General Characteristics

The gram-positive cocci are a very heterogenous group. Historically, the genus Staphylococcus was included with the genus Micrococcus in the family Micrococcaceae. However, molecular phylogenetic and chemical analysis has indicated that these two genera are not closely related. The Staphylococcus spp. has now been combined with the Bacillaceae, Planococcaceae, and Listeriaceae into the order Bacillales. There are approximately 39 species and 21 subspecies within the genus Staphylococcus. Several of the Micrococcus species are now reclassified into the genera Kocuria, Nesterenkonia, Kytococcus, and Dermacoccus. These genera have now been reorganized into two families, the Micrococcaceae and the Dermacoccaceae. The only other organism, Alloiococcus otitidis, that biochemically reacts similar to the families included in this chapter belongs to the family Carnobacteriaceae. The species described in this chapter are all catalase-positive, gram-positive cocci. The organisms are aerobic or facultative anaerobic with the exception of S. aureus subsp. anaerobius and S. sacchrolyticus, obligate anaerobes, and may be catalase negative. However, only those belonging to the genus Staphylococcus are of primary clinical significance. Staphylococcus are nonmotile and non-spore forming. Several of the coagulase-negative staphylococci (CoNS or non–Staphylococcus aureus) species listed may be encountered in clinical specimens. The CoNS have been subdivided into two groups based on their novobiocin susceptibility pattern. The CoNS group that demonstrates novobiocin susceptibility includes S. epidermidis, S. capitis, S. haemolyticus, S. hominis subsp. hominis, S. lugdunensis, S. saccharolyticus, S. warneri, and other species. The novobiocin resistant group consists of such species as S. cohnii, S. kloosii, S. saprophyticus, and S. xylosus. The skin colonizers Micrococcus sp., Kocuria sp. and Kytococcus sp. are easily confused with staphylococci. Occasionally, these genera will be associated with skin lesions and are more commonly isolated form immunocompromised patients.

Epidemiology

As outlined in Table 14-1, the staphylococci associated with infections in humans are colonizers of various skin and mucosal surfaces. There are three types of nasal carrier states associated with S. aureus: persistent carriers that harbor a single strain for an extended period of time, intermittent carriers that will harbor different strains over time, and then individuals that do not harbor any organisms or non-carriers. Because the carrier state is common among the human population, infections are frequently acquired when the colonizing strain gains entrance to a normally sterile site as a result of trauma or abrasion to the skin or mucosal surface. However, the traumatic event often may be so minor that it goes unnoticed. Health care workers have a high incidence of carrier state along with immunocompromised individuals, including those with insulin-dependent diabetes mellitus, long-term hemodialysis patients, and IV drug users. Vaginal carriage may be seen in premenopausal women.

Staphylococci are also transmitted from person to person. Upon transmission, the organisms may become established as part of the recipient’s normal flora and later introduced to sterile sites by trauma or invasive medical procedures, such as surgery. Person-to-person spread of staphylococci, particularly antimicrobial-resistant strains, occurs in hospitals and presents substantial infection control problems. However, more recently serious S. aureus infections have been encountered in the community setting as well.

Pathogenesis and Spectrum of Disease

Without question, S. aureus is the most virulent species of staphylococci encountered. A wide spectrum of factors, not all of which are completely understood, contribute to this organism’s ability to cause infections and disease. S. aureus and S. epidermidis produce a polysaccharide capsule that inhibits phagocytosis. The capsule, which is produced in various amounts by individual clinical isolates, may appear as a slime layer or biofilm, allowing the organisms to adhere to inorganic surfaces and circumventing the actions of antibiotics. The gram-positive cell wall chemical composition is also implicated in the mediation of pathogenesis. The peptidoglycan resembles the endotoxin effect of gram negatives by activating complement, interleukin 1 (IL-1), and acting as a chemotactic factor for the recruitment of PMNs. This cascade of events causes swelling and may lead to the exacerbation of tissue damage because of the additional virulent factors produced by the organisms. S. aureus produces a surface protein, known as protein A. This protein is bound to the cytoplasmic membrane of the organism and has a high affinity for the Fc receptor on IgG molecules as well as complement. This provides a mechanism for the organisms to bind the immune active molecules, decreasing the ability for clearance of the organism from the site of infection. Several toxins and enzymes mediate tissue invasion and survival at the infection site (Table 14-2). Cytotoxins alpha, beta, delta, and gamma are produced by a variety of species. Most strains of S. aureus produce alpha toxin, which disrupts the smooth muscle in blood vessels and is toxic to erythrocytes, leukocytes, hepatocytes, and platelets. Beta toxin, believed to work in conjunction with the alpha toxin, is a heat-labile sphingomyelinase, which catalyzes the hydrolysis of membrane phospholipids resulting in cell lysis. S. aureus, S. epidermidis, and S. haemolyticus have been identified as capable of producing Delta toxin, which is cytolytic to erythrocytes and demonstrates nonspecific membrane toxicity to other mammalian cells. Gamma toxin is produced by all strains of S. aureus and may actually function in association with the Panton-Valentine leukocidin (PVL). Elaboration of these factors is chiefly responsible for the various skin, wound, and deep tissue infections commonly caused by S. aureus. Many of these infections can rapidly become life threatening if not treated and managed appropriately.

Thirty to fifty percent of all S. aureus strains are capable of producing one of eight distinct serologic types of a heat-stable enterotoxin. The enterotoxins are resistant to hydrolysis by the gastric and intestinal enzymes. The toxins, which are often found in milk products, are associated with pseudomembranous enterocolitis and toxic shock syndrome, and they may exacerbate the normal immune response, resulting in further tissue damage and systemic pathology.

Localized skin or soft tissue infections (SSTIs) may involve hair follicles (i.e., folliculitis) and spread into the tissue causing boils (i.e., furuncles). More serious, deeper infections result when the furuncles coalesce to form carbuncles. Impetigo, the S. aureus skin infection involving the epidermis, is typified by the production of vesicles that rupture and crust over. Regardless of the initial site of infection, the invasive nature of this organism always presents a threat for deeper tissue invasion, bacteremia, and spread to one or more internal organs including the respiratory tract. Furthermore, these serious infections have emerged more frequently among the general population and are associated with strains that produce the PVL toxin. PVL is toxic to white blood cells, preventing clearance of the organism by the immune system. These serious soft tissue “community-associated” infections are frequently mediated by methicillin-resistant S. aureus (community-acquired MRSA or CA-MRSA).

S. aureus also produces toxin-mediated diseases, such as scalded skin syndrome and toxic shock syndrome. In these cases, the organisms may remain relatively localized, but production of potent toxins causes systemic or widespread effects. With scalded skin syndrome (Ritter’s disease), which usually afflicts neonates, the exfoliative toxin is a serine protease that splits the intracellular bridges of the epidermidis, resulting in extensive sloughing of epidermis to produce a burnlike effect on the patient. The toxic shock syndrome toxin (TSST-1), also referred to as pyrogenic exotoxin C, has several systemic effects, including fever, desquamation, and hypotension potentially leading to shock and death.

Other coagulase-positive or variable staphylococci are normal flora of a variety of animal species including dogs. These species include S. intermedius, S. pseudointermedius, and S. delphini. These organisms may be associated with skin infections in dogs, as well as invasive infections in immunocompromised humans or a result of a bite or scratch wound.

The coagulase-negative staphylococci, among which S. epidermidis is the most commonly encountered, are substantially less virulent than S. aureus and are opportunistic pathogens. Their prevalence as nosocomial pathogens is as much, if not more, related to medical procedures and practices than to the organism’s capacity to establish an infection. Infections with S. epidermidis and, less commonly, S. haemolyticus and S. lugdunensis usually involve implantation of medical devices (see Table 14-2). This kind of medical intervention allows invasion by these normally noninvasive organisms. Two organism characteristics that do enhance the likelihood of infection include production of a slime layer or biofilm-facilitating attachment to implanted medical devices and the ability to acquire resistance to most of the antimicrobial agents used in hospital environments. S. lugdunensis infections resemble S. aureus infections.

Although most coagulase-negative staphylococci are primarily associated with nosocomial infections, urinary tract infections caused by S. saprophyticus are clear exceptions. This organism is most frequently associated with community-acquired urinary tract infections in young, sexually active females but is not commonly associated with hospital-acquired infections or any infections at non–urinary tract sites. It is the second most common (following Escherichia coli) as the cause of urinary tract infections in young women.

Because coagulase-negative staphylococci are ubiquitous colonizers, they are frequently found as contaminants in clinical specimens. This fact, coupled with the emergence of these organisms as nosocomial pathogens, complicates laboratory interpretation of their clinical significance. When these organisms are isolated from clinical specimens, every effort should be made to substantiate their clinical relevance in a particular patient.

The Micrococcaceae and Dermacoccaceae are generally normal flora of the skin, some of the genera including Micrococcus, Kocuria, and Kytococcus spp. have been associated with infections such as endocarditis, pneumonia, sepsis, and skin infections in immunocompromised patients. What, if any, virulence factors are produced by the remaining genera within this group is not known. Because these organisms are rarely associated with infections in healthy individuals, they are probably of low virulence.

No special considerations are required for specimen collection and transport of the organisms discussed in this chapter. Refer to Table 5-1 for general information on specimen collection and transport.

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 14-1). 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.

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 LightCycler 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 thioglycollate, 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 organisms 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 14-2).

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 14-3).

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 14-3 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.

TABLE 14-3

Colonial Appearance and Characteristics on 5% Sheep Blood Agar

Organism	Appearance
Micrococcus spp. and related organisms*	Small to medium (1-2 µm); opaque, convex; nonhemolytic; wide variety of pigments (white, tan, yellow, orange, pink)
Staphylococcus aureus	Medium to large (0.5-1.5 µm); smooth, entire, slightly raised, low convex, opaque; most colonies pigmented creamy yellow; most colonies beta-hemolytic
S. epidermidis	Small to medium; opaque, gray-white colonies; most colonies nonhemolytic; slime-producing strains are extremely sticky and adhere to the agar surface
S. haemolyticus	Medium; smooth, butyrous, and opaque; beta-hemolytic
S. hominis	Medium to large; smooth, butyrous, and opaque; may be unpigmented or cream-yellow-orange
S. lugdunensis	Medium to large; smooth, glossy, entire edge with slightly domed center; unpigmented or cream to yellow-orange, may be β-hemolytic
S. warneri	Resembles S. lugdunensis
S. saprophyticus	Large; entire, very glossy, smooth, opaque, butyrous, convex; usually white but colonies can be yellow or orange
S. schleiferi	Medium to large; smooth, glossy, slightly convex with entire edges; unpigmented
S. intermedius	Large; slightly convex, entire, smooth, glossy, translucent; usually nonpigmented
S. hyicus	Large; slightly convex, entire, smooth, glossy, opaque; usually nonpigmented
S. capitis	Small to medium; smooth, slightly convex, glistening, entire, opaque; S. capitis subsp. urealyticus usually pigmented (yellow or yellow-orange); S. capitis subsp. capitis is nonpigmented
S. cohnii	Medium to large; convex, entire, circular, smooth, glistening, opaque; S. cohnii subsp. urealyticum usually pigmented (yellow or yellow-orange); S. cohnii subsp. cohnii is nonpigmented
S. simulans	Large; raised, circular, nonpigmented, entire, smooth, slightly glistening
S. auricularis	Small to medium; smooth, butyrous, convex, opaque, entire, slightly glistening; nonpigmented
S. xylosus	Large; raised to slightly convex, circular, smooth to rough, opaque, dull to glistening; some colonies pigmented yellow or yellow-orange
S. sciuri	Medium to large; raised, smooth, glistening, circular, opaque; most strains pigmented yellow in center of colonies
S. caprae	Small to medium; circular, entire, convex, opaque, glistening; nonpigmented

^*Includes Kytococcus, Nesterenkonia, Dermacoccus, Kocuria, and Arthrobacter.

Approach to Identification

The commercial systems for identification of Staphylococcus spp. and Micrococcus spp. are discussed in Chapter 13. 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 14-3) provides a presumptive identification. The Staphylococci spp. and Micrococci spp. are distinguishable from the related family Streptococcaceae (see Chapter 15) by the catalase test. Table 14-4 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 14-4; 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.

TABLE 14-4

Differentiation among Gram-Positive, Catalase-Positive Cocci

				Resistance to:
Organism	Catalase	Microdase (modified oxidase)	Aerotolerance	Bacitracin (0.04 U)a	Furazolidone (100 µg)a	Lysostaphin (200 µg/µL)
Staphylococcus	+b	−c	FA	R	S	S
Micrococcus (and related organisms)	+	+	Ad	S	R	Re
Macrococcus	+	+	±	+	S	S
Rothia	±	−	FA	R or S	R or S	R
Aerococcus	−f	−	FAg	S	S	R
Alloiococcus	±	−	A	ND	ND	ND
Enterococcus	−f	−	FA	R	S	R
Streptococcus	−	−	FA	+d	−	+

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