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

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

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.

Pathogenesis and Spectrum of Disease

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

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

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).

S. pyogenes infections are prone to progression with involvement of deeper tissues and organs, a characteristic that has earned the designation in general publications as the “flesh-eating bacteria.” Such systemic infections are life threatening. Additionally, even when infections remain localized, streptococcal pyrogenic exotoxins (SPEs) may be released and produce scarlet fever, which occurs in association with streptococcal pharyngitis and is manifested by a rash of the face and upper trunk. The SPEs are erythrogenic toxins produced by lysogenic strains. They are heat labile and rarely found in group C and G streptococci. The SPEs act as superantigens activating macrophages and T-helper cells inducing the release of powerful immune mediators including IL-1, IL-2, IL-6, TNF-alpha, TNF-beta, interferons, and cytokines, which induce shock and organ failure. Streptococcal toxic shock syndrome, typified by multisystem involvement including renal and respiratory failure, rash, and diarrhea, is a serious disease mediated by production of potent SPE.

Other complications that result from S. pyogenes infections are the poststreptococcal diseases rheumatic fever and acute glomerulonephritis. The poststreptococcal diseases are mediated by the presence of the M protein, not present in any other Lancefield groups. The M protein consists of two alpha helical polypeptides anchored in the cytoplasmic membrane of the organism and extending through the cell wall to the outer surface. The outer amino terminus of the protein is highly variable, consisting of greater than 100 serotypes. Class 1 M protein is associated with rheumatic fever, and class I or II is typically associated with glomerulonephritis. Rheumatic fever, which is manifested by fever, endocarditis (inflammation of heart muscle), subcutaneous nodules, and polyarthritis, usually follows respiratory tract infections and is thought to be mediated by antibodies produced against S. pyogenes M protein that cross-react with human heart tissue. Acute glomerulonephritis, characterized by edema, hypertension, hematuria, and proteinuria, can follow respiratory or cutaneous infections and is mediated by antigen-antibody complexes that deposit in glomeruli, where they initiate damage.

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. pyogenes is also a powerful modulator of the host immune system, preventing clearance of the infection. The M protein is able to bind beta globulin factor H, a regulatory protein of the alternate complement pathway involved in the degradation of C3b. The M protein also binds to fibrinogen blocking complement alternate pathway activation. In addition, all strains produce a C5a peptidase, which is a serine protease capable of inactivating the chemotactic factor for neutrophils and monocytes (C5a).

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.

Streptococcus Pneumoniae and Viridans Streptococci

S. pneumoniae contains the C polysaccharide unrelated to the Lancefield grouping and is still one of the leading causes of morbidity and mortality. The organism is the primary cause of bacterial pneumonia, meningitis, and otitis media. The antiphagocytic property of the polysaccharide capsule is associated with the organism’s virulence. There are more than 90 different serotypes of encapsulated strains of S. pneumoniae. Nonencapsulated strains are avirulent. The organism may harmlessly inhabit the upper respiratory tract with a 5% to 75% carriage rate in humans. S. pneumoniae is capable of spreading to the lungs, paranasal sinuses, and middle ear. In addition, this organism accesses the bloodstream and the meninges to cause acute, purulent, and often life-threatening infections.

S. pneumoniae is capable of mobilizing inflammatory cells mediated by its cell wall structure, including peptidoglycan, teichoic acids, and a pneumolysin. The pneumolysis activates the classical complement pathway. The pneumolysin mediates suppression of the oxidative burst in phagocytes providing for effective evasion of immune clearance. In addition, the organism contains phosphorylcholine within the cell wall, which binds receptors for platele- activating factor in endothelia cells, leukocytes, platelets, and tissue cells of the lungs and meninges providing for entry and spread of the organism.

The viridans (greening) streptococci and Abiotrophia spp. (formally known as nutritionally variant streptococci) are a heterogenous group consisting of alpha hemolytic and nonhemolytic species generally considered to be opportunistic pathogens of low virulence. The organisms colonize the gastrointestinal and genitourinary tract. These organisms are not known to produce any factors that facilitate invasion of the host. However, when access is gained, a transient bacteremia occurs and endocarditis and infections at other sites in compromised patients may result.

Enterococci

Enterococci, previously classified as group D streptococci, commonly colonizes the gastrointestinal tract. Greater than 29 species exist, including commensals that lack potent toxins and other well-defined virulence factors. Although virulence factors associated with enterococci are a topic of increasing research interest, little is known about the characteristics that have allowed these organisms to become a prominent cause of nosocomial infections. Enterococci are one of the most feared nosocomial pathogens isolated from the urinary tract, peritoneum, heart tissue, bacteremia, endocarditis, and intra-abdominal infections.

Compared with other clinically important gram-positive cocci, this genus is intrinsically more resistant to the antimicrobial agents commonly used in hospitals and is especially resistant to all currently available cephalosporins and aminoglycosides. In addition, these organisms are capable of acquiring and exchanging genes encoding resistance to antimicrobial agents. This genus is the first clinically relevant group of gram-positive cocci to acquire and disseminate resistance to vancomycin, the single cell–wall active agent available for use against gram-positive organisms resistant to beta-lactams (e.g., methicillin-resistant staphylococci). Spread of this troublesome resistance marker from enterococci to other clinically relevant organisms is a serious public health concern and appears to have occurred with the emergence of vancomycin-resistant S. aureus.

A wide variety of enterococcal species have been isolated from human infections, but E. faecalis and E. faecium still clearly predominate as the species most commonly encountered. E. faecalis and E. faecium have been isolated from the respiratory tract and the myocardium. Between these two species, E. faecalis is the most commonly encountered, but the incidence of E. faecium infections is on the rise in many hospitals, which is probably related in some way to the acquisition of resistance to vancomycin and other antimicrobial agents. Two additional species, E. gallinarum and E. casseliflavus, have been associated with intestinal infections.

Miscellaneous Other Gram-Positive Cocci

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.

Antigen detection screening methods are available for several streptococcal antigens. Detection of antigens is possible using latex agglutination or enzyme-linked immunosorbent assay (ELISA) technologies. These commercial kits have been reported to be very specific, but false-negative results may occur if specimens contain low numbers of S. pyogenes. Sensitivity has ranged from approximately 60% to greater than 95% depending on the methodology and other variables. Therefore, many microbiologists recommend collecting two throat swabs from each patient. If the first swab yields a positive result by a direct antigen method, the second swab can be discarded. However, for those specimens in which the rapid antigen test yielded a negative result, a blood agar plate or selective streptococcal blood agar plate should be inoculated with the second swab.

Several commercial antigen detection kits are available for diagnosis of neonatal sepsis and meningitis caused by group B streptococci. Developed for use with serum, urine, or cerebrospinal fluid (CSF), the best results have been achieved with CSF; false-positive results have been a problem using urine. Because neonates acquire S. agalactiae infection during passage through the colonized birth canal, direct detection of group B streptococcal antigen from vaginal swabs has also been attempted. However, direct extraction and latex particle agglutination have not been sensitive enough for use alone as a screening test.

Latex agglutination kits to detect the capsular polysaccharide antigen of the pneumococcus have also been developed for use with urine, serum, and CSF, although they are no longer commonly used in clinical microbiology laboratories.

Molecular Diagnostic Testing

Nucleic acid based testing is available and offers a rapid and increased specificity as compared to traditional identification schemes. Polymerase chain reaction is available for the detection of an internal sequence of the CAMP-factor (cfb gene) for group B streptococci. Analyte-specific reagents are available from Roche Applied Science (Indianapolis, Indiana) for the detection of the ptsI gene of group B and group A streptococci. The two groups are differentiated based on specificity of the sequences within the primer pairs for the assay. Gen-Probe Incorporated (San Diego, California) has developed several DNA probe assays for the differentiation of streptococci. The GASDirect test is a DNA probe hybridization assay for the detection of group streptococcal RNA from throat swabs. The ACCUPROBE group B Streptococcus assay is a hybridization protection assay that utilizes a DNA probe for the detection of 16 s ribosomal RNA sequences unique to Streptococcus agalactiae.

A fully integrated automated real-time PCR-based GeneXpert system has been developed by Cepheid (Sunnyvale, California). The system completely automates the sample preparation, DNA extraction, amplification, and detection of the target sequence within a closed system. The GeneXpert platform offers a qualitative assay for the detection of group B Streptococcus DNA directly from a swab.

Gram Stain

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

Organisms	Gram Stain from Thio Broth	Hemolysis α, β, or non^a	Cytochrome^b/ Catalase	Van	LAP	PVR	Gas in MRS Broth	Motility	on BE	in 6.5% NaCl Broth	Growth		Comments
Organisms	Gram Stain from Thio Broth	Hemolysis α, β, or non^a	Cytochrome^b/ Catalase	Van	LAP	PVR	Gas in MRS Broth	Motility	on BE	in 6.5% NaCl Broth	At 10° C	At 45° C	Comments
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	V^f	−	−	V^d	V^h	−	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	+	V^j
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 bacilli^k	α	–/–	R	−	NT	+	V	+	V	NT	+	Arginine positive

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Aug 25, 2016 | Posted by admin in MICROBIOLOGY | Comments Off

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Streptococcus, Enterococcus, and Similar Organisms

Streptococcus, Enterococcus, and Similar Organisms

General Characteristics

Epidemiology

Pathogenesis and Spectrum of Disease

Beta-Hemolytic Streptococci

Streptococcus Pneumoniae and Viridans Streptococci

Enterococci

Miscellaneous Other Gram-Positive Cocci

Laboratory Diagnosis

Specimen Collection and Transport

Specimen Processing

Direct Detection Methods

Antigen Detection

Molecular Diagnostic Testing

Gram Stain

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