Gram-Positive Cocci

BACTERIAL TAXONOMY (AN OVERVIEW)

The classic approach to the classification of bacteria is based on size and shape, later aided by reaction to Gram stain. As advances in microscope-making were made and simple biochemical tests became available, gradually the system became more refined. The science of taxonomy matured with the availability of the electron microscope and advances in molecular biology. Muller may have been the first biologist to attempt to classify bacteria in late 18th century, but his attempts were limited by lack of understanding of bacteria and crudeness of microscopes. Cohn, in late 19th century, made further advances, but serious attempts to classify bacteria were first made only in 20th century. Bergey’s Manual of Determinative Bacteriol- ogy was first published in 1923 and it instantly became the foundation of and the most authoritative source for bacterial taxonomy. Aided by inputs from the American Society for Microbiology, international societies for bacterial taxonomy and nomen- clature, and the International Journal of Systemic Bacteriology, Bergey’s manual has been greatly refined and its scope enlarged. Its 9th edition was published in 1994. Another remarkable contribution to bacterial taxonomy was made by the publication of Bergey’s Manual of Systematic Bacteriology, a five-volume set that examines bacterial taxonomy in greater detail. Students interested in bacterial taxonomy are encouraged to consult these highly authoritative sources.

Bacterial taxonomy, as it is, remains a complex branch of science that is not free from controversies. Several microbiologists have become famous simply by changing the names of certain species, including the names of some of the well- established species, often on trivial grounds. Authors respect modern advances but prefer a traditional approach. The tools currently available to a microbiologist for the delineation of species range from gene probes to old fashioned fermentation tests using Durham tubes. However, most modern pathogenic microbiology labs use a semi- or fully automated system, such as the Vitek or Biolog systems, for the iden- tification of bacteria and yeast-like fungi. For the sake of convenience, the authors of this book have used a simple approach to group clinically important bacteria based on their Gram reaction, shape, gas requirements, and some simple physiological

Figure 5.1.    Schema for the grouping of Gram-positive bacteria of medical importance

tests. An example of the basis for grouping Gram-positive bacteria is illustrated in the schema below (Fig. 5.1). Similar schema for grouping Gram-negative bacteria are illustrated in the relevant chapters. 

CLINICALLY IMPORTANT GRAM-POSITIVE COCCI 

From the perspective of pathogenic microbiology, species belonging to the following three genera are of clinical importance: Staphylococcus spp., Streptococcus spp., and Enterococcus spp. Members of a fourth taxa, Micrococcus spp., are closely related, but they are mostly harmless constituents of normal skin microbiota. Major differ- ences between Staphylococcus and Micrococcus spp. are summarized in Table 5.1. 

Staphylococcus Species 

Staphylococci are endogenous (found within the host) bacteria, commonly associ- ated with the skin, nose, ear, and throat. Staphylococcus aureus is the most important

Table 5.1    Some Differentiating Characteristics of Staphylococcus and Micrococcus spp

pathogen. Other species that may be incriminated in diseases include S. epidermidis, S. saprophyticus, S. haemolyticus, and S. lugdunensis, which are otherwise considered constituents of normal skin microbiota. Species of Staphylococcus that may colonize the human body but are not known to cause diseases are S. saccharolyticus, S. warneri, S. hominis, S. auricularis, S. xylosus, S. simulans, S. cohnii, and S. pasteuri. Approximately 30% of the general population and more than 50% of medical professionals are carriers of S. aureus, mostly in their nostrils or skin. 

Diseases 

Clinical conditions triggered by S. aureus and related species are summarized below. 

• Soft tissue infections such as folliculitis and cellulitis, abscess formation, and toxic shock syndrome 

• Bacteremia and endocarditis 

• Osteomyelitis 

• Pneumonia and empyema 

• Toxin-related food poisoning 

• Infections involving eyes, nose, throat, urethra, and vagina (in elderly women) 

Virulence Factors 

Toxins including hemolysins, leukocidins, enterotoxins A through E, and toxic shock syndrome toxin (TSST) may be produced by strains of S. aureus in human hosts. Extracellular enzymes including coagulase, catalase, hyaluronidase, lipases, and proteinases are also known to cause damage to the host. 

Laboratory Diagnosis 

Blood agar is an excellent isolation medium. Mannitol salt agar is a selective medium. Staphylococci are aerobic; they grow well at 35°C with visible colonies within 18–24 hours. The colonies are smooth, spherical, and opaque with a golden hue. Many strains cause β-hemolysis on blood agar

Table 5.2    Important Differentiating Characteristics of Staphylococcus spp. Other Than

S. aureus

Taxonomy 

The distinctive characteristics of S. aureus are the following: 

• Positive coagulase test 

• Positive thermonuclease test 

• Fermentation of mannitol 

Gene probes are also available for the identification of staphylococci. Differentiation with other Staphylococcus spp. requires elaborate physiological tests. Some of the relevant characteristics are summarized in Table 5.2. 

Antibiotic Sensitivity 

Staphylococci used to be very susceptible to penicillin. However, over time and with increased usage of beta lactam antibiotics, resistant strains have emerged as a major challenge. A group of such resistant strains are called MRSA (methicillin resistant Staphylococcus aureus). A sensitivity test can be helpful. Currently, vancomycin is a mainstay of therapy for serious infections but resistance to it is increasing world- wide. Newer agents such as daptomycin and linezolid are also being used as well as new cephalosporins that are the only beta lactams to cover MRSA, which include ceftaroline and ceftobiprole. 

Streptococcus Species 

Streptococci are catalase-negative, Gram-positive cocci that form short or long chains in situ. Several members of the genus Streptococcus constitute normal micro- biota of mouth and upper respiratory tract. These can also be isolated from gastro- intestinal tract and female genitourinary tract. Streptococcus mutans, S. salivarius, S. sanguis, and S. mitis are some of the nonpathogenic species, that are collectively referred to as Viridans streptococci. 

Diseases 

There are three pathogenic species: Streptococcus pneumoniae, S. pyogenes, and S. agalactiae. 

       Streptococcus pneumoniae, also referred to as pneumococcus, is known to cause pneumonia, otitis media (midear infection), sinusitis, meningitis, and bacteremia (septicemia). Most strains of S. pneumoniae are nonhemolytic. The common serotypes isolated from clinical specimens are referred to as serotype 6, 14, 18, and 19. This bacterium can also be isolated from the respiratory tract of apparently healthy individuals. Infections occur when pneumococci get into the lungs or bloodstream. Under normal circumstances nonspecific immune components, especially phagocytes and macrophages, keep these bacteria under control. Pneumococcal pneumonia is often accompanied by high fever and chill and the symptoms may be mistaken for viral infection. The cough may be productive and blood tinged. Children and the elderly are more prone to pneumococcal pneumonia. Ear infections are more common in children, but the sinusitis can affect adults as well. Bacteremia is more serious form of infection and not an uncommon complication in pneumonia. Bacteremia can also lead to endocarditis and meningitis. 

    Streptococcus pyogenes, also called Group A streptococcus, causes infections involving the upper respiratory tract (pharyngitis or strep throat) and mucocutaneous tissues, as well as skin infection, scarlet fever, and muscle infection (hence the term “flesh-eating bacteria”). The incubation period of strep throat infection is very short and the symptoms may include sore throat, fever, and headache. The cervical lymph nodes may be affected. In some cases, strep throat infection may lead to scarlet fever, characterized by body rashes that may heal in about 1 week. The skin infection may begin with exposure of bruised skin to S. pyogenes either through direct contact or through inanimate objects (fomite). Pus-filled vesicles develop and eventually rupture and crust over. Another form of skin infection, called necrotizing fasciitis, involves deeper subcutaneous tissue and results in a severe destruction of the muscles. The disease often develops into a systemic infection and eventual death in many cases. Infection with this bacterium is also known to lead to secondary, or post streptococcal glomerulonephritis (PSGN), which is characterized by inflammation due to immune complex formation in glomeruli, resulting in hematuria (blood in urine) and protein- uria (high protein concentration in urine). Another secondary consequence of S. pyogenes infection is rheumatic fever. This usually occurs 2–3 weeks after acute pharyngitis with S. pyogenes. It may involve the heart (resulting in damaged heart valves), joints (multiple joint arthritis), central nervous system, and skin. 

Streptococcus agalactiae, also called Group B Streptococcus, is normally present in the gastrointestinal tract. Isolation of this bacterium from the respiratory tract of apparently healthy individuals is not uncommon. In addition, mostly due to poor personal hygiene, S. agalactiae can contaminate the vaginal area without causing any clinical symptom in the woman. This bacterium can cause neonatal pneumonia, bacteremia, and meningitis. Infections in adults are usually less severe, limited to fever and malaise. Cases of upper respiratory tract infection due to Group B Streptococcus in those who perform oral sex on women are not uncommon. 

Virulence Factors 

Streptococcal virulence factors can be divided into two categories: 

1. Extracellular toxins: These include Streptolysin-S, Streptolysin-O, strep- tokinase, and pyrogenic exotoxins. Streptolysin-S is mostly produced by members of Group A, C, and G. Streptolysin-O destroys hemoglobin and suppresses chemotaxis. It can also cause lysis of leukocytes and erythrocytes. Streptokinase causes lysis of blood clots and facilitates the spread of bacteria. 

Streptococcal pyrogenic exotoxins A, B, and C are believed to be responsible for the rashes in scarlet fever and for the symptoms of toxic shock syndrome. A rise in the distribution of streptococcal pyrogenic toxin- producing strains is believed to be associated with the rise in Group A streptococcal invasive infections. 

2. Cell-associated factors: The cell-associated factors include M protein, which helps bacterium in evading host’s immune system, and lipoteichoic acid (mostly associated with S. pneumoniae), which facilitates adherence. Capsules as seen in S. pneumoniae protect the pathogen from the host’s defenses and also from the effects of antibiotics and other chemicals. 

Lab Diagnosis 

       Appropriate clinical specimens, such as throat swab, sputum, or blood, must be obtained under aseptic conditions. Often, Gram staining can provide useful clues. Pathogenic streptococci require complex media for growth. Best results are obtained with blood agar, which also helps in the detection of hemolysis. Brain heart infusion agar is also good. Incubation is done at 35°C in aerobic conditions for 24 hours. Colonies of S. pyogenes are very small, translucent, and surrounded by a clear, large, and nearly transparent zone caused by β-hemolysis. Colonies of S. agalactiae are relatively larger, flatter, and creamy in appearance. The β-hemolysis zone in the case of S. agalactiae is much smaller. Colonies of S. pneumoniae are initially dome- shaped and mucoid or watery with none to slight α-hemolysis. Colonies of Viridans streptococci are much smaller and opaque with a strong α-hemolysis. 

Immunological tests including enzyme linked immunosorbent assay (ELISA) and agglutination tests aimed at group-specific carbohydrates are useful in detecting Group A streptococci directly in the throat swab. A number of commercial latex agglutination kits are available for the rapid detection of S. pyogenes, S. pneumoniae, and S. agalactiae. 

Taxonomy 

Several physiological characteristics are taken into account for the delineation of the Streptococcus spp. In addition, the patterns of hemolysis and Lancefield classifica- tion of Beta hemolytic streptococci are helpful. 

1. Alpha (α) hemolysis: Characterized by partial destruction of erythrocytes accompanied by greenish/brownish tinge around the colony (e.g., Viridans streptococci). 

2. Beta (β)-hemolysis: Characterized by total destruction of erythrocytes and a clear zone around the colony (e.g., S. pyogenes). 

3. Gamma (γ) hemolysis: Characterized by an absence of hemolysis (e.g., S. pneumoniae). 

Like Enterococcus and Lactobacillus spp., streptococci are catalase negative (do not split H2O2 into O2 and H2O). 

The Lancefield classification of beta hemolytic Streptococcus spp. is shown in Table 5.3. 

Viridans Streptococci 

Viridans streptococci include hemolytic and nonhemolytic species that constitute normal microbiota of mouth and upper respiratory tract. Hemolytic species are known to produce only α-hemolysis. A short list of important Viridans streptococci based on CDC classification is given below: 

• Streptococcus mutans 

• S. salivarius

Table 5.3 Lancefield Classification of Beta Hemolytic Streptococcus spp. Based on Antigenic Properties

• S. sanguis 

• S. mitis 

• S. anginosus 

Viridans streptococci can be identified on the basis of the utilization of mannitol, sorbitol, arginine, and hydrolysis of esculin. Streptococcus mutans strains are almost always positive with mannitol, sorbitol, and esculin, while S. mitis are always negative. Streptococcus sanguis is positive with arginine and esculin while S. salivarius is positive only on esculin. Most Viridans streptococci are negative in urease test. 

Antibiotic Sensitivity 

Initially, streptococci were quite sensitive to penicillin. Cephalosporins and erythromycin are used in penicillin-sensitive hosts. In some cases, a combination of ampicillin and an aminoglycoside is indicated. Cases of rheumatic fever may need long periods of medication. Other effective agents include vancomycin and levofloxacin. However, instances of resistance to any of these antibiotics, including newer macrolides (azithromycin and clarithromycin), have been noted in recent years. Pneumococcal vaccine is effective in the prevention of S. pneumoniae infections, although patients may be susceptible to strains not included in the vaccine. 

GRAM-POSITIVE COCCI RELATED TO STREPTOCOCCUS SPECIES 

Aerococcus viridans 

Involvement of Aerococcus viridans in human cases has been reported, albeit sparingly. This bacterium is normally present in the environment and is often encountered as a constituent of resident microbiota in humans. It has been occasionally reported as the causal agent in cases of bacteremia, meningitis, endocarditis, septic arthritis, and urinary tract infection. 

Gamella Species 

    Human cases of infection by Gamella spp. are rare. Isolation of one of the Gamella spp., G. hemolysans, has been reported from the upper respiratory tract and incriminated in subacute endocarditis. Like G. hemolysans, another species, G. morbillorum, too has been occasionally isolated from the genitourinary and respiratory tracts, blood, and abscess swabs. It has been, at least on rare occasions, incriminated in the cases of endocarditis and brain abscess. It must be noted that because of their similarities, these are perhaps frequently misidentified as Viridans streptococci. Therefore, the possibility of Gamella spp. being more frequently involved in the causation of human diseases cannot be ruled out. 

Pediococcus Species

Eight species of the genus Pediococcus have been isolated from clinical specimens, though most of them sparingly. Of these, P. acidilactici is of a relatively greater clinical significance. It has been reported as the causal agent of septicemia. It has been also isolated, with uncertain causality, from the cases of leukemia and neutropenia. In addition, Pediococcus acidilactici has been noted in several cases of bone marrow transplants undergoing digestive system decontamination therapy with gen- tamicin, vancomycin, and colistin.

Enterococcus Species

Members of the genus Enterococcus were previously called Group D streptococci. However, based on detailed physiological and genetic tests, these are now placed in a different genus, Enterococcus. Enterococci are normally present in the human intestine. Enterococcus faecalis is the main pathogenic species that accounts for a large number of cases of urinary tract infection (UTI). Cases of bacteremia have also been attributed to this bacterium. Endocarditis can be seen as a secondary complication in cases of bacteremia and can be difficult to cure.

Laboratory Diagnosis

Enterococcus faecalis grows well on blood agar and chocolate agar. A selective medium especially formulated for this pathogen is also available. Most strains of E. faecalis are catalase negative. Enterococci can be differentiated from other Group

Figure 5.2. A Gram-stained smear from a clinical specimen showing Gram-positive cocci ofEnterococcus.

D streptococci by their ability to hydrolyze pyrrolidonyl arylamidase and grow in media containing 6.5% sodium chloride. Enterococci are Gram-positive cocci. They are often seen singly, but occasionally form a short chain (Fig. 5.2).

Antibiotic Sensitivity

Enterococcus faecalis is notoriously resistant to most antibiotics. A combined therapy using an aminoglycoside and ampicillin or vancomycin has proven effective in some cases. Some of the newer agents being used for MRSA, such as linezolid and daptomycin, also have efficacy against enterococcus.