dealing with viridans streptococci in the clinical laboratory: the continuing challenge

Clinical Microbiology Newsletter Vol. 15, No. 10 May 15,1993

Dealing with Viridans Streptococci in the Clinical Laboratory: The Continuing Challenge Kathryn L. Ruoff, Ph.D. Microbiology Laboratory Massachusetts General Hospital Boston, MA 02114

The interest shown by oral microbiologists in the viridans streptococci has rarely been evidenced by medical microbiologists, most of whom consider these organisms a group of unexciting, fairly avirulent streptococci that need be examined only to the extent of ruling out their identity as pneumococci. While everyone agrees that viridans streptococci can cause serious infections (i.e., endocarditis), their role as isolates flom clinical specimens including normally sterile body fluids like blood or CSF is often that of a trouble- some contaminant. This article will at- tempt to review the current status of the viridans streptococci in an effort to guide clinical microbiologists in their handling of these organisms in the laboratory.

The Viridans Group Streptococci included in the viridans

group are often alpha-hemolytic, but non-hemolytic and even beta-hemolytic strains can also he found among these organisms. Although some viridans strains produce Lancefield antigens, these antigens are not necessarily corre- lated with a given species. Physiologi- cal testing has, therefore, traditionally been used for differentiation of viridans streptococci to the species level. Spe- cies included in the viridans group are vancomycin-susceptible and produce the enzyme leucine aminopeptidase

(LAP), but do not produce the enzyme pyrrolidonyl arylamidase (PYR). Other genera that may be misidentified as viridans streptococci, like Leuconostoc, P ediococcus, Aerococcus, Lactococ cus, and Gemella can usually be differentiated from streptococci by the tests men- tioned above (1). Table 1 summarizes some basic characteristics of the major viridans species or species groups; more detailed information on each species appears below.

The Streptococcus mutans Group S. mutans is one of a group of at

least seven physiologically similar but genetically distinct species. This non- hemolytic cariogenic Streptococcus can be isolated from the dental plaque of most humans, but some individuals har- bor the related species Streptococcus so- brinus, Streptococcus rattus, or Streptococcus cricetus instead of S. mutans. Other species, isolated only from animals, are named Streptococcus ferus, Streptococcus macacae, or Streptococcus downei. In addition to causing dental car- ies, S. mutans is an agent of endocarditis. This species can form eilher coccoid or rod-shaped cells, depending on growth conditions, and is very active biochemi- cally. Using sucrose as a substrate, S. mutans swains produce a glucose-containing extracellular polysaccharide. Produc- tion of this polysaccharide causes the formation of adhexent colonies on sucrose-containing agar (2).

The Streptococcus salivarius Group S. salivarius and Streptococcus ves-

tibularis have been documented as hu-

man isolates of this group; other related species are Streptococcus intestinalis and Streptococcus thermophilus. The dorsal surface of the tongue seems to be the preferred habitat of S. salivarius. This non-hemolytic viridans species may produce the enzyme urease and forms unique domed or "gumdrop" colonies on sucrose-containing agar due to the production of extracellular polysaccharide (2). Although S. salivarius is rarely isolated from endocarditis cases, it has been described as an agent of bacteremia in cancer patients (3).

Streptococcus bovis Traditionally, S. bovis was consid-

ered as a non-hemolytic, non-enterococcal group D Streptococcus. In view of the nonspecificity of the group D anti- gen (found in the genera Streptococcus, Enterococcus, and P ediococcus) and

In This Issue

Viridans Streptococci . . . . . . . . . . . 73 How to deal with the challenge that these organisms present to the laboratory

Making Your Laboratory Safe against Drug-Resistant Tuberculosis . . . . . . . . . . . . . . . . . . . 76 Procedures that can be implemented in the laboratory to minimize the risk of exposure to multi-drug-resistant strains of Mycobacterium tuberculosis

CMNEEJ 15(10)73-80,1993 EIsovi©r 0196-4399D3~$0.00 + 06.00

TABLE 1. Differentiation of commonly isolated viridans streptococci" Species VP Arsinine Esculin Mannitol Sorbitol S. mutans group + _b + ~_ + S. salivarius group +c _ + . . . . S. bovis + _ + +/_d _

"S. milled" group + + +/- +/- -

S. sanguls group - +e + f _ _

S. mitis . . . . . alnformation in this table is based on the review by Coykendall (2) and other publications listed in the

reference section. bs. rattus is arginine-positive.

C s. vestibularis is variable. S. vestibularis is also alpha-he~nolytic, unlike the non-hemolytic S. salivarius.

'iS. boris biotype I is positive; biotype II is negative.

eS. crista may be negative. f S. crista is negative, and S. parasanguis may be negative.

the fact that other distinsuishing characteristics of group D streptococci (i.e., a positive bile esculin reaction) can be found among viridans strains, S. bovis might be better included with the viridans group. S. bovis is physiologically similar to S. salivarius (see Table 1) and the two species may be confused with one another. S. bov/s swains are differentiated into two biotypes (desig- nated I and ll), with biotype I distin- guished by its ability to ferment mannitol and form large watery colonies on sucrose agar due to polysaccharide production (2).

The "Streptococcus mUleri" Group The name "Streptococcus milleri"

was originally used to describe non- hemolytic streptococci from oral infections. It later became apparent that there were physiological similarities between these strains and other previously described non-hemolytic isolates and beta-hemolytic "minute colony-forming" streptococci with Lancefield group antigens (A, C, F, or G). Facklam (4) suggested that the non-beta-hemolytic "S. milleri" strains be divided into separate species on the basis of physiological traits; he later revised his nomenclature and included small colony-forming beta-hemolytic strains in his scheme (5). The 1980s brought a proposal by Coykendall and colleagues

i

(6) to include all "S. milleri" strains in a single species, S. anginosus, but later publications supported the division of these ~ganisms into three separate species (7). Table 2 summarizes these no- menclatural changes.

The current species of the "S. milleri" group and key tests for identifying them are displayed in Table 3. The differential parameters are not necessarily traditional tests for viridans streptococcal identification and conse- quently are not all contained in most commercially available identification systems. Organisms belonging to the "S. milleri" group may be found as nor- mal flora of the oral cavity and upper respiratory and intestinal tracts. Under the right circumstances they can, however, participate in serious purulent infections.

has a taxonomic history that is en- meshed with that of Streptococcus mitis, to the extent that this original type, strain of S. mitis was really a member of the S. sanguis group. It now appears that these streptococci, which may produce Lancefield's group H or W anti- gen, are divisible into at least four groups with species status. These include S. sanguis, Streptococcus gor- donii (2), Streptococcus parasanguis (8), and Streptococcus crista (9), a newly named species characterized by cells with lateral tufts of fibrils, Many S. sanguis group swains produce extracellular polysaccharide when grown on sucrose-containing media.

The Streptococcus sanguis Group The alpha-hemolytic species S. san-

guis, also referred to as S. sanguis I in Facidam's nomenclature scheme (4),

Streptococcus mitis Strains of this alpha-hemolytic spe-

cies, some of which produce extracellular polysaccharide, have also been referred to as Streptococcus mitior and Streptococcus sanguis II. Although a description of a similar species, Strepto- coccus oralis, was published in the early 1980s, Coykendall feels that S. oralis strains should really be included in the species S. mitis (2). Cell wall ribitol teichoic acids, a feature of both S. mitis and nutritionally variant streptococcal strains, set both of these groups of streptococci apart from other viridans isolates. Nutritionally variant streptococci can be differentiated from S. mitis by their fastidious growth requirements and their production of pyrrolidonyl arylamidase, an enzyme absent from virtually all viri "dans strains (1).

Other Viridans Streptococci Facklam (4), in a study of viridans

TABLE 2. Nomenctatural history of the "S. railleri" group Date Nomenclature Reference 1956 S. milleri Guthof (20) 1977 S. anginosua-conatellatus, S. MG-intermedius Facldam (4) 1984 S. anginosus, S. constellatus, S. intermedius Facklam (5) 1987 S. ang/nosus Coykendall et al. (6) 1991 S. an$inosus, S" constellatus, S. intermedius Whiley and Beishton(7 )

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streptococci from clinical specimens, found a small number (less than 1% of 1200 isolates) of strains identified as Streptococcus acidominimus or Strepto- coccus uberis. Both of these species hy- drolyze hippurate, unlike other viridans strains, and were originally described as bovine isolates. Streptococcus morbillorum, accounting for approximately 4% of the isolates in Facklam's study, was found to belong to the genus Ge- mella, and is currently known as Ge- mella morbillorum.

Clinical Significance of Viridans Streptococci

Viridans slreptococci are generally associated with subacute bacterial endocarditis, and dam suggest that extraceUular polysaccharide production may be a viru- lence factor in this infection. Attempts to identify especially virulent species have been hampered by inadequate and non-uni- form identification methods and changing taxonomy. Viridans streptococci have been noted as emerging pathogens in neo- nates (10) and cancer patients (3).

In certain cases, associations between a given species and certain types of infection or other diseases have been noted. Bacteremia with S. bovis, an in- habitant of the intestine, has been associated with concurrent colonic cancer. In a review of approximately 250 previously reported cases, Klein and colleagues (11) found that approximately 40% of S. bovis patients also had evi- dence of colonic disease. At the Massa- chusetts General Hospital (12) we found a similar incidence of colonic disease in a smaller sample of S. bovis bacteremia patients. However, when we identified the isolates to the biotype level using the API Rapid Strep kit, we found a much stronger association between biotype I bacteremia and colonic disease and endocarditis (70 and 90%, respectively) than with biotype II (15% for both colonic disease and endocarditis).

"Streptococcus milleri" group isolates have been associated with purulent infections of the oral, abdominal, and thoracic cavities, the central nerv- ous system, and other body sites (13). Using the most current taxonomy for determining the species of "S. milleri" group strains, Whiley and associates (14) found that whereas S. anginosus

TABLE 3. Current classification of the"S, mUleri" group according to Whiley and Beighton (7) Enzymatic activity S. anginosus S. constellatus S. intermedius

Beta-D-fucosidase - - +

Beta-N-acetylglucosaminidase - - +

Beta-N-g alactosaminidase - - +

Sialidase - - + Beta-galactosidase - - + Beta-glucosidase + - v a

Hyaluronidase - + + av, 16 to 84% of strains positive.

and S. constellatus were isolated from infections of numerous body sites, Streptococcus intermedius was more often found in liver and brain ab- scesses. S. anginosus was the predomi- nant species isolated from genito- urinary and gastrointestinal sites. Clearly defined, universally accepted taxonomy and identification methods will allow more specific associations between species and types of infections to be made, and will enhance the study of the pathogenic strategies of these op- portunistic pathogens.

The role of viridans streptococci as contaminants in blood and cerebrospi- nat fluid has been documented. Nachamkin and Dalton (15) noted that viridans sla'ains could be isolated from lumbar puncture skin sites and that these organisms appeared to be contaminants in 81% of 43 patients with positive CSF cultures. Approximately 80% of isolates from blood cultures of 86 patients were judged to be clinically insignificant by Swenson and Rubin (16). Similar observations were made by Hamoudi and coworkers (17) in a pediatric population.

Handling Viridans Streptococci in the Clinical L a b o r a t o r y

In most cases, microbiological data alone can guide laboratory personnel in determining whether a given viridans isolate is significant enough to identify and test for susceptibility to antimicrobial agents. When there is a question as to significance of an isolate, the laboratory should seek guidance from the medical staff. Slrains isolated repeat- edly and in pure culture from normally sterile body fluids or sites should be identified. These include isolates from blood, CSF, other fluids, tissue specimens, and prosthetic devices. Occasion-

ally, species identification of strains that appear to be contaminants can also provide meaningful information for the clinician. For example, antimicrobial therapy can probably be considered ade- quate in a patient undergoing treatment for endocarditis if a solitary follow-up blood culture isolate is shown to differ from the original strain causing the infection.

Viridans strains present in mixed cultures likely to be contaminated with respiratory, gastrointestinal, or skin flora need not be identified to the species level. In some instances, however, viridans isolates may be significant even when present with other organisms (e.g., "S. milleri" present with an- aerobes or other bacteria). Viridans isolates from virtually any specimen type might be significant in immuno- compromised patients; in these cases consultation with the physician as to the extent of identification is desirable.

When isolates are significant enough to identify, they should also be tested for antimicrobial susceptibility using blood-supplemented media according to NCCLS standards. While viridans strains are usually susceptible to beta-lactams and other agents, occa- sional reports of penicillin-resistant isolates have appeared (1). A test for vancomycin susceptibility is essential to rule out vancomycin-resistant genera that closely resemble viridans streptococci.

Conventional media for viridans streptococcal identification are described by Facklam and Washington (1), who suggest using a minimum number of tests for identification to the species group level. Commercially available kit-generated identifications do not always agree with one another

Clinical Microbiology Newsletter 15:10,1993 © 1993 Elsevier Science Publishing Co., Inc. 0196-4399/93/$0.00 + O6.00 75

or with conventional method identifications, and to make matters more confus- ing, different products use different nomenclature systems. Hinnebusch and colleagues (18) found varying levels of agreement (50 to 74%) between five commercially available products and conventional identifications. The authors noted that supplemental testing was often required with these systems, which included API Rapid Strep, Bax- ter MicroScan Pos ID Panel, BBL Minitek Differential Identification Sys- tem, IDS R a p i d STR System, and Vitek GPI. A new product, the b ioMtr- ieux Rapid ID 32 Strep, was recently reported to be quite accurate at viridans identification, but additional tests were also often required (19).

Even if idenlifications generated by currently available products are less than 100% accurate, they still may be valuable for biotyping isolates from a single patient in order to assess efficacy of treatment. Although there are no methods now that are both simple and completely accurate for identifying viridans streptococci, we can expect the situation to change for the better as our knowledge of the taxonomy and pathogenic potentials of these bacteria expands.

References 1. Facklam, R.R. and J.A. Washington lI.

1991. Streptococcus and rd.at~ catalase- negative gram-lxm~five cocci, p. 238--257. In A. Balows et al. (ed.), Manual of clinical microbiology. American Society foe Microbiology, Washington, DC.

2. Coykendall, A.L 1989. Classificatioa and identification of the viridans streptococci. Clin. MicrobioL Rev. 2:315-318.

3. Awada, A. et al. 1992. Streptococcal

and enterococcal bacteremia patients with cancer. Clin. Infect. Dis. 15:33-48.

4. Facklam, R.R. 1977. Physiological differentiation of viridans streptococci. J. Clin. Microbioi. 5:184-201.

5. Facklam, R.R. 1984. The major differ- ences in the American and British Strep- tococcus taxonomy schemes with special reference to Streptococcus milleri. Eur. J. Clin. Microbiol. 3:91-93.

6. Coykendall, A. L., P. M. Wesbescher, and K. B. Gustafson. 1987. "Streptococ- cus milleri," Streptococcus constellatus, and Streptococcus intermedius are later synonyms of Streptococcus anginosus. Int. J. Syst. Bacteriol. 37:222-228.

7. Whiley, R.A. and D. Beighton. 1991. Emended descriptions and recognition of Streptococcus constellatus, Strepto- coccus intermedius, and Streptococcus anginosus as distinct species. Int. J. Syst. B acteriol. 41:1-5.

8. Whiley, R.A. et al. 1990. Streptococcus parasanguis sp. nov. FEMS Microbiol. Lett. 68:115-122.

9. Handley, P. 1991. Streptococcus crista sp. nov., a viridans streptococcus with tufted fibrils, isolated from the human oral cavity and throat. Int. J. Syst. Bacte- riol. 41:543--547.

10. Broughton, R.A., R. Krafka, and C.J. Baker. 1981. Non-group D alpha-hemolytic streptococci: new neonatal pathogens. J. Pediatr. 99:450--454.

11. Klein, R.S. et al. 1987. Lack of association of Streptococcus bovis with non- colonic gastrointestinal carcinoma. Am. J. Gaslroenterol. 82:540-543.

12. Ruoff, K.L. et al. 1989. Bacteremia with Streptococcus bovis and Strepto- coccus salivarius: clinical correlates of more accurate identification of isolates. J. Clin. Microbiol. 27:305-308.

13. Gossling, J. 1988. O e c ~ c e and patho-

genicity o t ~iac Streptococcu,~' rrUilert group. Row. Infect. Dis. 10:257--285.

14. Whiley, R.A et al. 1992. Streptococcus' intermech'u~, Slreptococc&~ constellatus, and Streptococcus angiru)sus (the Streptococcus milleri group): association with different body sites and clinical infectiol~s. J. Clin. Microbiol. 30:243-24-~

15. Nachamkin, 1. and H.P. Dalton. 1983. The clinical significance of streptococcal species isolated from cerebrospinal fluid. Am. J Clin. Pathol. 79:195--199o

16. Swenson, F.J. and S.J. RubhL 1982o Clinical significance of viridans streptococci isolated from blood cultures. J. Clin. Microbiot. 15:7Z~--727.

17. Hamoudi, A. C. 1990. Clinical rele- vance of vh-idans and nonhemolytic streptococci isolated from blood and cerebrospinal fluid in a pediatric population. AJn. J Clin. Pathol. 93:270-2721

18. Hinnebusch, C.J., D.M. Nikolai, and D.A. Bruckner. 1991. Comparison of API Rapid STREP, Baxter MicroScan Rapid Pos ID Panel, BBL Minitek Dif-. ferential Identification System, IDS RapiD STR System, and Vitek GPI to conventional biochtmfical tests for identification of viridans streptococci. Am. J. Clin. Pathol. 96:459-463.

19. Freney, J. mal. 1992. Description and evaluation of the semiautomated 4-hour Rapid ID 32 Strep method for identification of streptococci and members of related genera. J. Clin. Microbiol. 30:2657-2661

20. Guthof, O. 1956. Uber pathogene "ver- grunende Streptokokken." Streptokok- ken-Befunde bei dentogenen Abszessen und Infiltraten in Bereich der Mund- hohle. Zentralb. Bakteriol. Parasitenkd. Infektionskt. Hyg. Abt. 1 Orig. Reihe A 166:553-5f~,

Editorial

Making Your Lab Safe against Multi-Drug- Resistant Mycobacterium tuberculosis

Joan Barenfanger, M.D., D.Sc. Director of Microbiology Division Memorial Medical Center Southern Illinois University School of

Medicine Springfield, IL 62781

Directors often "inherit" a hospital laboratory and assume that their facili- ties are satisfactory when, in reality, the obligation of fulfilling mandatory safety regulations has been shuffled to a back burner. The Infection Control Committee assumes the safety officer is aware of and full'filing OSHA and state guidelines; the safety officer assumes Engineering is doing this; Engineering assumes the laboratory director is doing this, and so on. This is a serious prob-

lem, but it has now become deadly serious with the arrival of increasing num- bers of patients with tuberculosis and strains of Mycobacterium tuberculosis that are multi-drug resistant (MDR).

Increasing Rates of T u b e r c u l o s i s Although globally one out o f three

people harbors M. tuberculosis, the rates of tuberculosis in the U.S. had been declining in the previous decades (1, 2). However , since 1985 there has

76 0196-4399/93/$0.00 + 06.00 © 1993 Elsevier Science Publishing Co., Inc. Clinical Microbiology Newsletter 15:10,1993

dealing with viridans streptococci in the clinical laboratory: the continuing challenge

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