Direct Detection Methods

 Microscopy

 The majority of the genera included within this chapter produce spherical, gram-positive cells. However, some of the species within the Micrococcaceae or Dermacoccaceae exhibit rod-shaped cells and are motile. During cell division, the organisms divide along both longitudinal and horizontal planes, forming pairs, tetrads, and, ultimately, irregular clusters (Figure 1). Gram stains should be performed on young cultures, because very old cells may lose their ability to retain crystal violet and may appear gram variable or gram negative. Staphylococci appear as gram-positive cocci, usually in clusters. Micrococci typically appear as gram-positive cocci in tetrads, rather than large clusters. The additional related genera (i.e., Kytococcus, Nesterenkonia, Dermacoccus, Arthrobacter, and Kocuria) resemble the staphylococci microscopically.

Fig1. Gram stain of Staphylococcus aureus from blood agar. 

 Nucleic Acid Testing

 Several rapid nucleic acid amplification methods have been developed including the Staphylo Resist (plus) (Amplex Diagnostics, Gars-Bahnhof, Germany) and StaphPlex Panel (Qiagen). These methods are PCR amplification approaches capable of detecting methicillin resistant staphylococci from clinical swabs. The assays detect the mecA gene (which encodes the methicillin resistance) in conjunction with a species-specific target gene. Caution should be used in the interpretation of these results, as several species of staphylococci may reside in the normal flora including methicillin-resistant CoNS causing false positives.

Single-locus amplification is available in several test systems, including the BD Gene OHM MRSA assay (BD, Franklin Lakes, New Jersey), Genotype MRSA Direct and Geno-Quick MRSA (Hain Lifescience, Xpert MRSA (Cepheid, Sunnyvale, California), and the Roche Light Cycler MRSA (Roche, Basel, Switzerland). These methods utilize a set of oligonucleotide primers that bind to the downstream sequence of the staphylococcal cassette chromosome region encoding the mec region (SCCmec) and the flanking open reading frame (orfX). This allows for amplification of the nucleic acid region that indicates antibiotic resistance coupled with a species-specific marker. However, presence of the amplicon does not ensure the presence of or the absence of methicillin resistant S. aureus. This is due to the variability associated with chromosomal recombination within the cassette region that may include partial or full deletion or exchange of antibiotic genes within the cassette. For this reason, it is recommended that positive nucleic acid based testing be utilized as a preliminary result and confirmatory culture and antimicrobial sensitivity testing is recommended.

Cultivation

Media of Choice

 The organisms will grow on 5% sheep blood and chocolate agars. They also grow well in broth-blood culture systems and common nutrient broths, such as thioglycolate, dextrose broth, and brain-heart infusion.

Selective media can also be used to isolate staphylococci from clinical material. Phenylethyl alcohol (PEA) or Columbia colistin-nalidixic acid (CNA) agars may be used to eliminate contamination by gram-negative organ isms in heavily contaminated specimens such as feces. In addition, mannitol salt agar may be used for this purpose. This agar contains a high concentration of salt (10%), the sugar mannitol, and phenol red as the pH indicator. S. aureus ferments mannitol and produces a yellow halo on this media as a result of acid production altering the pH (Figure 2).

Fig2. A, Yellow colonies of S. aureus fermenting mannitol as evident by the yellow color of the agar. B, White colonies of S. epidermidis, no-mannitol fermenting, as evident by the original pink color of the agar. (Photos courtesy of Malissa Tille, Sioux Falls, South Dakota.)

CHROMagar (originally invented by Alain Rambach) is a selective and differential media for the identification of methicillin-resistant Staphylococcus aureus. The media are now available from a variety of manufacturers. These media are becoming more widely used for the direct detection of nasal colonization. The medium is selective because it contains cefoxitin, and MRSA is resistant to this antibiotic. The addition of chromogenic substrates hydrolyzed by the organisms produce a mauve-colored colony, allowing for the identification of the organisms. Other organisms will hydrolyze various chromogenic substances within the media, resulting in a variety of colored colonies from white to blue to green (Figure 3).

Fig3.  CHROMagar for the identification of MRSA isolates  through the selective and differential formation of mauve-colored  colonies. (Photo courtesy of Avera Regional Laboratory, Sioux Falls,  South Dakota.)

Incubation Conditions and Duration

 Visible growth on 5% sheep blood and chocolate agars incubated at 35° C in carbon dioxide (CO2) or ambient air usually occurs within 24 hours of inoculation. Mannitol salt agar and other selective media may require incubation for at least 48 to 72 hours before growth is detected.

 Colonial Appearance

 Table 1 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis) of each genus and various staphylococcal species on 5% sheep blood agar. Growth on chocolate agar is similar. S. aureus yields colonies surrounded by a yellow halo on mannitol salt agar. In addition, small colony variants of S. aureus appear as small pinpoint, nonhemolytic and nonpigmented colonies on blood agar. Small colony variants (SCVs) may result from limited nutrients or other selective pressures and may revert to the normal S. aureus phenotype following subculture. However, other staphylococci (particularly S. saprophyticus) may also ferment mannitol and thus resemble S. aureus on this medium.

Table1.  Colonial Appearance and Characteristics on 5% Sheep Blood Agar

Approach to Identification

 Most commercial systems are successful in the identification of S. aureus, S epidermidis, and S. saprophyticus. The identification of the other species varies from system to system. In addition, automated systems may not correctly identify nutritionally variant forms such as small colony variants and other unusual isolates.

Gram stains are used in the clinical laboratory as the initial presumptive identification method for all gram-positive cocci. Microscopic along with macroscopic colonial morphology (see Table 1) provides a presumptive identification. The Staphylococci spp. and Micrococci spp. are distinguishable from the related family Streptococcaceae by the catalase test. Table 2 shows how the catalase-positive, gram-positive cocci can be differentiated. Because they may show a pseudocatalase reaction—that is, they may appear to be catalase positive—Aerococcus and Enterococcus are included in Table 2; Rothia (formerly Stomatococcus) is included for the same reason. Once an organism has been characterized as a gram-positive, catalase-positive, coccoid bacterium, complete identification may involve a series of tests, including (1) atmospheric requirements, (2) resistance to 0.04 U of bacitracin (Taxo A disk) and furazolidone, and (3) possession of cytochrome C as determined by the microdase (modified oxidase) test. However, in the busy setting of many clinical laboratories, microbiologists proceed immediately to a coagulase test based on recognition of a staphylococcal-like colony and a positive catalase test.

Table2. Differentiation among Gram-Positive, Catalase-Positive Cocci

Microdase disks, a modified oxidase test, are available commercially (Remel, Inc., Lenexa, Kansas). The test is used for differentiating Micrococcus spp. from Staphylococcus spp. A visible amount of growth from an 18- to 24-hour-old culture is smeared on the disk; Micrococcus spp. turn blue within 2 minutes . A variety of tests including the formation of acid from carbohydrates followed by tests for glycosidases, hydrolases, and peptidases are used for species identification and are included in a variety of identification panels.

Both for bacitracin and for furazolidone resistance, disk tests are used (Figure 4). A 0.04-U bacitracin impregnated disk and a 100-µg furazolidone-impregnated disk, both available from Becton Dickinson and Company, are placed on the surface of a 5% sheep blood agar streaked in three directions with a cotton-tipped swab that has been dipped in a bacterial suspension prepared to match the turbidity of the 0.5 McFarland standard (i.e., the same as that used in preparing inoculum for disk diffusion susceptibility tests ). The tests are then interpreted based on the inhibition or sensitivity of the bacteria by measuring the zone of inhibition present around the disk.

Additional rapid identification systems are available for presumptive screening for the detection of clumping factor A, a cell wall-associated adhesin for fibrinogen and protein A.

Comments Regarding Specific Organisms

 Micrococcus spp. and related genera are (1) not lysed with lysostaphin, (2) resistant to the antibiotic furazolidone, (3) susceptible to 0.04 U of bacitracin, and (4) microdase positive; they usually will only grow aerobically. In contrast, staphylococci are (1) lysed with lysostaphin, (2) resistant to 0.04 U of bacitracin, (3) susceptible to furazolidone, (4) microdase-negative, and (5) facultatively anaerobic.

Once an isolate is identified as, or strongly suspected to be, a species of staphylococci, a test for coagulase production is performed to separate S. aureus from the other species collectively referred to as coagulase-negative staphylococci (Figure 5).

Fig5. Staphylococcal identification to species. (Based on the methods in Hébert GA, Crowder CG, Hancock GA, et al: Characteristics  of coagulase-negative Staphylococci that help differentiate these species and other members of the family Micrococcaceae, J Clin Microbiol  26:1939, 1988.)

The enzyme coagulase produced by S. aureus binds plasma fibrinogen and activates a cascade of reactions causing plasma to clot. An organism can produce two types of coagulase, referred to as bound and free (see Procedure 13-13 for further information on coagulase tests). Bound coagulase, or clumping factor, is detected using a rapid slide test (i.e., the slide coagulase test), in which a positive test is indicated when the organisms agglutinate on a glass slide when mixed with plasma . Most, but not all, strains of S. aureus produce clumping factor and thus are readily detected by this slide test. Approximately 10% to 15% of strains may give a negative slide coagulase test as a result of the masking by capsular polysaccharides. In addition, false positives may occur as a result of auto agglutination when colonies are grown on media with high salt concentrations.

Isolates suspected of being S. aureus but failing to produce bound coagulase must be tested for production of extracellular (i.e., free) coagulase because S. lugdunen sis and S. schleiferi may give a positive slide coagulase test. This test, referred to as the tube coagulase test, is per formed by inoculation of a tube containing plasma and incubating at 35° C. Production of the enzyme results in a clot formation within 1 to 4 hours of inoculation . Some strains produce fibrinolysin, dis solve the clot after 4 hours of incubation at 35° C, and may appear to be negative if allowed to incubate longer than 4 hours. Because citrate-utilizing organisms may yield false-positive results, plasma containing ethylene diaminetetraacetic (EDTA) rather than citrate should be used.

Various commercial systems are available that substitute for the conventional coagulase tests previously described. Latex agglutination procedures that detect clumping factor and protein A and passive hemagglutination tests capable detecting clumping factor are no longer used as extensively because they often fail to detect methicillin-resistant S. aureus strains, which are being isolated from an increasing number of community acquired infections. In addition, the recent third generation assays that include monoclonal antibodies to the capsular polysaccharide serotypes 5 and 8 or other molecules have a higher sensitivity but are not as specific.

False-positive reactions occur in the presence of some CoNS species such as S. haemolyticus, S. hominis, and S. saprophyticus.

In addition to the screening tests previously described, a variety molecular testing methodologies have been developed for the rapid identification of staphylococci. Accuprobe is a commercially available DNA probe assay available for the confirmation of an identification of S. aureus (Gen-Probe, Inc., San Diego, California). S. aureus may also be identified by amplification of the nuc gene, which encodes a thermostable nuclease. The amplified DNA product is approximately a 270 bp fragment. The detection limit for successful isolation and amplification is less than 10 colony-forming units or 0.69 pg of DNA. This method is highly specific. Additional amplification assays have been developed that detect species-specific genes or chromosomal sequences including 16S and 23SrRNA genes and spacer regions, elongation factor (tuf), DNA gyrase (gyrA), superoxide dismutase (sodA), glyceraldehyde-3-phosphate dehydrogenase gene (gap), and a heat shock protein (HSP60/ GroE). MRSA has been identified using the staphylococcal insertion sequence IS431.

A qualitative nucleic acid hybridization assay that targets rRNA sequences in S. aureus and CoNS has been developed by bioMerieux. The hybridization assay is based on the binding of a peptide nucleic acid (PNA) labeled with a fluorescent dye to S. aureus in a blood smear prepared from a positive blood culture bottle.

Table3 provides the results for various tests used to differentiate the coagulase-positive staphylococci; S. intermedius is an important agent isolated from dog bite wound infections and may be misidentified as S. aureus if only coagulase testing is performed. Microbiologists should perform additional confirmatory tests in cases in which coagulase-positive staphylococci are isolated from dog bite infections. Otherwise, catalase-positive, gram-positive cocci in clusters from a white to yellow, creamy, opaque colony on blood agar that is slide coagulase-positive and tube coagulase-positive in 4 hours may be presumptively identified as S. aureus.

Table 3. Differentiation among the Most Clinically Significant Coagulase-Positive Staphylococci

Most laboratories do not identify the coagulase negative staphylococci to species. However, exceptions may include isolates from normally sterile sites (blood, joint fluid, or cerebrospinal fluid [CSF]), isolates from prosthetic devices, catheters, and shunts; and isolates from urinary tract infections that may be S. saprophyticus.

The coagulase-negative staphylococci may be identified based on the criteria shown in Figure 5 and Tables 3 through 7 . Isolates not identified to species are reported as “coagulase-negative staphylococci.”

It is particularly important to differentiate S. lugdunensis from other coagulase-negative staphylococci from sterile sites because there are different interpretive criteria for susceptibility to oxacillin for this organism. S. lugdunensis is positive for both the 2-hour PYR and ornithine decarboxylase tests.

Table4. Differentiation among Coagulase-Negative, PYR-Negative, Novobiocin-Resistant Staphylococci

Table 5. Differentiation among Coagulase-Negative, PYR-Negative, Novobiocin-Susceptible, Alkaline  Phosphatase-Negative Staphylococci

Table 6. Differentiation of Coagulase-Negative, PYR-Positive, Novobiocin-Susceptible, Alkaline Phosphatase-Negative Staphylococci

Table 7 . Differentiation of Coagulase-Negative, PYR Positive, Novobiocin-Susceptible, Alkaline Phosphatase–Positive  Staphylococci

 Serodiagnosis

 Serologic testing for antibodies associated with infections with Staphylococcal organisms is not clinically relevant due to low specificity and cross-reacting antibodies. Anti bodies to teichoic acid, a major cell wall component of gram-positive bacteria, are usually produced in long standing or deep-seated staphylococcal infections, such as osteomyelitis. This procedure is usually performed in reference laboratories. However, the clinical utility of performing this assay is, at best, uncertain. The identification of protective antibodies in toxin-mediated syndromes such as toxic shock syndrome and staphylococcal scalded skin syndrome may be absent or present at very low levels. However, seroconversion following the onset of symptoms and during convalescence may be observed. Various kits are available for the detection of staphylococcal toxins in foods or patient specimens that may be helpful in clinical diagnosis. Additional assays for the detection of other staphylococcal proteins are being examined for their clinical utility in identifying staphylococcal infections.