however,
clinical isolates resistant to these agents have been reported (25, 95, 197).
While
most
kytococcal isolates have been susceptible to carbapenems, gentamicin,
ciprofloxacin,
tetracycline,
rifampin, and glycopeptides, kytococci are usually resistant to penicillin G,
cephalosporins,
and oxacillin (not mecA- based) (25, 95, 174).
An antibiotic regimen that
has
been suggested for the treatment of infection by members of both families is a
combination
of vancomycin with rifampin and gentamicin (199). R.
mucilaginosa appears to
be
variable in its antimicrobial susceptibility (197, 200).
The observation that R.
mucilaginosa
exhibits poor to no growth on Mueller-Hinton agar makes
susceptibility testing
a
challenge for clinical laboratories. A supplementation with 5% sheep blood and
incubation
in
6% CO2 may enhance susceptibility testing (197, 200).
Alloiococcus
A.
otitis has been reported as susceptible to ampicillin, cefotaxime,
tetracycline, and
vancomycin
but resistant to macrolides, azithromycin, and co-trimoxazole (10, 29, 46).
In a
ddition
to gentamicin-resistant isolates, some A. otitis isolates with
intermediate levels of
resistance
to β-lactams have been reported, although they are β-lactamase negative.
EVALUATION, INTERPRETATION, AND REPORTING OF
RESULTS Back to top
Distinguishing
contaminants and colonizers from staphylococcal and “micrococcal” isolates
causing
infection continues to be an important challenge for laboratorians and
clinicians. It is
imperative
to have an appreciation of the quality of the specimen under consideration.
Clinical
features and the results of other investigations should be taken into account
during
the
interpretative process. There is no replacement for good communication between
laboratory
staff and primary physicians.
Cultivation
and identification of the causative pathogen to the species level represent the
“gold
standard” for the diagnosis of staphylococcal infections. The most critical
step when
interpreting
a culture that is suspect for staphylococci is to distinguish between S.
aureus
and other species. Due to the importance of the report of “S.
aureus” and, in
particular,
of “MRSa” for prognosis, therapy, hospital hygiene, and infection control, any
uncertainty
regarding the species identification or the susceptibility to oxacillin
(cefoxitin)
should
be investigated via a second independent method. Considering costs and
rapidity,
applicable
routine proceedings may comprise the use of respective latex agglutination
assays
combined
with the use of automated systems for identification and susceptibility
testing. In
case
of doubt, further tests, preferentially nucleic acid-based approaches, should
be applied.
However,
the need for additional procedures must be weighed against delay in informing
the
physician
concerning a preliminary S. aureus identification as the putative
causative agent.
Species-level
identification of CoNS associated with infection should be considered, though
it
is
still a matter for debate because species-level identification of CoNS rarely
leads to an
intervention
or to changes in therapy. To rule out S. saprophyticus for urine
isolates, the
novobiocin
test could be used. Isolates from deep-tissue infections and blood cultures of
patients
with suspected endocarditis should be differentiated to the species level,
since the
identification
of S. lugdunensis raises the index of suspicion for aggressive disease.
To rule
out S.
lugdunensis for invasive and sterile-site isolates, its positivity for
pyrrolidonase and
ornithine
decarboxylase could be considered (Tables 2 and 3).
Other CoNS have also been
increasingly
described as causative pathogens of severe infection. Ribotyping and/or PFGE
should
be used when determination of genetic relatedness is required. Contaminants and
colonizing
CoNS do not require susceptibility testing or identification. Several reports
suggest
that
cultures of colonially indistinguishable CoNS may contain multiple different
strains (67).
Extending
the incubation period of the initial cultures to 72 h may enable different
colony
types
to be more readily identified.
Unless
there is strong evidence to the contrary, isolation of S. aureus from a
sterile-site
culture
(such as aspirated pus, blood, or cerebrospinal fluid) should be considered
clinically
significant.
Contamination of high-quality samples by S. aureus is rare, and further
samples
should
be taken if there is clinical doubt. Interpreting the isolation of S. aureus
from
specimens
contaminated with elements of a normal microbiota requires consideration of
setting,
clinical features, and recent interventions. Quantitative culture may be
helpful, for
example,
when interpreting a bronchoalveolar lavage or urine sample. In persons with S.
aureus
endocarditis, bacteremia is continuous and associated with higher
loads, while in
cases
with transient bacteremia (e.g., manipulations with mucous membrane trauma)
bacteremia
is associated with lower loads and typically has a short duration. Because
quantitative
blood cultures are not routinely performed, the time between incubation onset
and
growth detection (defined as the time to positivity) may provide additional
information in
continuous
blood culture-monitoring systems. Rapid growth of S. aureus (within 14 h
after
the
initiation of incubation) has been associated with a high likelihood of
endovascular
infection,
delayed clearance, and complications (98).
Isolation of S. aureus from surgical
wounds
and other sites such as ulcers may represent infection or colonization, and the
clinician
’s response to the culture report should be guided by a bedside assessment of
signs
and
symptoms of infection. Colonization alone is an insufficient reason to treat,
unless the
patient
is colonized by MRSA and decolonization is undertaken as part of a specific
infection
control
policy.
Compared
to S. aureus, interpreting the significance of cultures that are
positive for CoNS is
more
challenging. CoNS are an important cause of nosocomial bloodstream infections,
but
they
are also the most common contaminants of blood cultures (79).
It is obvious that
samples
taken from colonized sites will contain members of these species. Blood samples
or
biopsy
specimens taken without careful skin cleaning and disinfection will become
contaminated
with the microbiota of the skin or mucous membranes. However, even careful
attention
to collection techniques will not prevent all episodes of contamination.
Interpretation
of blood cultures positive for CoNS requires knowledge of the presence of
prosthetic
material in the intravascular compartment, risk factors for true CoNS sepsis
such
as
prematurity or the presence of an impaired immune system, and clinical features
of
sepsis.
Factors helpful in distinguishing between true positive and contaminated
cultures
taken
from a patient with clinical features of infection include (i) isolation of a
strain in pure
culture
from the infected site or body fluid and (ii) the repeated isolation of the
same strain
or
combination of strains over the course of the infection (103, 186).
An algorithm to reduce
misclassification
of nosocomial bloodstream infections due to CoNS was defined as at least
two
blood cultures positive for CoNS within 5 days or one positive blood culture
plus clinical
evidence
of infection (26).
The
presence of the same CoNS strain on an intravenous catheter tip and in a blood
culture
is
supportive evidence for intravenous catheter-associated bacteremia. Measurement
of
differential
time to positivity between blood cultures drawn through the central venous
catheter
and those drawn from the peripheral vein was reported as highly sensitive and
specific
for the in situ diagnosis of catheter-related bloodstream infection in patients
with
short-
and long-term catheters; however, other studies did not show that this method
was of
major
diagnostic value (149, 153, 165). Isolation of CoNS from peritoneal dialysis fluid or
cerebrospinal
fluid taken from ventricular shunts in a patient suspected of having infection
is
usually
significant. While most contaminated clinical specimens produce mixed cultures
of
different
strains and/or species, some infections may be attributable to more than one
strain
or
species (67).
For
patients with complicated cases, who probably have a true CoNS infection, it is
advisable
to
develop a sampling strategy. This is particularly pertinent when dealing with
patients with
low-grade
infection associated with implanted prosthetic material such as a joint
replacement
or
vascular graft. Samples should include those from each anatomical layer or
region, and
fresh
instruments should be used to gather deep-site samples (11).
Susceptibility
testing, and in particular the detection of methicillin resistance, should
always
be
carried out following the identification of S. aureus. For surveillance
cultures to detect
MRSA
colonization, susceptibility testing beyond the determination of methicillin
resistance
may
only be needed for patients undergoing decolonization to predict the success of
mupirocin
therapy. All CoNS associated with true infection require susceptibility
testing.
The
considerations of clinical significance discussed for CoNS are also appropriate
for
members
of theMicrococcaceae and Dermacoccaceae families; however, the
criteria used for
distinguishing
etiologically relevant isolates from contaminants and colonizers, respectively,
should
be applied much more strictly. Since species of the Kytococcus genus are
resistant to
some
β-lactams, they should be (if clinically relevant) carefully distinguished from
other
“micrococci” that are
usually susceptible.
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