Streptococcus and Enterococcus belong to the family Streptococcaceae. Members of both genera are catalase-negative, gram-positive cocci that are usually arranged in pairs or chains (Figure 15-1). A negative catalase test result differentiates the streptococci and enterococci from the staphylococci. Weak false-positive catalase reactions can be seen when growth is taken from media containing blood. Compared with other gram-positive cocci, the cells of enterococci and some streptococci appear somewhat more elongated than spherical. The streptococcal cells are more likely to appear in chains when grown in broth cultures.

Most members of the genera Streptococcus and Enterococcus behave as facultative anaerobes. Because they  grow in the presence of oxygen but are unable to use oxygen for respiration, they may be considered

aerotolerant anaerobes. Carbohydrates are metabolize fermentatively, with lactic acid the major end product; gas is not produced. Some species are capnophilic, requiring increased concentration of CO2,whereas the growth of others is stimulated by increased CO2,Growth is poor on nutrient media such as trypticase soy agar.

On media enriched with blood or serum, however, growth is more pronounced. The colonies are usually small and somewhat transparent. The role of the streptococci and enterococci in disease has been known for more than 100 years. The range of infections caused by these organisms is wide and well studied. As we have seen with other organisms, however, the roles in disease of the previously unknown or poorly characterized species and the saprophytes are becoming more prominent.


Cell-Wall Structure

The streptococci possess a typical gram-positive cell wall consisting of peptidoglycan and teichoic acid.

 Most streptococci, except for many of the viridans group, have a layer of a group of common C carbohydrate (polysaccharide), which can be used to classify an isolate serologically. A schematic diagram of the

streptococcal cell wall is shown in Figure 15-2. Other cell-wall antigens are present in specific C carbohydrate groups and are explained in the discussions of the individual groups. Some species can produce a type-specific polysaccharide capsule as well.


Classification Schemes

Several different approaches to classification of the catalase-negative, gram-positive cocci have been used. Four commonly used classification schemes are (1) hemolytic pattern on sheep red blood cell agar; (2) physiologic characteristics; (3) serologic grouping or typing of C carbohydrate (Lancefield classification), capsular polysaccharide, or surface protein, such as the M protein of Streptococcus pyogenes; and (4) biochemical characteristics. The identification process for a streptococcal isolate in the clinical laboratory may use features from each scheme Hemolytic Patterns The clinical laboratory scientist often makes an initial classification of the streptococci based on the hemolytic pattern of the isolate grown on sheep blood agar (SBA). Although hemolysis patterns can be helpful during the initial workup of an isolate, it must be kept in mind that many species of streptococci may show variable hemolytic patterns. The types of hemolysis possible are outlined in Table 15-1. When lysis of the red blood cells (RBCs) in the agar surrounding the colony is complete, the resulting area is clear; this is referred to as ~hemolysis (Figure 15-3). Partial lysis of the RBCs results in a greenish discoloration of the area surrounding the colony and is termed a-hemolysis (Figure 15-4).

When the RBCs immediately surrounding the colony are unaffected, the bacteria are termed nonhemolytic.

Some references term this result y-hemolysis. Because no lysis of the RBCs occurs, however, the term yhemolysis is confusing and is not recommended. Some isolates belonging to the viridans group produce what is called wide-zone or a-prime hemolysis. The colonies are surrounded by a very small zone of no hemolysis and then a wider zone of ~-hemolysis. This reaction may be mistaken for ~-hemolysis at first glance. The use of a dissecting microscope or the scanning objective shows the narrow zone of intact RBCs and

the wider zone of complete hemolysis.


Physiologic Characteristics

Streptococci have also been classified according to physiologic characteristics. This classification divides the species into four groups: pyogenic streptococci, lactococci, enterococci, and viridans streptococci. The

pyogenic streptococci are those that produce pus; these organisms are mostly l3-hemolytic and constitute the majority of the Lancefield groups. The lactococci, members of the genus Lactococcus, are non hemolytic organisms with Lancefield group N antigen and are often found in dairy products. The enterococci comprise

species found as part of the normal flora of the human intestine; this group of organisms belongs to

the genus Enterococcus.

The viridans streptococci are widely found as normal flora in the upper respiratory tract of humans. Most strains lack a C carbohydrate and are not part of the Lancefield classification; however, some have A, C, F, G, or N antigen. The viridans streptococci are a-hemolytic or nonhemolytic and are often seen as opportunistic pathogens. For the most part this physiologic classification is historical. Nevertheless the terms enterococci and viridans streptococci remain and are still used to describe clinical isolates.


The Lancefield Classification Scheme

The most commonly used classification scheme was developed in the 1930s by Rebecca Lancefield. The Lancefield classification is based on extraction of C carbohydrate from the streptococcal cell wall by placing the organisms in dilute acid and heating for 10 minutes. The soluble antigen is used to immunize rabbits to obtain antisera to the various C carbohydrate groups. After first recognizing the antigen in l3-hemolytic streptococci, Lancefield was able to divide the streptococci into serologic groups, designated by letters. Organisms in group A possess the same antigenic C carbohydrate; those in group B have the same C carbohydrate, and so on.

The classic Lancefield serologic grouping has been most significant in classifying and identifying l3-hemolytic streptococci. During the past several decades, however, since DNA relatedness has been applied to the classification and identification of a-hemolytic streptococcal species, investigators have found no correlation between genetic relationships and streptococcal group antigens. Contrary to group B l3-hemolytic streptococci, in which there is only one species identified, a-hemolytic streptococci as a whole are phenotypically and genotypically diverse and therefore difficult to characterize.

Streptococcal species other than those that produce l3-hemolysis possess C carbohydrate. Some are found as normal flora in animals or as animal pathogens, and others may be found in both humans and animals. The Lancefield groups seen in human infections are A, B, C, D, F, G, and N, although not all

members of these groups cause human infection. The classification of Streptococcus and Enterococcus species is shown in Table 15-2.


Biochemical Identification

Biochemical identification can be performed even by small laboratories. Although definitive identification requires a large number of biochemical characteristics or perhaps serologic methods, presumptive identification

can be accomplished relatively easily with a few key tests and characteristics (Figure 15-5). Initial biochemical tests performed are often selected based on the hemolytic reaction of the isolate. Presumptive identification, in the great majority of cases, possesses a high enough rate of accuracy to be useful to the clinician and does not require the exhaustive additional tests that are needed to meet the criteria for definitive identification, especially for species in groups A, B, and D as well as Streptococcus pneumoniae and Enterococcus. Speciation of the viridans streptococci, however, does require a considerable increase in the number of tests. Several multitest commercial kits are available, such as Remel’s IDSRapIDSTR (Lenexa, Kan) shown in Figure 15-6.In selecting an identification scheme or kit, the clinical laboratory scientist must evaluate the needs of the clinicians and patient population served, the cost of an expanded identification scheme, the resources and abilities of the laboratory, and the usefulness of the data obtained. Table 15-3 outlines the biochemical characteristics used to presumptively identify selected members of the family Streptococcaceae and similar organisms.

Bacitracin Susceptibility

Historically bacitracin susceptibility has been used as an inexpensive test to presumptively identify S. pyagenes (group A streptococci). Procedure 15-1 describes the bacitracin susceptibility test. I3-hemolytic streptococcus colonies are subcultured to an SBA plate, and a filter paper disk impregnated with 0.04 units of bacitracin is added. The plate is incubated at 35° C overnight; any zone of inhibition is interpreted as sensitive and is a presumptive identification of group A streptococcus (Figure 15-7). The pyrrolidonyl-a-naphthylamide PYR test, discussed in the following section, is a more specific rapid test for group A streptococcus.

Susceptibility to trimethoprim and sulfamethoxazole (SXT) can be used in conjunction with that of

bacitracin to improve the accuracy of group A identification.

Groups A and B are resistant to SXT,whereas groups other than A and B are sensitive. Figure 15-8 shows the resistance of group A ~-hemolytic streptococci to SXT.The pattern shown by S. pyogenes (group A) is susceptibility to bacitracin and resistance to SXT. The pattern shown by Streptococcus agalactiae (group B) is resistance to both bacitracin and SXT.If the SBAcontains SXT,a bacitracin disk may be placed directly on the primary inoculum. Growth of most interfering respiratory flora will be inhibited, but S.

pyogenes and S. agalactiae will grow. This method is helpful to screen for group A streptococci in throat cultures. The SBA containing SXT is inoculated with the throat swab, and a bacitracin disk is placed onto the agar. Those B-hemolytic colonies that grow and are susceptible to bacitracin are presumptively identified as S. pyogenes.


A test that is used to presumptively identify group B streptococci is the CAMP test. “CAMP” is an acronym based on the first letters of the surnames of the individuals who first described the reaction: Christie, Atkins, and Munch-Petersen. The “CAMP” test can be performed three ways. One is with the use of a B-Iysin-producing strain of Staphylococcus aureus; another is with the use of a disk impregnated with the I)B-lysin. Both methods take advantage of the enhanced hemolysis occurring when the B-lysin and the hemolysin produced by group B streptococci are in contact.

In the assay utilizing S. aureus, the unknown streptococcal isolate is inoculated perpendicularly to the S. aureus inoculum. The result is a characteristic arrowhead-shaped hemolysis pattern (Figure 15-9).

Procedure 15-2 describes how to perform the CAMP test. When a disk containing B-lysin is used, the enhanced

hemolysis is not a typical arrowhead shape because the disk is round. A third method, the rapid CAMP test (or spot CAMP test), involves placing a drop of extracted B-lysin on the area of confluent growth of the suspected group B streptococci. After incubation at 35° C for at least 20 minutes, enhanced hemolysis is observed (Figure 15-10).


Hippurate Hydrolysis

A useful test to differentiate S. agalactiae from other I)B-hemolytic streptococci is hippurate hydrolysis. S. agalactiae possess the enzyme hippuricase (also called hippurate hydrolase), which hydrolyzes sodium hippurate to form sodium benzoate and glycine. Hydrolysis can be detected by adding ninhydrin,

which reacts with glycine to form a purple color. A 2-hour rapid test is available; Procedure 15-3 describes the test.

PYR Hydrolysis

The PYR hydrolysis test provides a high probability for the presumptive differentiation of the B-hemolytic group A streptococci and the nonhemolytic group 0 enterococci from the other streptococcal species

 Figure 15-11). The test is as specific as bile esculin and salt tolerance broth (see Bile Esculin and Salt Tolerance later in this chapter) for Enterococcus spp. and is more specific than bacitracin for S. pyogenes. Procedure 15-4 describes the PYR hydrolysis test.

The PYR test detects the activity of L-pyrrolicdonyl arylamidase, also called pyrrolidonyl aminopeptidase. The PYR test takes advantage of the fact that S.

pyogenes and Enterococcus spp. are able to hydrolyze the substrate PYR. Several commercial systems are available. Substrates used in the PYR test are Lpyrrolidonyl- I)-naphthylamine and L-pyroglutamic acicd- B-naphthylamide. Following hydrolysis of the substrate by the peptidase, the resulting I)B-naphthylamine produces a red color on the addition of 0.01% cinnamalcde hyde reagent (0.01 %, p-dimethylaminocinnamaldehyde [OMACA]. The substrate can be impregnated into filter paper disks, which are moistened and inoculated with the suspected isolate. The BBL Dry Slide PYR test (BO, Sparks, Md) is not premoistened before inocu- lation. After 2 to 5 minutes to allow for hydrolysis, the cinnamaldehyde reagent is dropped onto the filter paper. A pink or cherry-red color appears within 1 minute if the reaction is positive. A negative reaction is indicated by no color change. The genera that are PYR BACITRACIN



To differentiate S. pyogenes from other ~-hemolytic groups


Group A streptococci are susceptible to low levels (0.04 units) of bacitracin, whereas other groups are resistant. Rare strains of group A streptococci are resistant (approximately 1%), whereas some strains of groups B, C, and G streptococci are sensitive (5% to 10%).Sensitivity to bacitracin presumptively identifies an isolate as S. pyogenes. This procedure was designed for use only with pure cultures.


Isolated colonies on sheep blood agar Media 5% sheep blood agar plate


Bacitracin disk (0.04 units)


۱٫ Streak surface of agar plate to obtain isolated colonies.

۲٫ Aseptically place bacitracin disk onto inoculated surface. Press down gently on the disk to ensure complete contact with the agar surface.

۳٫ Invert and incubate plates at 35° C for 18 to 24 hours.


Positive result (susceptible) = Any zone of inhibition around the bacitracin Negative result (resistant) = Uniform lawn of growth up to the edge of the disk (Figure 15-7)


positive include Enterococcus, Aerococcus, and Gemella. The only member of Streptococcus that is PYR positive is S. pyogenes.

Leucine Aminopeptidase Leucine aminopeptidase (LAP) is a peptidase that hydrolyzes peptide bonds adjacent to a free amino group. Because LAPreacts most quickly with leucine, it is called leucine aminopeptidase. The substrate, leucine- I3-naphthylamide, is hydrolyzed to I3-naphthylamine. After the addition of DMACA,a red color develops.

Rapid commercial tests performed on filter paper disks are available. The LAP test is often used with the PYR test and is most helpful in differentiating Aerococcus and Leuconostoc organisms from other gram-positive cocci.



To differentiate S. agalactiae from other ~-hemolytic streptococci Principle

S. agalactiae produces a CAMP factor that enhances the lysis of sheep red cells by staphylococcal I3-Iysin.

A positive reaction can be observed in 5 to 6 hours with incubation in CO2(18 hours with incubation in ambient air).


۱٫ Isolated colonies on sheep blood agar

۲٫ f)-lysin-producing S. aureus on blood agar Medium

Sheep blood agar plate


۱٫ Inoculate S. aureus along a line down the center of the agar plate.

۲٫ Inoculate the streptococcal isolate along a thin line

۲ cm long and perpendicular to, but not touching, the S. aureus streak.

۳٫ Incubate plate at 35° C for 18 hours. Interpretation Positive results =Arrowhead-shaped area of enhanced hemolysis where the two streaks (staphylococcal and streptococcal) approach each other (see Figure 15-9)

Negative result = No enhanced hemolysis CAMP inhibition reaction = Inhibition of hemolysis by S. aureus where the two streaks approach each other. This reaction is characteristic of Arcanobacterium



Positive: S. agalactiae

Negative: S. pyogenes


Streptococcus, Enterococcus, and Pediococcus spp. Are LAP positive, and Aerococcus and Leuconostoc spp. Are LAP negative.

Vogues-Proskauer Test

The Vogues-Proskauer (VP) test is used to distinguish the small-colony-forming I3-hemolytic anginosus group containing groups A or C antigens from largecolony- forming pyogenic strains with the same antigens. The VP test detects acetoin production from glucose. A heavy suspension of bacteria is made in VP broth. After about 6 hours of incubation at 35° C, a few drops of 5%a-naphthol and 40% potassium hydroxide are added. The tube is shaken to increase the concentration of dissolved oxygen, and the broth is incubated at room temperature for 30 minutes. The formation of a red or pink color is a positive reaction.

Members of the anginosus group are positive.



To differentiate S. agalactiae from other ~-hemolytic streptococci


The enzyme hippuricase hydrolyzes hippuric acid to form sodium benzoate and glycine. Subsequent addition of ninhydrin results in the release of ammonia from the oxidative deamination of the a-amino group in glycine as well as the reduced form of ninhydrin, hydrindantin. The ammonia reacts with residual ninhydrin and hydrindantin to produce a purple-colored complex. Some isolates of group D streptococci also hydrolyze hippurate; however, these isolates are less likely to be I3-hemolytic, and their colony morphology is different from that of group B streptococci.

An isolate that is hippurate positive and bile esculin negative has a very high probability of being S. agalactiae.


Isolated colonies on sheep blood agar.



To differentiate S. agalactiae from other ~-hemolytic streptococci


The enzyme hippuricase hydrolyzes hippuric acid to form sodium benzoate and glycine. Subsequent addition of ninhydrin results in the release of ammonia from the oxidative deamination of the a-amino group in glycine as well as the reduced form of ninhydrin, hydrindantin. The ammonia reacts with residual ninhydrin and hydrindantin to produce a purple-colored complex. Some isolates of group D streptococci also hydrolyze hippurate; however, these isolates are less likely to be I3-hemolytic, and their colony morphology is different from that of group B streptococci.

An isolate that is hippurate positive and bile esculin negative has a very high probability of being S. agalactiae.


Isolated colonies on sheep blood agar


Sodium hippurate (1 %)

Sodium hippurate

Distilled water

۱ g

۱۰۰ mL

Dispense 0.5-mL aliquots in small capped vials.

Store at _200 C. Storage life is 6 months.

Ninhydrin reagent:


Acetone-butanol mixture (1:1)

۳٫۵ g


Store at room temperature. Storage life is 12 months.


۱٫ Inoculate the solution of sodium hippurate heavily with colonies 18 to 24 hours old until a milky suspension is obtained.

۲٫ Incubate tube for 2 hours at 350 C.

۳٫ Add 0.2 mL of ninhydrin reagent.

۴٫ Mix and incubate for 10 to 15 minutes.


Positive result = Deep purple color (indicates hippurate hydrolysis)

Negative result = No color change (or very slight purple color)


Positive: S. agalactiae

Negative: S. pyogenes



To differentiate those gram-positive cocci that will hydrolyze the substrate L-pyrrolidonyl-a naphthylamide (pYR) from those that are PYRnegative


PYR-impregnated disks serve as the substrate to produce a-naphthylamine, which is detected in the presence of D-dimethylaminocinnamaldehyde by the production of a red color.


Isolated colonies on sheep blood agar


۱٫ Lightly moisten a PYR-impregnated disk with sterile water.

۲٫ Using a sterile loop applicator, rub one or more isolated colonies on the surface of the disk.

۳٫ NOTE:Incubation time and temperature varies

slightly by manufacturer. Incubate as indicated in

the manufacturer’s instructions (2 to 15 minutes).

۴٫ Add a drop of color developer, and observer for a

red color on the disk within 5 minutes.


Positive result =Red color

Negative result =Colorless


Positive: Enterococcus faecalis

Negative: Streptococcus agalactiae


The p-D-Glucuronidase (BGUR) test detects the action of BGUR,an enzyme found in isolates of largecolony- forming p-hemolytic group C and G streptococci but not in the small-colony-forming p-hemolytic anginosus group. Several commercially prepared rapid assays are available.

Bile Esculin and Salt Tolerance The two tests that have been mainstays in identification schemes for the non-p-hemolytic, catalase-negative, gram-positive cocci are the bile esculin hydrolysis test (procedure 15-5) and the salttolerance test. The bile esculin test is a two-step test; bacteria must grow in the presence of 40% bile and be able to hydrolyze esculin to produce a positive reaction. Hydrolysis of esculin results in esculetin, which reacts with ferric citrate or ferric ammonium citrate in the medium to form a black precipitate in the agar, indicating a positive reaction (Figure 15-12). Group D streptococci and Enterococcus spp. are positive.

Organisms positive for bile esculin are separated into group D streptococci or Enterococcus by the salttolerance test (procedure 15-6). Growth in 6.5% sodium chloride broth is used to identify Enterococcus and Aerococcus organisms. Some species of Pediococcus and Leuconostoc spp. grow in 6.5% NaCI broth when incubated for 24 hours. Group D streptococci, however, do not grow in a 6.5% NaCI broth.



To differentiate group Dstreptococci and Enterococcus from other gram-positive cocci Principle Group D streptococci and Enterococcus grow in the presence of bile and also hydrolyze esculin to esculetin and glucose. Esculetin diffuses into the agar and combines with ferric citrate in the medium to give a black complex.


Isolated colonies on sheep blood agar


Bile esculin agar


۱٫ Pick one or two isolated colonies from the sheep blood agar plate and inoculate to bile esculin agar


۲٫ Incubate at 35° C for 18 to 24 hours. NOTEA: positive result is often seen within 4 hours. Anegative result should be incubated for an additional 24-hour period.


Positive result = Blackening of the agar slant

Negative result = No blackening of the agar (NOTE: Growth alone does not constitute a positive result)


Positive: Group D Streptococcus

Negative: Viridans Streptococcus



To differentiate gram-positive cocci that will grow in 6.5% NaCI from those that are inhibited by this salt concentration


Enterococcus, Aerococcus, and some species of Pediococcus and Leuconostoc can withstand a higher salt concentration than other gram-positive cocci.


Isolated colonies on sheep blood agar Medium 6.5% NaCI broth (nutrient broth base)


۱٫ Pick one or two isolated colonies from the blood agar plate and lightly inoculate 5 mL of NaCI broth.

۲٫ Incubate at 35° C for 3 days. Check for growth daily.


Positive result = Turbidity

Negative result = No turbidity


Positive: Enterococcus faecalis

Negative: Viridans Streptococcus

Optochin Susceptibility and Bile Solubility

In the optochin test, a filter paper disk containing optochin (ethylhydrocuprein hydrochloride) is added to the surface of an SBA plate that has just been inoculated with an a-hemolytic streptococcus. The plate is incubated overnight at 35° C in a CO2incubator. A zone of inhibition greater than 14 mm with a 6-mm disk and a zone of inhibition greater than 16 mm with a 10-mm disk are considered susceptible and a presumptive identification of S. pneumoniae (Figure 15-13). Isolates producing smaller zones should be tested for bile solubility to confirm their identity.

A characteristic that correlates well with optochin susceptibility is bile solubility. The test for bile solubility takes advantage of the very active autocatalytic enzyme amidase, which S. pneumoniae possesses. Under the influence of a bile salt or detergent, the organism’s cell wall lyses during cell division. A suspension of S. pneumoniae in a solution of sodium deoxycholate lyses and the solution becomes clear.

Other a-hemolytic organisms do not undergo lysis,and the solution remains cloudy. Suspensions of bacteria made in saline serve as negative controls.


Noncultural Identification


Identification of streptococci, particularly group A, can be made by the detection of the group-specific antigen from either isolated colonies or, in some cases, a direct clinical specimen such as a throat swab. Identification from isolated colonies can be accomplished byextracting the C carbohydrate by means of acid or heat. The extract containing the specific group carbohydrate is then used in a capillary precipitin test or a slide agglutination test. In the capillary precipitin test, antiserum to the specific group carbohydrate is overlayed

by the solution containing the streptococcal extract.

After 5 to 10 minutes the interface between the extraction solution and the antiserum is examined carefully for a white precipitate, which indicates a positive reaction. Each extract can be tested with a number of antisera specific to the various group antigens.

The slide agglutination test, an easier to perform alternative method, uses a carrier particle, such as latex, for the group-specific antibody. When an antibodyantigen reaction occurs, it is seen macroscopically as agglutination of the particles (Figure 15-14). These immunoassays give a definitive identification as to the Lancefield group the isolate belongs to, but when the group comprises several species the methods do not give a species identification. Of course if the results indicate that the isolate belongs to group B, it can be identified as S. agalactiae. The cost of these methods is higher than that of the standard cultural approaches; however, the results are often quicker and more accurate.

More than two dozen different antigen-detection products are commercially available, many of which use slide agglutination or an enzyme-linked immunosorbent assay (ELISA)system to detect group A streptococci from throat swabs. These are designed primarily for use in a clinic or physician’s office. Although a positive finding allows the primary care provider to treat for S. pyogenes infections without waiting for culture results, a negative result necessitates a throat culture because the sensitivity of direct detection methods for group A streptococci is not high enough to ensure that the negative result is not a false-negative. Additionally, the cost of using direct detection methods can be significantly higher than that of culture only. Ifthe direct test is negative and a subsequent culture is performed to confirm the result, the total cost is almost twice as much as a culture only. The direct detection method for group A streptococci can be a valuable diagnostic tool in the right context; however, the cost and sensitivity must be weighed carefully against the desired and actual results.

Nucleic Acid Probes

Nucleic acid probe assays are commercially available and offer rapid results and increased specificity compared with conventional identifications methods.

However, nucleic acid probe assays are more costly.

The LightCycier (Roche Applied Science, Indianapolis, Ind) is a real-time polymerase chain reaction (PCR) instrument that can identify group A and group B streptococci as well as groups C and G. The assay detects the db gene, which encodes the CAMP-factor protein of group Bstreptococci and the pts! (phosphotransferase) gene of group A streptococci. Groups C and G have a pts! gene sequence similar to group A streptococci, but the instrument is able to distinguish among the three groups. Compared with bacterial throat cultures for group A streptococci, the LightCycler has a sensitivity of 93% and a specificity of 98%. It should be noted that the LightCycler had more positive results (58 of 384) than cultures (55 of 384).

The AccuProbe pneumococcus test (Gen-Probe, San Diego, CaliO uses a DNA probe to detect the 16s Rrna sequence of S. pneumoniae. The less than 1% difference in the rRNA sequence between S. pneumoniae and S. oratis and S. mitis raises questions about the specificity of this assay. The Smart Cycler (Cepheid, Sunnyvale, CaliO real-time PCR instrument also provides kits for the detection of streptococci. This instrument has fourchannel optics, allowing the detection of several targets in one sample (multiplex).

Susceptibility Testing

Despite widespread use, penicillin is the drug of choice in treating most streptococcal infections. However, penicillin-resistant S. pneumoniae and viridans group isolates have been reported worldwide, and the incidence of penicillin-resistant S. pneumoniae seems to be increasing. Erythromycin and narrow-spectrum cephalosporins are alternatives, although erythromycin resistance began being reported in the early 1990s.

Multiple-drug-resistant pneumococci have also been reported.

Vancomycin is an effective antimicrobial for treating infections caused by gram-positive organisms. Until the mid-1980s resistance to vancomycin was rarely seen in clinical isolates. Vancomycin resistance is now being seen more commonly, although it is still not widespread. Gram-positive isolates are generally routinely tested for vancomycin susceptibility. Table 15-3 lists the usual patterns of vancomycin susceptibility for a number of gram-positive cocci. In particular, Pediococcus and Leuconostoc spp. are resistant to vancomycin. Some streptococci and a significant number of enterococci demonstrate resistance to this antimicrobial agent. Because of the fastidious nature of the streptococci, antimicrobial susceptibility testing is not normally performed. As their susceptibility patterns have become less predictable, however, more laboratories have been forced to offer antimicrobial susceptibility testing. Fastidious bacteria do not grow well in most standardized procedures for antimicrobial susceptibility testing. In the case of S. pneumoniae and other streptococci, the Clinical and Laboratory Standards Institute (CLS!) , formerly known as the National Committee for Clinical Laboratory Standards (NCCLS), has established procedures for both disk diffusion and broth dilution assays. Commercial

methods, such as the Etest (AB Biodisk, Solna, Sweden), have been given Food and Drug Administration (FDA) approval.



Antigenic Structure

S. pyogenes has a cell-wall structure similar to that of other streptococci and gram-positive bacteria.

The group antigen is unique, placing the organism in Lancefield group A. M protein, an antigen not found in the other Lancefield groups, is attached to the peptidoglycan of the cell wall and extends to the cell surface. The M protein is essential for virulence.

Virulence Factors

The best-defined virulence factor in S. pyogenes is M protein, encoded by the emm genes. More than80 different serotypes of M protein exist, identified as M1, M2, and so on. Resistance to infection with S. pyogenes appears to be related to the presence of typespecific antibodies to the M protein. This means that an individual with antibodies against M5 is protected from infection by S. pyogenes with the M5 protein but remains unprotected against infection with the roughly 80 remaining M protein serotypes. The M protein molecule causes the streptococcal cell to resist phagocytosis and also plays a role in adherence of the bacterial cell to mucosal cells. Additional virulence factors associated with group A streptococci are fibronectin-binding protein (protein F); lipoteichoic acid; hyaluronic acid capsule; and extracellular products, including hemolysins, toxins, and enzymes. Lipoteichoic acid and protein F are adhesion molecules that mediate adherence to host epithelial cells. Lipoteichoic acid, which is affixed to proteins on the bacterial surface, in concert with M proteins and fibronectin-binding protein secures the attachment of streptococci to the oral mucosal cells. The hyaluronic acid capsule of S. pyogenes is weakly immunogenic.

The capsule prevents opsonized phagocytosis by neutrophils or macro phages. The capsule also allows the bacterium to mask its antigens and remain unrecognized by its host.

Other products produced by S. pyogenes are streptolysin 0, streptolysin S, deoxyribonuclease (DNase), streptokinase, hyaluronidase, and erythrogenic toxin.

Although all these products have been postulated to play a role in virulence, the exact role each has in

infection is not clear. S. pyogenes secretes four different DNases: A, B, C, and D. All strains produce at least one DNase; the most common is DNase B. These enzymes are antigenic, and antibodies to DNase can be detected following infection.

Ahemolysin responsible for hemolysis on SBAplates incubated anaerobically is streptolysin 0 (SLO). The “0” refers to the fact that this hemolysin is oxygen labile. It is active only in the reduced form, which is achieved in an anaerobic environment. Streptolysin 0 lyses leukocytes, platelets, and other cells as well as RBCs. Streptolysin 0 is highly immunogenic, and the infected individual readily forms antibodies to the hemolysin. These antibodies can be measured in the anti-streptolysin-O (ASO) test to determine whether an individual has had a recent infection with S. pyogenes.

Streptolysin S is oxygen stable, lyses leukocytes, and is nonimmunogenic. The hemolysis seen around colonies that have been incubated aerobically is due to streptolysin S. Filtrates of group A streptococci cause the lysis of fibrin clots through the action of streptokinase on plasminogen. The plasminogen is converted into a protease (plasmin), which lyses the fibrin. Antibodies

to streptokinase can be detected following infection but are not specific indicators of group A infection because groups C and G also form streptokinase.

Hyaluronidase, or spreading factor, is an enzyme that solubilizes the ground substance of mammalian connective tissues (hyaluronic acid). It was postulated that the bacteria use this enzyme to separate the tissue and then spread the infection. No real evidence exists, however, that hyaluronidase favors the spread of S. pyogenes through the tissues.

Some strains of S. pyogenes cause a red spreading rash, referred to as scarlet fever, caused by streptococcal pyrogenic exotoxins (Spes), formerly called erythrogenic toxins. The three immunologically distinct types are SpeA, SpeB, and SpeC. These toxins function as superantigens. Streptococcal superantigens belong to a family of highly mitogenic proteins secreted individually or in certain combinations by many S. pyogenes strains. These proteins share the ability to

stimulate T-lymphocyte cell proliferation by interaction with class II major histocompatibility  complex (MHC) molecules on antigen presenting cells and specific V f3-chains of the T-cell receptor. This interaction results in the production of interleukin-1, tumor necrotizing factor, and other cytokines that appear to mediate the disease processes associated with these toxins. Clinical Infections Infections resulting from S. pyogenes include pharyngitis, scarlet fever, skin or pyodermal infections, and other septic infections. In addition, rheumatic fever and acute glomerulonephritis (AGN) may occur as a result of infection with S. pyogenes.

Bacterial Pharyngitis

The most common clinical manifestations of group A streptococcal infection are pharyngitis and tonsillitis.

Most cases of bacterial pharyngitis are due to S. pyogenes. Other groups, particularly C and G, have the capability to produce significant acute pharyngitis but are less commonly seen.

“Strep throat” is most commonly seen in children between 5 and 15 years of age. After an incubation

period of 1 to 4 days, an abrupt onset of illness ensues, with sore throat, malaise, fever, and headache. Nausea, vomiting, and abdominal pain are not unusual. The tonsils and pharynx are inflamed. The cervical lymph nodes are swollen and tender. The disease ranges in intensity, however, and all these symptoms may not be seen. In fact, it is not unusual to isolate a nearly pure culture of S. pyogenes from the throat of a child with a fever who complains only of a mild sore throat.

The symptoms subside within 3 to 5 days unless complications, such as peritonsillar abscesses, occur.

The disease is spread by droplets and close contact.

Although clinical criteria have been proposed, the diagnosis of streptococcal sore throat relies on a throat culture or direct antigen detection. About one third of those complainingof sore throat have a throat culture positive for S. pyogenes.

Pyodermal Infections

Skin or pyodermal infections with group Astreptococci result in the syndrome of impetigo, cellulitis, erysipelas, wound infection, or arthritis. Impetigo, a localized skin disease, begins as small vesicles that progress to weeping lesions. The lesions crust over after several days.

Impetigo is usually seen in young children (2 to 5 years) and affects exposed areas of the skin. Inoculation of the organism occurs through minor abrasions or insect bites. Erysipelas is an uncommonly seen infection of the skin and subcutaneous tissues most often occurring in elderly patients. It is characterized by an acute spreading skin lesion that is intensely erythematous with a plainly demarcated but irregular edge. Cellulitis may develop following deeper invasion by streptococci.

The infection can be serious, even life threatening, with bacteremia or sepsis. In patients with peripheral vascular disease or diabetes, cellulitis may lead to gangrene.

Infection with strains of S. pyogenes that produce Spes may result in scarlet fever.Strains of S. pyogenes infected with the temperate bacteriophage Tl2 produce streptococcal pyrogenic exotoxins. Scarlet fever, which appears within 1 to 2 days following bacterial infection, is characterized by a diffuse red rash that appears on the upper chest and spreads to the trunk and extremities. The rash disappears over the next 5 to 7 days and is followed by desquamation.

Necrotizing Fasciitis

Group A streptococcus has been associated with necrotizing fasciitis (NF), an invasive infection characterized by a rapidly progressing inflammation and necrosis of the skin, subcutaneous fat, and fascia. Although relatively

uncommon, NF is a life-threatening infection.

Mortality and morbidity may be prevented if early intervention is administered to the affected individual; the mortality rate may reach greater than 70% if left untreated. Many different bacteria can cause destruction of the soft tissue in this manner, a clinical feature that has been described as “flesh-eating disease.”

Depending on which organisms are cultured, NF may be categorized as type 1, 2, or 3. A polymicrobial infection from which aerobic and anaerobic bacteria are recovered is categorized as type 1 NF. Type 2 NF consists of only group A streptococci. Type 3 is gas gangrene or clostridial myonecrosis. A variant of NF type 1 is saltwater NF, in which an apparently minor skin wound is contaminated with saltwater containing a Vibrio sp.

Cases of NF were described as far back as the eighteenth century, but the term was not conceived until 1952. In addition to flesh-eating bacteria syndrome, other terms for NF have included suppurative fasciitis, hospital gangrene, and necrotizing erysipelas. Antigenic NF may occur as a result of trauma, such as burns and lacerations. In most cases NF occurs in an individual with an underlying illness who has suffered trauma to

the skin. The break in the skin may become the portal of entry for the bacteria. Necrotizing fasciitis infections

caused by group A streptococci, however, occur in young, healthy individuals, and in many cases a break in the skin that served as portal of entry is not found.

Streptococcal Toxic Shock Syndrome

An increase in streptococcal toxic shock syndrome (STSS) cases has been reported in recent years. STSS is a condition in which the entire organ system shuts down, leading to death. The exact portal of infection is unknown for most STSS cases, although minor injuries or surgeries have been implicated. The initial streptococcal infection is often severe (e.g., pharyngitis, peritonitis, cellulitis, wound infections), and the symptoms that develop are similar to those of staphylococcal toxic shock syndrome. Patients are often bacteremic and have NF. Group A streptococcus associated with STSS produces an Spe, notably SpeA. It has been proposed that these toxins play a major role in the pathogenesis of this disease. Other virulence factors, such as SLO and various cell-wall antigens, can also cause toxic shock.


Post-Streptococcal Sequelae

Two serious complications of group A streptococcal disease are rheumatic fever and AGN. Rheumatic fever is a complication that typically follows S. pyogenes pharyngitis. It is characterized by fever and inflammation of the heart, joints, blood vessels, and subcutaneous tissues. Attacks usually begin within a month after infection. The most serious result is chronic, progressive damage to the heart valves. Repeated infections may produce further valve damage. By 1980 many clinicians considered rheumatic fever eradicated. A resurgence of rheumatic fever occurred during the late 1980s, however, and it is once again a significant problem.

The cause of this resurgence is not completely clear, but it appears to be a result of several factors, including clinical management of bacterial pharyngitis, epidemiologic changes, and bacteriologic factors.

Acute rheumatic fever and its chronic sequelae, rheumatic heart disease, remain problematic in developing countries and in some poor inhabitant populations in industrialized countries.

The pathogenesis of rheumatic fever is poorly understood. Several theories have been proposed, including antigenic cross-reactivity between streptococcal antigens and heart tissue, direct toxicity resulting from bacterial

exotoxins, and actual invasion of the heart tissues by the organism. Most evidence favors cross-reactivity as being responsible for the effects.

In contrast to rheumatic fever, AGN sometimes occurs after a cutaneous or pharyngeal infection. It is more common in children than in adults. The pathogenesis appears to be immunologically mediated.

Circulating immune complexes are found in the serum of patients with AGN, and it is postulated that these antigen-antibody complexes deposit in the glomeruli.

Complement is subsequently fixed, and an inflammatory response causes damage to the glomeruli, resulting in impairment of kidney function.


Antimicrobial Therapy

The group A streptococci are susceptible to penicillin, which remains the drug of choice. For patients allergic to penicillin, erythromycin can be used. For patients who have a history of rheumatic fever, prophylactic doses of penicillin are given to prevent any recurrent infections that might cause additional damage to the heart valves.


Laboratory Diagnosis

An essential step in the diagnosis of streptococcal pharyngitis is proper sampling. The tongue should be depressed and the swab rubbed over the posterior pharynx and each tonsillar area. If exudate is present, it should also be touched with the swab. Care should be taken to avoid the tongue and uvula. Examination of Gram stains of upper respiratory specimens or skin swabs is of little value because these areas have considerable amounts of gram-positive cocci as part of the normal bacterial flora.

Transport media are not required for normal conditions. The organism is resistant to drying and can be recovered from swabs several hours after collection.

An SBA plate is inoculated and streaked for isolation. Incubation should be at 35° C either in ambient air or under anaerobic conditions. Studies have shown that the normal respiratory flora tend to overgrow the ~-hemolytic streptococci when incubated in increased CO2, Several selective media, such as SBA containing trimethoprim-sulfamethoxazole, have been recommended for better recovery of B-hemolytic streptococci from throat cultures. The plate is observed after 24 hours for the presence of B-hemolytic colonies. If none are found, incubation should continue for an additional 24 hours before the culture is reported as negative. Colonies of S. pyogenes on SBAare small, transparent, and smooth with a well-defined area of !)-hemolysis. A Gram stain will reveal gram-positive cocci with some short chains. Suspect colonies can be Lancefield-typed using serologic methods, which will give a definitive identification, or biochemical tests can be performed.

The correlation between the presumptive identification using biochemical methods and the definitive serologic method is high. Therefore many laboratories use the less-expensive biochemical methods. A key test that should be done is bacitracin susceptibility or PYR hydrolysis. S. pyogenes is susceptible to bacitracin and hydrolyzes PYR, whereas the other !)-hemolytic groups are resistant to bacitracin and are PYR negative.

When the origin of an isolate is not the throat (i.e., blood, sputum), additional tests should be part of the early identification scheme. In this case hippurate hydrolysis or the CAMP test, bile esculin test, and NaCI broth growth should also be included. The reactions shown by some of the catalase-negative, gram-positive cocci to various biochemical tests are outlined in Table 15-3. Some immunologic tests used to detect past infection with S. pyogenes include ASO, anti-DNase, antistreptokinase, and anti-hyaluronidase titers.


Streptococcus agalactiae Antigenic Structure

All strains of S. agalactiae have the group B-specific antigen, an acid-stable polysaccharide located in the cell wall. Additionally, there are three major serotypes: I, II,and III.These type-specific antigens are capsular polysaccharides and can be detected by precipitin tests.

The terminal position in the repeating unit is composed of sialic acid, an important virulence factor.

Virulence Factors

The capsule is an important virulence factor in group B streptococcal infections. Antibodies against the typespecific antigens protect mice against strains of S.

agalac-tiae with the homologous polysaccharide in the capsule. The capsule prevents phagocytosis but is ineffective after opsonization. Sialic acid appears to be the most significant component of the capsule. Studies

with mutant strains of S. agalactiae showed that loss of capsular sialic acid was associated with loss of virulence.

The capsular sialic acid appears to be a critical virulence determinant. It is postulated that the sialic acid in the surface of the bacterial cell inhibits activation of the alternative pathway of complement. Other products produced by S. agalactiae include a hemolysin, the CAMP factor, neuraminidase, deoxyribonuclease, hyaluronidase, and protease. No evidence exists that any of these products plays a role in the virulence of this organism.

Clinical Infections

Group B streptococci have been known for many years as the cause of mastitis in cattle. It was not until Lancefield defined streptococcal classification that their role in human disease was recognized. S. agalactiae is a significant cause of invasive disease in the newborn. Two clinical syndromes are used to describe neonatal group B streptococcal disease: early-onset infection (less than 7 days old) and late-onset infection (at least 7 days old). Early-onset disease accounts for about 80% of the clinical cases in newborns and is caused by vertical transmission of the organism from the mother. Colonization of the vagina and rectal area with group B streptococci is found in 10% to 30% of pregnant women.

Most infections of infants occur in the first 3 days after birth, usually within 24 hours. This infection is commonly associated with obstetric complications, prolonged rupture of membranes, and premature birth.

It is often a pneumonia or meningitis with bacteremia.

The mortality rate is high, and death usually occurs if treatment is not started quickly. The most important determining factor seems to be the presence of group B streptococci in the vagina of the mother. It is recommended that all pregnant women be screened for group B streptococci at 35 to 37 weeks’ gestation.

Late-onset infection occurs between 1 week and 3 months after birth and usually presents as meningitis.

This infection is uncommonly associated with obstetric complications. Also, the organism is rarely found in the mother’s vagina prior to birth. The mortality rate is considerably less than that of early-onset disease, but it is high enough to be of serious concern.

The incidence of group B streptococcal infections drops dramatically after the neonatal period. In adults infection affects two types of patients. The first is young, previously healthy women who become ill after childbirth or abortion; endometritis and wound infections are most common. The second type of patient is the elderly person with a serious underlying disease or immunodeficiency.

The drug of choice for treating group B streptococci infections is penicillin, although this group is less susceptible to penicillin than are group A streptococci.

The clinical response to antimicrobial therapy is often poor despite the heavy doses given. Some clinicians recommend a combination of ampicillin and an aminoglycoside for treating group B streptococci infections.


Laboratory Diagnosis

The group B streptococci grow on SBA as grayish white mucoid colonies surrounded by a small zone of [)- hemolysis (Figure 15-15). Group B streptococci are gram-positive cocci that form short chains in clinical specimens and longer chains in culture. Presumptive identification is based on biochemical reactions. The

most useful tests are hippurate hydrolysis and the CAMP test. These tests enable the organism to be readily differentiated from other j3-hemolytic streptococcal isolates. Figure 15-16 demonstrates how bacitracin and the CAMP test can be used to differentiate Streptococcus. The definitive identification can be made by extracting the group antigen and reacting it

with specific anti-group B antisera in a precipitin or agglutination procedure. Detection of group B streptococci in expectant women is done by collecting vaginal and rectal material with swabs between 35 and 37 weeks of gestation. Samples are inoculated into selective broth such as Todd-Hewitt broth containing either 10 flgfmL of colistin and 15 ~lgfmL nalidixic acid (Urn broth; Becton Dickinson, Cockeysville, Md). Gentamicin, 8 flgfmL and 15 flgfmL nalidixic acid may also be used (Hardy Diagnostics, Santa Maria, CaliQ. The inoculated media are incubated at room temperature for 18 to 24 hours before being subcultured to SBA. Cultures are examined for colonies resembling group B streptococci after 24 hours of incubation at 35° C in an incubator containing 5% CO2, Cultures that do not show colonies resembling group B streptococci are incubated for an additional 24 hours.

Other Streptococcal Groups

 Groups C and G

In the most recent reclassification of I)-hemolytic streptococci, isolates from humans that belong to groups A, C, or G are subdivided into large- and small-colony forms. The large-colony-forming isolates group A (s. pyogenes), C, and G are classified as pyogenic streptococci. The large-colony-forming I)-hemolytic isolates

with group C and G antigens belong to the same subspecies, S. dysgalactiae subsp. equisimilis. However,

there have been reports of blood culture isolates in which S. dysgalactiae subsp. equisimilis were identified with group A antigen and not group C or G antigens.

The small-colony-forming j3-hemolytic isolates with group C and G antigens belong to the anginosus or Streptococcus milleri spp. group. Most members of the S. milleri spp. group are a-hemolytic and are considered part of the viridans group.

S. equisimilis isolates were recovered from the upper respiratory tract, vagina, and skin of humans and were

thought to be uncommon in domestic animals. Meanwhile a bovine bacterium, S. dysgalactiae, was reported to be identical to S. equisimilis except for the lack of 11- hemolysis. In reviving the nomenclature designations of these two species after they were ignored for several years, investigators demonstrated that S. dysgalactiae, S. equisimilis, and streptococci belonging to group G and L showed high levels of DNA-DNA relatedness and therefore belonged to a single species, S. dysgalactiae. Because several antigenic groups are included within the species, serotyping of S. dysgalactiae to determine species is extremely complex. Among the group C streptococci are five taxa that have been differentiated: (1) human I)-hemolytic isolates; (2) porcine, previously named S. equisimilis; (3) bovine, n-hemolytic; (4) equine, I)-hemolytic, belonging to the two subspecies of S. equi (s. equi subsp. equi and S. equi subsp. zooepidemicus); and (5) the human small colony-forming I)-hemolytic isolates, which belong to the S. milleri group. In group G, three taxa were described: (1) human, large-colony-forming, I3-hemolytic isolates, which belong to S. dysgalactiae; (2) human, small-colonyforming, f3-hemolytic, which belong to the S. milleri group; and (3) the bovine, canine, and feline f3-hemolytic isolates, which belong to S. canis. Finally group L f3 hemolyticstreptococci also belong to S. dysgalactiae. Clinical infections from groups C and G streptococci have been reported in the literature, although these are uncommon human pathogens; group Cstreptococci account for less than 1% of all bacteremias.

Most infections, including endocarditis, meningitis, and primary bacteremia, are community acquired and occur in patients with underlying illnesses. Cases of NF or myositis have also been reported.


Group D

The group 0 streptococci include S. bovis and S. equinus. Until the mid-1980s, the group 0 streptococci were subdivided into the enterococcal and nonenterococcal groups, with the understanding that those found in the intestinal tract were part of the enterococcal group. Both groups were bile esculin positive, but the nonenterococcal organisms would not grow in a nutrient broth with 6.5% NaCI. As more became known about the molecular characteristics of each of these subgroups, however, the enterococcal group was placed in a new genus, Enterococcus, but the nonenterococcal group remained part of the group 0 streptococci. Although S. bovis is considered a nonenterococcal isolate, it can be found in the intestinal tract.

The group 0 streptococci can produce bacterial endocarditis, urinary tract infections (UTls), and other

diseases, such as abscesses and wound infections. An association has been made between bacteremia resulting from S. bovis and the presence of gastrointestinal tumors. Isolation of S. bovis from a blood culture may be the first indication that the patient has an occult tumor. It is important to distinguish the group 0 streptococci from Enterococcus because group 0 is susceptible to penicillin, whereas Enterococcus is usually resistant.

The group 0 streptococci can be presumptively identified as indicated in Table 15-3.The differentiation of nonhemolytic streptococci is outlined in Figure 15-17. Normally hemolysis is absent (Figure 15-18), although isolates can be a- or ~-hemolytic on occasion. A key reaction of this group is that it is positive for bile esculin but fails to grow in 6.5%NaCIbroth. In addition, it can be separated from Enterococcus with the PYR test; group 0 is negative, but Enterococcus is positive.

Serotyping should be done to identify an isolate as

S. bovis because it cannot be positively distinguished from some of the viridans group on the basis of biochemical tests alone.


Enterococci were previously classified as group 0 streptococci. This group consists of gram-positive cocci that are natural inhabitants of the intestinal tracts of humans and animals. The commonly identified species in clinical specimens are E. faecalis and E. faecium. Other species such as E. durans, E. avium, E. casseliflavus, E. gal/inarum, and E. raffinosus are observed occasionally. All species produce the cell wall associated teichoic acid antigen, also referred to as the group 0 antigen in the Lancefield classification system.

Most enterococci are nonhemolytic or a-hemolytic, although some strains show f3-hemolysis. It should be noted that enterococci sometimes exhibit a pseudocatalase reaction: weak bubbling in catalase test.

Identification of the different species is based on biochemical characteristics. Unlike streptococci, enterococci have the ability to grow under extreme conditions, for example, in the presence of bile or 6.5% NaClor at 45°Cor alkaline pH. The ability of enterococci to hydrolyze PYRis useful for differentiating them from the group 0 streptococci (Figure 15-19).


Virulence Factors

The virulence factors that contribute to the pathogenicity of enterococci are not completely understood.

The enterococci have a survival advantage over other organisms in the fact that they can grow in extreme conditions and are resistant to multiple antimicrobial agents. The extracellular surface protein, extracellular serine protease, and gelatinase of E. faecalis are thought to playa role in the adherence and colonization of the species to heart valves and renal epithelial cells. E. faecalis also produces a two subunit toxin, termed cytolysin. This toxin shows similarity to bacteriocins produced by gram-positive bacteria and is

expressed by a quorum-sensing mechanism.

Clinical Infections Enterococci are frequent causes of nosocomial infections.

Of these, UTls are the most common, followed by bacteremia. Urinary tract infection is often associated with urinary catheterization or other urologic manipulations.

Prolonged hospitalization is a risk factor for acquiring enterococcal bacteremia. Bacteremia is often observed in hemodialysis patients, immunocompromised patients with a serious underlying disease, or patients who have undergone a prior surgical procedure.

Endocarditis from enterococci is seen mainly in elderly patients with prosthetic valves or valvular heart disease. Enterococci account for about 5% to 10% of infections in patients with bacterial endocarditis.

Although frequently isolated from intraabdominal or pelvic wound infections, their role in these infections remains contentious. In burn patients, enterococcal wound infection and sepsis resulting from contaminated xenografts have been reported. Rare cases of enterococcal infection of the central nervous system in patients who have had neurosurgery or head trauma and in immunosuppressed patients with enterococcal bacteremia have been reported. Respiratory tract infections from enterococci are also rare and have been reported in severely ill patients who have had prolonged antimicrobial therapy.

Laboratory Diagnosis

Standard procedures for collecting and transport of blood, urine, or wound specimens should be followed.

The specimens should be cultured as soon as possible with minimum delay. Trypticase soy or brain-heart infusion agar supplemented with 5% sheep blood is routinely used to culture enterococci. Enterococci grow well at 35° C in the presence of CO2 but do not require a high level of CO2 for growth. If the clinical specimen is obtained from a contaminated site or is likely to contain gram-negative organisms, selective media containing bile-esculin azide, colistin-nalidixic acid agar, phenylethyl alcohol agar, media with chromogenic substrates, or cephalexin-aztreonamarabinose agar should be used for isolation of enterococci.

Enterococcus spp. are identified based on their ability to (1) produce acid in carbohydrate broth, (2) hydrolyze arginine, (3) tolerate 0.04% tellurite, (4) utilize pyruvate, (5) produce acid in methyl u.-Dglucopyranoside

(MGP), (6) grow in the presence of 100-lAgefrotomycin acid disk, and (7) exhibit motility. Table 15-4 shows the differentiation of the enterococci species based on these phenotypic and biochemical characteristics. E. faecalis is easily identified by its ability to grown in the presence of tellurite.

Commercially available kits may be useful for identification of E. faecalis, but they are not adequate for other enterococci.

Molecular typing methods, such as pulsed-field gel electrophoresis, contour-clamped homogeneous

electric-field electrophoresis, ribotyping and PCR-based typing methods have been used mainly to type enterococcal species in epidemiologic studies and investigations of vancomycin-resistant enterococci (VRE).


Antimicrobial Resistance

Enterococci show resistance to several of the commonly used antimicrobial agents, so differentiation from Streptococcus and susceptibility testing are important.

Enterococci have intrinsic or acquired resistance to several antimicrobial agents, including aminoglycosides, I3-Iactams, and glycopeptides. Resistance of enterococci to glycopeptides such as vancomycin and teicoplanin were first described in late 1980s. Of the six vancomycin-resistance phenotypes (VanA to VanE and VanG), VanA and VanS phenotype are most frequently encountered. The VanAphenotype is acquired, inducible, carried on a transposon ([nI546), and characterized by high-level resistance to vancomycin (minimal inhibitory concentration [MIC] >32 mg/L) and teicoplanin (MIC >16 mg/L), whereas the VanS phenotype is chromosomal mediated and characterized by variable levels of resistance to vancomycin and susceptibility to teicoplanin.

VanC phenotype is characterized by low levels of resistance to vancomycin (MIC 8 to 16 mg/L) and

susceptibility to teicoplanin and has been described in only one strain of E. gallinarum. VanD phenotype has been described in a few strains of E. faecium and is characterized by low-level resistance to vancomycin with susceptibility or intermediate resistance to teicoplanin. VanE phenotype has been described in two E. faecalis strains, and VanG phenotype has been described in a few E. faecalis strains from Australia and Canada. The VanE and VanG phenotypes are similar to VanC in that they are characterized by low levels of vancomycin resistance. Vancomycin containing agar has been used for screening of VRE colonization and for infection control in hospitals. Susceptibility testing of the enterococcal isolate should be performed only if the isolate is clinically significant.


 Streptococcus pneumoniae Antigenic Structure

Also known as the pneumococcus, S. pneumoniae is isolated from a variety of infections. The cell wall of S.

pneumoniae contains an antigen, referred to as Csubstance, which is similar to the C carbohydrate of the various Lancefield groups. A ~-globulin in human serum, called the C-reactive protein, reacts with C substance to form a precipitate. This is a chemical reaction reaction and not an antigen antibody combination. The amount of C-reactive protein increases during inflammation and infection. The pneumococcus can express one of approximately 80 different capsular types based on chemical variations of the capsular polysaccharide.

Isolates from certain sources, for example, cases of lobar pneumonia, show a predominance of a particular capsular type or types. The capsule is antigenic and can be identified with appropriate antisera.

In the presence of specific anticapsular serum, the capsule swells (Quellung reaction). This reaction not only allows for identification of S. pneumoniae but serves to serotype the isolate specifically as well.


Virulence Factors

The characteristic of S. pneumoniae that is clearly associated with virulence is the capsular polysaccharide.

Laboratory strains that have lost the ability to produce a capsule are nonpathogenic. In addition, opsonization of the capsule renders the organism nonvirulent. Several toxins are produced, including a hemolysin, an immunoglobulin A protease, neuraminidase, and hyaluronidase. None of these has been shown to have a role in disease production.


Clinical Infections

S. pneumoniae is a common isolate in the clinical microbiology laboratory. It is an important human pathogen, causing pneumonia, sinusitis, otitis media, bacteremia, and meningitis. S. pneumoniae is the most frequently encountered isolate in children under the age of 3 years with recurrent otitis media. S. pneumoniae, the number one cause of bacterial pneumonia, is especially prevalent in elderly persons as well as in patients with underlying disease. Of the more than 80 capsular serotypes, about a dozen account for most pneumococcal pneumonia cases.

For an individual to contract pneumococcal pneumonia, the organism must be present in the nasopharynx and the individual must be deficient in specific circulating antibody against the capsular type of the colonizing strain of S. pneumoniae. Pneumonia resulting from S. pneumoniae is not usually a primary infection but rather a result of disturbance of the normal defense barriers. Predisposing conditions such as alcoholism, anesthesia, malnutrition, and viral infections of the upper respiratory tract can lead to pneumococcal disease in the form of lobar pneumonia.

Pneumococcal pneumonia is characterized by sudden onset with chills, dyspnea, and cough. The infection begins with aspiration of respiratory secretions, which often contain pneumococci. The infecting organisms in the alveoli stimulate an outpouring of edema fluid, which serves to facilitate the spread of the organism to adjacent alveoli. The process stops when the fluid reaches fibrous septa that separate the major lung lobes. This accounts for the “lobar” distribution of the infection-hence the name. Most isolates from pneumococcal lobar pneumonia are types 1,2, and 3. Pneumonia may be complicated by a pleural effusion that is usually sterile (empyema). The laboratory may receive fluid from a pleural aspirate for culture.

An infected effusion contains many white cells and pneumococci, which are visible on Gram stain. Even with antimicrobial therapy, the mortality is relatively high (5% to 10%); without therapy, however, the mortality rate approaches 50%.

Pneumococcus causes bacterial meningitis in all age groups. Meningitis usually follows other infections with

S. pneumoniae, such as otitis media or pneumonia. The course of the disease is rapid, and the mortality rate is near 40%. Direct smears of the cerebrospinal fluid (CSF) often reveal leukocytes and numerous gram-positive

cocci in pairs. Pneumococci may also be involved in other infections, such as endocarditis, peritonitis, and bacteremia. Bacteremia often occurs during the course of a serious infection. Consequently samples for blood culture are often taken simultaneously with sputum or a fluid aspirate.

A vaccine containing the polysaccharide capsular material of the 23 most commonly encountered types is available. It is recommended for those at risk of developing pneumococcal disease (e.g., asplenic individuals and elderly patients with cardiac or pulmonary disease). The vaccine has been successful in reducing the incidence and severity of pneumococcal disease. A pediatric vaccine was recently approved that contains fewer antigenic types.


Antimicrobial Resistance

Because most isolates are susceptible, pneumococcal infections are usually treated with penicillin. Over the last three decades, however, S. pneumoniae has become increasingly resistant to penicillin, and these isolates are generally treated with erythromycin or chloramphenicol. Moreover, penicillin-resistant pneumococci are reported to show resistance to other classes of drugs, such as B-Iactams, macrolides, and tetracyclines. The susceptibility of S. pneumoniae to penicillin and macrolides has also varied over time, geographic region, and country. Resistance to extended-spectrum cephalosporins such as ceftriaxone and cefotaxime has also been on the rise, although these agents have been used successfully in the treatment of serious infections caused by penicillinresistant pneumococci.

Mutlidrug-resistant S. pneumoniae, defined as pneumococcal isolates resistant in vitro to two or more classes of antimicrobial agents, have been reported and can occur in the presence or absence of penicillin resistance. Pneumococcal strains that exhibit resistance to various antimicrobial agents such as erythromy erythromycin, tetracycline, chloramphenicol, and trimethoprimsulfamethoxazole have emerged.


Laboratory Diagnosis

The cells characteristically seen on Gram stain appear as gram-positive cocci in pairs (diplococci). The ends of the cells are slightly pointed, giving them an oval or lancet shape (Figure 15-20). The cocci may occur singly,

in pairs, or in short chains but most often are seen as pairs. As the culture ages, the Gram stain reaction becomes variable, and gram-negative cells are seen. The capsule can be demonstrated by using a capsule stain.

The nutritional requirements of S. pneumoniae are complex. Media such as brain-heart infusion agar, trypticase soy agar with 5% sheep RBCs, or chocolate agar are necessary for good growth. Some isolates require increased CO2 for growth during primary isolation. The organism can use a wide range of carbohydrates. Isolates produce a large zone of a-hemolysis surrounding the colonies. Young cultures have a round, glistening, wet, mucoid, dome-shaped appearance (Figure 15-21).As the colonies become older, autolytic changes result in a collapse of each colony’s center, giving it the appearance of a coin with a raised rim.

The tendency of S. pneumoniae to undergo autolysis can make it difficult to keep isolates alive. Clinical isolates and stock cultures require frequent subculturing (every 2 to 3 days) to ensure viability. The colonies may closely resemble those of the viridans streptococci, but a presumptive differentiation is not difficult to make.

The greatest concern in the laboratory diagnosis is distinguishing S. pneumoniae from the viridans streptococci.

Procedures commonly used to accomplish this are optochin susceptibility, bile solubility, and the

Quellung reaction; optochin susceptibility is the most commonly used procedure. Optochin susceptibility takes advantage of the fact that S. pneumoniae is susceptible to optochin, whereas other a-hemolytic species are resistant.

The bile solubility test evaluates the ability of S. pneumoniae to lyse in the presence of bile salts. It correlates with optochin susceptibility; that is, an isolate that is S. pneumoniae will be optochin susceptible and bile soluble. This test also differentiates pneumococcus from the viridans streptococci.

Although currently seldom used, the Quellung reaction identifies an isolate as S. pneumoniae and determines its capsular type. This reaction can be used to identify the organism directly from sputum, CSF,and other sources. The anti-pneumococcal serum is mixed with the material (clinical specimen or isolated colony) along with methylene blue and then examined using the oil-immersion objective. A positive reaction occurs when the pneumococci are mixed with homologous capsular antiserum; the capsule becomes more refractile and swollen.


Viridans Streptococci

The viridans streptococci are constituents of the normal flora of the upper respiratory tract, the female genital tract, and the gastrointestinal tract. The term viridans means “green.” Many species in this group produce ahemolysis, showing a green discoloration on SBA.A few species are nonhemolytic, and rarely are they 13- hemolytic. Viridans streptococci are fastidious, with some strains requiring CO2for growth. The current classification assigns streptococci species in the viridans group to one of the four groups: anginosus group (s.

anginosus, S. constellatus, and S. intermedius); mitis group (s. sanguis, S. parasanguis, S. gordonii, S. crista, S. infantis, S. mitis, S. ora/is, and S. peroris); mutans group (s. criceti, S. downei, S. macacae, S. mutans, S. rattus, and S. sobrinus); and salivarius groups (s. salivarius, S. thermophilus, and S, vestibu/aris). Organisms of the anginosus group may possess the Lancefield group A, C, F,G, or N antigen and in some instances may not be groupable. The organisms may also cross-react with other grouping sera. Thus identification using the

Lancefield sera is of little value.


Virulence Factors

Virulence factors that characterize the pathogenicity of viridans streptococci have not been well established.

A polysaccharide capsule and cytolysin have been identified in some members of the anginosus group. Besides these, extracellular dextran and cell surface-associated proteins (adhesins) have been implicated in adherence and colonization of these organisms in endocarditis.


Clinical Infections

The viridans streptococci are oropharyngeal commensals that are regarded as opportunistic pathogens.

Although their virulence is low, they cause disease if host defenses are compromised. Viridans streptococci are the most common cause of subacute bacterial endocarditis. Transient bacteremia is associated with endocarditis. Viridans streptococcal bacteremia is more common in children than in adults and is usually more prevalent in patients with hematologic malignancies.

Fatal cases have been characterized by fulminant cardiovascular collapse or meningitis. Those isolates that cause endocarditis produce dextran, which may allow the organism to adhere to the damaged vascular endothelium. Generally the course of endocarditis is very slow; symptoms may be present for weeks or months. Individuals whose heart valves have been damaged by rheumatic fever are especially susceptible to endocarditis resulting from viridans streptococci. Besides bloodstream infections, oral infections such as gingivitis and dental caries are caused by viridans streptococci. They have also been implicated in meningitis, dental caries, abscesses, osteomyelitis, and empyema. Although viridans streptococci are frequently isolated in association with other bacteria from bronchial brushing, their role in bacterial pneumonia is unclear. The anginosus group of the viridans streptococci has been frequently associated with abscess formation.

Certain members of the viridans group streptococci have been associated with skin and soft tissue

infections; S. intermedius group (s. intermedius, S. constellatus, and S. anginosus) have been isolated from bacteremic patients associated with invasive pyogenic infections and the tendency to form abscesses.

Treatment of viridans streptococci infections is with penicillin. Although some resistant strains have been reported, most remain susceptible.


Laboratory Diagnosis

Viridans streptococci show typical Streptococcus characteristics on Gram stain. The colonies are small and are surrounded by a zone of a-hemolysis; some isolates are nonhemolytic. The differentiation of viridans streptococci from S. pneumoniae, also a-hemolytic, is based on optochin sensitivity and bile solubility tests. The hemolytic patterns and some of the biochemical tests that distinguish members of the viridans group, namely, acid production in carbohydrate broth, hydrolysis of esculin, hydrolysis of arginine, and production of acetoin (VP test) are shown in Table 15-5. The lack of p-hemolysis separates the viridans streptococci from groups A, S, C, and G.

Two groups that might be confused with the viridans streptococci are Enterococcus and group 0 Streptococcus. Growth in 6.5% NaCl broth differentiates the viridans group from Enterococcus because the viridans streptococci fail to grow (Figure 15-22).Some strains of S. salivarius can be misidentified as S. bovis, however, because a significant number of S. salivarius isolates are bile esculin positive.

S. milleri has been isolated from abscesses and other pyogenic infections, but it is rarely involved in endocarditis.

Asingle name, S. anginosus, has been proposed for those organisms belonging to the S. milleri group. The S. milleri group is composed of strains that may have A, C, F, G, or no Lancefield antigen. These are minute colony types showing a-, p-, or no hemolysis.

When they are p-hemolytic, the zone size is several times the size of the colony. When they are growing in pure culture or in high concentration, a characteristic sweet odor, honeysuckle or butterscotch, may be present. Positive VP and negative PYR test results identify a ~-hemolytic isolate as S. milleri.

The speciation of members of the viridans streptococci group involves performing a considerable number of biochemical tests, making it difficult for identifying isolates to the species level without the use of a commercial system developed specifically for that purpose.

Several commercial systems identify the viridans streptococci, but no one system can identify all possible species. Also, such systems are relatively expensive.


Nutritionally Variant Streptococci

Nutritionally variant streptococci (NVS) were first described in 1961. These bacteria grow as satellite colonies around other bacteria and require suIfydryl compounds for growth. NVS are part of the human oral and gastrointestinal flora. Phylogenetic analysis using 16S rRNA revealed that NVS were genetically distinct from streptococci. They are now classified within the genera Granulicatella and Abiotrophia. Most of the species are not groupable by the Lancefield system; however, strains with group antigens A, F, H, L, and N have been reported. Virulence Factors The mechanisms by which NVS cause infection are not completely understood. Studies in rats suggest that the fibronectin-binding capacity of the Abiotrophia spp. may be important in infectivity of damaged heart tissue.

Clinical Infections

NVSare a significant cause of bacteremia, endocarditis, and otitis media. Endocarditis resulting from NVSis difficult to treat because of their increased tolerance to antimicrobial agents. Surgery is usually required to effect a cure. Few cases of osteomyelitis, endophthalmitis after cataract extraction, brain abscess, chronic sinusitis, septic arthritis, and meningitis attributable to NVS have been reported.


Laboratory Diagnosis

Nutritionally variant streptococci should be suspected when gram-positive cocci resembling streptococci are observed in positive blood cultures that subsequently fail to grow on subculture. It would be appropriate to use an SBAplate with a S. aureus streak and examine it for satellitism by NYS.Alternatively media can be supplemented with 10 mg/L pyridoxal hydrochloride to confirm presence of NYS.

Biochemical characteristics that differentiate species within the genera Granulicatella and Abiotrophia (G. adiacens, G. elegans, G. balaenopterae, A. defective, and A. adjacens) are production of a-and ~-galactosidase, production of I3-glucuronidase, hippurate hydrolysis, arginine hydrolysis, and acid production from trehalose and starch.


Streptococcus-like Organisms

The genera Aerococcus, Gemella, Lactococcus, Leuconostoc, Pediococcus consist of streptococcus-like organisms that resemble viridans and viridans streptococci. These have been isolated in clinical specimens and associated with infections similar to those caused by enterococci and streptococci. These organisms are frequently identified when antimicrobial susceptibility testing of a “streptococcal” isolate reveals it to be vancomycin resistant. The vancomycin-resistant, gram-positive

cocci are likely to be Leuconostoc or Pediococcus. Aerococcus is normally susceptible to vancomycin.



Aerococcus is a common airborne organism. It is widespread and an opportunistic pathogen associated with bacteremia, endocarditis, and UTls in immunocompromised patients. Aerococci resemble viridans streptococci on culture but are microscopically similar to staphylococci in that they occur as tetrads or clusters.

These organisms sometimes show a weak catalase or pseudocatalase reaction. They grow in the presence of 6.5%NaCIand can easily be confused with enterococci.

Two species, A. viridans, and A. urinae, have been associated with infection. Some strains of A. viridans are bile esculin positive and PYR positive. A. urinae is bile esculin negative and PYRnegative.



Gemella isolates are similar in colonial morphology and habitat to the viridans streptococci. Gemella spp. produce a-hemolysis or are non hemolytic. The bacteria easily decolorize on Gram staining and

Therefore appear as gram-negative cocci in pairs, tetrads, clusters, or short chains. Gemella spp. have been isolated from cases of endocarditis, wounds, and abscesses.

The following four species have been associated with clinical infections: G. haemolysans, G. morbillorum (formerly Streptococcus morbillorum), G. bergeriae, and G. sanguinis.



Lactococcus spp. are gram-positive cocci that occur singly, in pairs, or in chains and are physiologically similar to enterococci. On SBA these organisms produce a-hemolysis or are nonhemolytic. These organisms  were previously classified as group Nstreptococci.

Lactococcus spp. have been isolated from patients with UTls and endocarditis. Production of acid from carbohydrates is useful in distinguishing Lactococcus spp.

from enterococci. These microorganisms also do not react with the genetic probe in the AccuProbe Enterococcus Culture Confirmation test (Gen-probe, San Diego, Calif).



The genus Leuconostoc consists of catalase-negative, gram-positive microorganisms with irregular coccoid morphology (Figure 15-23). These organisms share several phenotypic and biochemical characteristics with Lactobacillus, viridans streptococci (Figure 15-24), Pediococcus, and Enterococcus spp. and are sometimes misidentified. Some species cross-react with the

Lancefield group D antiserum. These microorganisms are intrinsically resistant to vancomycin. In nature they are frequently found on plant surfaces, on vegetables, and in milk products. They have only recently been recognized as opportunistic pathogens in patients who are immunocompromised or treated for underlying disease with vancomycin. These microorganisms have been isolated from cases of meningitis, bacteremia, UTls, and pulmonary infections. Species associated with infection include L. citreum, L. cremoris, L. dextranicum, L. lactis, L. mesenteroides, and L. pseudomesenteroides. Biochemical identification is based on presence of catalase, absence of PYR and LAP activities, hydrolysis of esculin in the presence of bile, growth in the presence of 6.5% NaCI, and production of gas from glucose metabolism.



Members of the genus Pediococcus are facultatively anaerobic, gram-positive cocci (arranged in pairs, tetrads, and clusters) that can grow at 45° C. They may be misidentified as viridans streptococci or enterococci.

Like Leuconostoc, Pediococcus spp. are intrinsically resistant to vancomycin. The following species have been associated with infection: P acidilactici, P damnosus, P dextrinicus, P parvulus, and P pentasaceus. Pediococcus spp. have been associated with patients who have underlying gastrointestinal abnormalities or who have previously undergone abdominal surgery. The organisms have also been associated with bacteremia, abscess formation, and meningitis. Biochemical characteristics used to identify them include a positive bile esculin, the presence of LAP activity, and absence of PYR activity. The organisms do not produce gas from glucose. Some of the strains are able to grow in the presence of 6.5% NaCI.

In addition to the preceding genera, a few species of Globicatella, Helcococcus, and Alloiococcus have been reported to cause clinically significant infections.

Globicatella and Helcococcus resemble Aerococcus. Globicatella sanguis has been associated with sepsis, meningitis, bacteremia, and UTls. G. sanguis is a-hemolytic, PYR positive, LAP negative, and vancomycin

susceptible. Helcococcus kunzii has been isolated from wound infections and is frequently misidentified

as A. viridans. Alloiococcus otitidis has been associated with otitis media in children. AIIOiococci are non hemolytic but may show an a-hemolytic pattern after prolonged incubation. These organisms are PYR and LAP positive but grow slowly in the presence of 6.5% NaCI.



The organisms included in the family Streptococcaceae and the Streptococcus-like organisms are gram-positive cocci usually arranged in pairs or chains that are catalase negative. Most are aerotolerant anaerobes that derive their energy from fermentation. The growth requirements can be complex, with the use of blood or enriched medium necessary for their isolation. Molecular studies have led to numerous taxonomic changes to the family Streptococcaceae and related organisms. Identification methods may include conventional physiological testing and molecular diagnostic procedures. The role of streptococci and enterococci in human disease ranges from well-established and common to rare, but their significance is increasing. Antimicrobial resistance in streptococcal species is also emerging and is becoming a significant public health concern.



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