Chapter 15 1. Describe the general characteristics of Streptococcus spp. and Enterococcus spp., including oxygenation, microscopic Gram-staining characteristics, and macroscopic appearance on blood agar. 2. Explain the Lancefield classification system for Streptococcus spp. 3. Identify the clinical infections associated with Streptococcus spp., Enterococcus spp., and related gram-positive cocci. 4. Describe the patterns of hemolysis for clinically significant species of Streptococci and Enterococci. 5. Explain the chemical principles for isolation of Streptococcus spp. and Enterococcus spp. on selective and differential media; include 5% sheep blood agar and Enterococcosel agar. 6. Compare and contrast streptolysin O and streptolysin S, including oxygen stability, immunogenicity, and appearance on blood agar. 7. Describe the major significance of serologic testing procedures for Anti streptolysin O and Anti streptolysin S, in combination with anti-DNase for diagnosis of poststreptococcal sequelae. 8. Explain the activity for the virulence factors of Streptococcus pyogenes and the pathogenic effects of each including M protein, hyaluronic acid capsule, streptokinase, F protein, hylauronidase, and the streptococcal pyrogenic exotoxins. 9. Explain the significance of S. agalactiae (group B) in perinatal infections. 10. Identify the two major virulence factors associated with S. pneumoniae, and describe their effect on the pathogenesis of the infection. 11. Describe the colony morphology, clinical significance, and laboratory techniques for the identification and recovery of the nutritionally variant streptococci. 12. List the appropriate clinical specimens for isolation of the individual Streptococcus spp., Enterococcus and Aerococcus viridans, Alloiococcus otitidis, Gemella, Leuconostoc, and Pediocococus. 13. Identify a clinical isolate based on the results from standard laboratory diagnostic procedures. Many of these organisms are commonly found as part of normal human flora and are encountered in clinical specimens as contaminants or as components of mixed cultures with minimal or unknown clinical significance (Table 15-1). However, when these organisms gain access to normally sterile sites, they can cause life-threatening infections. Other organisms, most notably Streptococcus pneumoniae and Streptococcus pyogenes, are notorious pathogens. Although S. pneumoniae can be found as part of the normal upper respiratory flora, this organism is also the leading cause of bacterial pneumonia and meningitis. Similarly, although S. pyogenes may be carried in the upper respiratory tract of humans, it is rarely considered to be normal flora and should be deemed clinically important whenever it is encountered. At the other extreme, organisms such as Leuconostoc spp. and Pediococcus spp. usually are only capable of causing infections in severely compromised patients. TABLE 15-1 Many of the organisms listed in Table 15-1 are spread person to person by various means and subsequently establish a state of colonization or carriage; infections may then develop when colonizing strains gain entrance to normally sterile sites. In some instances, this may involve trauma (medically or non-medically induced) to skin or mucosal surfaces or, as in the case of S. pneumoniae pneumonia, may result from aspiration into the lungs of organisms colonizing the upper respiratory tract. The capacity of the organisms listed in Table 15-2 to produce disease and the spectrum of infections they cause vary widely with the different genera and species. TABLE 15-2 Pathogenesis and Spectrum of Disease S. pyogenes, the most clinically important Lancefield group A, produces several factors that contribute to its virulence; it is one of the most aggressive pathogens encountered in clinical microbiology laboratories. Among these factors are streptolysin O and S, which not only contribute to virulence but are also responsible for the beta-hemolytic pattern on blood agar plates used as a guide to identify this species. Streptolysin S is an oxygen stable, nonimmunogenic hemolysin capable of lysing erythrocytes, leukocytes, and platelets in the presence of room air. Streptolysin O is immunogenic, capable of lysing the same cells and cultured cells, is broken down by oxygen, and will produce hemolysis only in the absence of room air. Streptolysin O is also inhibited by the cholesterol in skin lipids resulting in the absence of the development of protective antibodies associated with skin infection. The infections caused by S. pyogenes may be localized or systemic; other problems may arise as a result of the host’s antibody response to the infections caused by these organisms. Localized infections include acute pharyngitis, for which S. pyogenes is the most common bacterial etiology, and skin infections, such as impetigo and erysipelas (see Chapter 76 for more information on skin and soft tissue infections). The organism adheres and invades the epithelial cells through the mediation of various proteins and enzymes. Internalization of the organism is believed to be important for persistent and deep tissue infections. Additional virulence factors are included in Table 15-2. S. agalactiae, group B, infections usually are associated with neonates and are acquired before or during the birthing process (see Table 15-2). The organism is known to cause septicemia, pneumonia, and meningitis in newborns. Although the virulence factors associated with the other beta-hemolytic streptococci have not been definitively identified, groups C, G, and F streptococci cause infections similar to those associated with S. pyogenes (i.e., skin and soft tissue infections and bacteremia) but are less commonly encountered, often involve compromised patients, and do not produce postinfection sequelae. The other genera listed in Table 15-2 are of low virulence and are almost exclusively associated with infections involving compromised hosts. A possible exception is the association of Alloiococcus otitidis with chronic otitis media in children. Certain intrinsic features, such as resistance to vancomycin among Leuconostoc spp. and Pediococcus spp., may contribute to the ability of these organisms to survive in the hospital environment. However, whenever they are encountered, strong consideration must be given to their clinical relevance and potential as contaminants. These organisms can also challenge many identification schemes used for gram-positive cocci, and they may be readily misidentified as viridans streptococci. 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. All the genera described in this chapter are gram-positive cocci. Microscopically, streptococci are round or oval-shaped, occasionally forming elongated cells that resemble pleomorphic corynebacteria or lactobacilli. They may appear gram-negative if cultures are old or if the patient has been treated with antibiotics. Gemella haemolysans is easily decolorized. S. pneumoniae is typically lancet-shaped and occurs singly, in pairs, or in short chains (Figure 15-1). Growth in broth should be used for determination of cellular morphology if there is a question regarding staining characteristics from solid media. In fact, the genera described in this chapter are subdivided based on whether they have a “strep”-like Gram stain or a “staph”-like Gram stain. For example, Streptococcus and Abiotrophia growing in broth form long chains of cocci (Figure 15-2), whereas Aerococcus, Gemella, and Pediococcus grow as large, spherical cocci arranged in tetrads or pairs or as individual cells. Leuconostoc may elongate to form coccobacilli, although cocci are the primary morphology. The cellular arrangements of the genera in this chapter are noted in Tables 15-3 and 15-4. TABLE 15-3 Differentiation of Catalase-Negative, Gram-Positive Coccoid Organisms Primarily in Chains
Streptococcus, Enterococcus, and Similar Organisms
Epidemiology
Organism
Habitat (reservoir)
Mode of Transmission
Streptococcus pyogenes
(group A)
Normal flora: Not considered normal flora
Inhabits skin and upper respiratory tract of humans, carried on nasal, pharyngeal, and, sometimes, anal mucosa; presence in specimens is almost always considered clinically significant
Direct contact: person to person
Indirect contact: aerosolized droplets from coughs or sneezes
Streptococcus agalactiae
(group B)
Normal flora: female genital tract and lower gastrointestinal tract
Occasional colonizer of upper respiratory tract
Endogenous strain: gaining access to sterile site(s) probable
Direct contact: person to person from mother in utero or during delivery; or nosocomial transmission by unwashed hands of mother or health care personnel
Groups C, F, and G beta-hemolytic streptococci
Normal flora:
Skin
Nasopharynx
Gastrointestinal tract
Genital tract
Endogenous strain: gain access to sterile site
Direct contact: person to person
Streptococcus pneumoniae
Colonizer of nasopharynx
Direct contact: person to person with contaminated respiratory secretions
Viridans streptococci
Normal flora:
Oral cavity
Gastrointestinal tract
Female genital tract
Endogenous strain: gain access to sterile site; most notably results from dental manipulations
Enterococcus spp.
Normal flora:
Humans, animals, and birds
E. faecalis and E. faecium) are normal flora of the human gastrointestinal tract and female genitourinary tract
Colonizers
Endogenous strain: gain access to sterile sites
Direct contact: person to person
Contaminated medical
equipment; immunocompromised patients are at risk of developing infections with antibiotic resistant strains
Abiotrophia spp. (nutritionally variant streptococci)
Normal flora:
Oral cavity
Endogenous strains: gain access to normally sterile sites
Leuconostoc spp.
Plants, vegetables, dairy products
Mode of transmission for the miscellaneous gram-positive cocci listed is unknown; most are likely to transiently colonize the gastrointestinal tract after ingestion; from that site they gain access to sterile sites, usually in compromised patients; all are rarely associated with human infections
Lactococcus spp. (group N)
Foods and vegetation
Globicatella sp.
Uncertain
Pediococcus spp.
Foods and vegetation
Aerococcus spp.
Environmental; occasionally found on skin
Gemella spp.
Normal flora of human oral cavity and upper respiratory tract
Helcococcus sp.
Uncertain
Alloiococcus otitidis
Occasionally isolated from human sources, but natural habitat is unknown
Uncertain; rarely implicated in infections
Pathogenesis and Spectrum of Disease
Organism
Virulence Factors
Spectrum of Diseases and Infections
Streptococcus pyogenes
Protein F mediates epithelial cell attachment (fibronectin binding); hyaluronic acid capsule inhibits phagocytosis; M protein is antiphagocytic (> 100 serotypes); produces several enzymes and hemolysins that contribute to tissue invasion and destruction, including streptolysin O, streptolysin S, streptokinase, DNase, and hyaluronidase. Streptococcal pyrogenic exotoxins (Spes)mediate production of rash (i.e., scarlet fever) or multisystem effects that may result in death; C5a Peptidase-destroying complement chemotactic factors.
Acute pharyngitis, impetigo, cellulitis, erysipelas, necrotizing fasciitis and myositis, bacteremia with potential for infection in any of several organs, pneumonia, scarlet fever, streptococcal toxic shock syndrome
Cross-reactions of antibodies produced against streptococcal antigens and human heart tissue
Rheumatic fever
Deposition of antibody-streptococcal antigen complexes in kidney results in damage to glomeruli
Acute, poststreptococcal glomerulonephritis
Streptococcus agalactiae
Uncertain; capsular material interferes with phagocytic activity and complement cascade activation
Infections most commonly involve neonates and infants, often preceded by premature rupture of mother’s membranes; transient vaginal carriage in 10%-30% of females; infections often present as multisystem problems, including sepsis, fever, meningitis, respiratory distress, lethargy, and hypotension; infections may be classified as early onset (occur within first 5 days of life) or late onset (occur 7 days to 3 months after birth); infections in adults usually involve postpartum infections such as endometritis, which can lead to pelvic abscesses and septic shock; infections in other adults usually reflect compromised state of the patient and include bacteremia, pneumonia, endocarditis, arthritis, osteomyelitis, and skin and soft tissue infections
Groups C, F, and G beta-hemolytic streptococci
None have been definitively identified, but likely include factors similar to those produced by S. pyogenes and S. agalactiae
Cause similar types of acute infections in adults as described for S. pyogenes and S. agalactiae, but usually involve compromised patients; a notable proportion of infections caused by group G streptococci occur in patients with underlying malignancies; group C organisms occasionally have been associated with acute pharyngitis
Streptococcus pneumoniae
Polysaccharide capsule that inhibits phagocytosis is primary virulence factor; pneumolysin has various effects on host cells, and several other factors likely are involved in eliciting a strong cellular response by the host; secretory IgA protease
A leading cause of meningitis and pneumonia with or without bacteremia; also causes sinusitis and otitis media
Viridans streptococci
Generally considered to be of low virulence; production of extracellular complex polysaccharides (e.g., glucans and dextrans) enhance attachment to host cell surfaces, such as cardiac endothelial cells or tooth surfaces in the case of dental caries
Slowly evolving (subacute) endocarditis, particularly in patients with previously damaged heart valves; bacteremia and infections of other sterile sites do occur in immunocompromised patients; meningitis can develop in patients suffering trauma or defects that allow upper respiratory flora to gain access to the central nervous system; S. mutans plays a key role in the development of dental caries
Enterococcus spp.
Little is known about virulence; adhesions, cytolysins, and other metabolic capabilities may allow these organisms to proliferate as nosocomial pathogens; multidrug resistance also contributes to proliferation
Most infections are nosocomial and include urinary tract infections, bacteremia, endocarditis, mixed infections of abdomen and pelvis, wounds, and occasionally, ocular infections; CNS and respiratory infections are rare
Abiotrophia spp. (nutritionally variant streptococci)
Unknown
Endocarditis; rarely encountered in infections of other sterile sites
Leuconostoc spp., Lactococcus spp., Globicatella sp., Pediococcus spp., Aerococcus spp., Gemella spp., Helcococcus sp.
Facklamia spp.
Ignavigranum ruoffiae
Dolosigranulum pigrum
Dolosicoccus paucivorans
Unknown; probably of low virulence; opportunistic organisms that require impaired host defenses to establish infection; intrinsic resistance to certain antimicrobial agents (e.g., Leuconostoc spp. and Pediococcus spp. resistant to vancomycin) may enhance survival of some species in the hospital setting
Whenever encountered in clinical specimens, these organisms should first be considered as probable contaminants; Aerococcus urinae is notably associated with urinary tract infections
Alloiococcus sp.
Unknown
Chronic otitis media in children
Beta-Hemolytic Streptococci
Miscellaneous Other Gram-Positive Cocci
Laboratory Diagnosis
Specimen Collection and Transport
Direct Detection Methods
Antigen Detection
Gram Stain
Organisms
Gram Stain from Thio Broth
Hemolysis α, β, or nona
Cytochromeb/ Catalase
Van
LAP
PVR
Gas in MRS Broth
Motility
on BE
in 6.5% NaCl Broth
Growth
Comments
At 10° C
At 45° C
Leuconostoc
cb, pr, ch
α, non
–/–
R
V
–
+
−
V
V
V
V
Enterococcus Vancomycin R
c, ch
α, β, or non
–/–c
R
+
+
−
−
+
+
+
+
Streptococcus (all)
c, ch
α, β, or non
–/–
S
+e
Vf
−
−
Vd
Vh
−
V
S. agalactiae
c, ch
β, non
–/–
S
+
−
NT
−
−
V
NT
NT
S. bovis
c, ch
α, non
–/–
S
+
−
−
−
+
−
−
+
Viridans streptococci
c, ch
α, non
–/–
S
+
−
−
−
−
−
−
V
S. urinalis
c, pr, ch
non
–/–
S
+
+
–
–
+
+
–
+
Abiotrophia
c, ch
α, non
–/–
S
V
V
–
–
–
–
–
V
Satellitism around S. aureus
Granulicatella
c, pr, ch
α
–/–
S
+
+
–
–
NT
–
–
V
Satellitism around S. aureus
Lactococcus
cb, ch
α, non
–/–
S
+
V
–
–
+
V
+
Vj
Dolosicoccus paucivorans
c, pr, ch
α
–/–
S
–
+
–
–
–
–
–
–
Globicatella sanguinis
c, ch, pr
α, non
–/–
S
–
V
–
–
+
+
+
V
Vagococcus
c, ch
α, non
–/–
S
+
+
−
+
+
V
+
V
Lactobacillus
cb, ch
α, non
–/–
V
V
−
V
−
V
V
+
V
Weissella confusa
Elongated bacillik
α
–/–
R
−
NT
+
V
+
V
NT
+
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