Oct 13, 1989 - salivarius, Todd-Hewitt, and thioglycolate media, all of which were obtained from Difco. Preparation of M. luteus suspension. M. luteus AH-47 ...
JOURNAL OF CLINICAL MICROBIOLOGY, July 1990, p. 1623-1627 0095-1137/90/071623-05$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 28, No. 7
Production of Bacteriolytic Activity in the Oral Cavity by Nutritionally Variant Streptococci RAFFAELLO POMPEI,l* EMANUELA
CAREDDA,l VINCENZO PIRAS,2 CORRADO SERRA,'
LUCIO PINTUS3 Istituto di Microbiologia,' Clinica Odontoiatrica,2 and Istituto di Igiene, Università degli Studi di Cagliari, Cagliari, Italy AND
Received 13 October 1989/Accepted 3 April 1990
Microorganisms from the oral flora were examined for the production of bacteriolytic substances. Among human viridans group streptococci, only one group of strains with thiol-dependent properties was shown to secrete enzymes with bacteriolytic activity on heat-killed cells of Micrococcus luteus on double-layer nutrient agar plates. By morphology, culture requirements, and biochemical properties, they were found to conform to descriptions of nutritionally variant streptococci (NVS). Bacteriolytic activity was shown to be a constant property of all of the human oral NVS isolated and a property of some reference strains of NVS from clinical sources. No other known species of viridans group streptococci demonstrated bacteriolytic activity. Analysis of bacteriolytic activity could be a useful tool for both the isolation and identification of this fastidious group of microorganisms.
A variety of antagonistic substances have been shown to be produced by streptococci of the saprophytic oral flora. Lactic and acetic acids, hydrogen peroxide, cyanosulfur compounds, dextranase (20, 23, 26), and a variety of bacteriocins (9, 18, 27) are produced and released in human saliva by different species of streptococci. Their presence is believed to play an important role in the maintenance of the state of equilibrium in oral and plaque ecologies (42). Recently, the production of bacteriolytic enzymes has been described in some microorganisms. These microorganisms, such as staphylococci, enterococci, lactobacilli, and gram-negative bacteria (17, 28, 29), can occasionally be found in the human oral flora. Bacteriolytic enzymes have been recognized as lysozyme-like proteins, and they have been ascribed a possible role in the pathogenicity of the strains that produce them (33). In addition, since it has been demonstrated that the production of such bacteriolytic enzymes is a constant characteristic of some species and that the enzymes excreted can be species specific (34, 35), the possibility of using the analysis of the bacteriolytic patterns of some strains for both species separation and identification has been suggested (40). For a long time it has been known that lysozyme is present in most animal secretions and human saliva (41). Its role in oral ecology has been examined extensively, but contrasting results have been obtained by different researchers on the real importance of this protein in the regulation of microbial multiplication in the oral cavity (15, 19, 22, 31). Recently, it has been claimed that lysozyme could play a role in the mechanisms of adhesiveness of oral streptococci to hydroxyapatite (31) and that its effect is different on different microorganisms.
No extensive studies on the bacteriolytic enzymes of the oral saprophytic flora have been carried out. In this study we examined the most important species of both plaque and salivary flora, paying particular attention to streptococci, for the production of lysozyme-like substances and found that only one group of microorganisms, which shows characteristics similar to those of the nutritionally variant streptococci (NVS) but not to those of other streptococci among the *
Corresponding author.
major species that normally colonize the human oral cavity, this property.
possesses
MATERIALS AND METHODS Specimen collection. Samples were taken from the oral cavities of 20 apparently healthy young human subjects of both sexes. Sterile cotton swabs were used to obtain specimens from the occlusal surface of molar teeth, supragingival plaque, saliva, the tongue, and the cheek mucosae. The cotton swabs were dispersed in 3 ml of sterile saline, and 0.1 ml of a 1/10 dilution of the suspension was immediately cultured on Micrococcus luteus double-layer agar plates. Preparation of medium for M. luteus double-layer agar plates. Medium for the M. luteus double-layer agar plates was made with 15 ml of a base layer of Columbia blood base agar (CA; Difco Laboratories, Detroit, Mich.) and a top layer of 5 ml of CA containing heat-killed cells of M. luteus AH-47 (39) to reach a final optical density of 4 optical units. In some experiments, CA medium was substituted by mitis salivarius, Todd-Hewitt, and thioglycolate media, all of which were obtained from Difco. Preparation of M. luteus suspension. M. luteus AH-47 was grown in brain heart infusion agar (Difco) plates for 5 days at 30°C. Then, the cells were collected, washed twice with sterile saline, and sterilized by autoclaving them for 15 min at 121°C. The heat-kiled M. luteus suspension was adjusted to an optical density of 200 and was kept in a refrigerator until use. A commercial preparation of Micrococcus lysodeikticus (M-0128; Sigma Chemical Co., St. Louis, Mo.), some wild-type strains of Micrococcus roseus and Micrococcus lylae, a strain of Staphylococcus aureus (ATCC 25923), and a strain of Enterococcus faecium (ATCC 9790) were also used and prepared as described above. Culture media. Sheep blood (5%) was added to CA medium in some experiments to detect hemolysis. When needed, L-cysteine hydrochloride (200 ,ug/ml; BDH, Poole, England), pyridoxal phosphate (20 ptg/ml; Sigma), and IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.) were added to the media. Bacterial strains. Streptococcus morbillorum, Peptostreptococcus, and Peptococcus strains were kindly provided by M. G. Menozzi, Parma, Italy. Streptococcus mutans strains 1623
1624
J. CLIN. MICROBIOL.
POMPEI ET AL.
TABLE 1. Incidence of isolation of bacteriolytic microorganisms from different sites of the oral cavity % Lytic colonies isolated in the following growth conditions and media: Aerobiosis
Isolation site
Anaerobiosis
Columbia base + cysteine
Mitis salivarius + cysteine
Columbia base + cysteine
Mitis salivarius + cysteine
1.5 4.2 1.0 0.2 0.5
0.8 1.5 1.2
1.8 10.7 1.5 0.4 0.8
0.9 4.5 1.0 0.2 0.5
Supragingival plaque Molar tooth surface Saliva Tongue mucosa Cheek mucosa
0.1 0.4
a The incidence of isolation of lytic colonies was calculated as a percentage of the total microbial flora grown on M. luteus double-layer agar plates. Each value is the mean for samples from 20 subjects.
were in part from the State Serum Institute, Copenhagen, Denmark, and in part from the Institute of Microbiology of the University of Siena, Siena, Italy. The species Streptococcus mitis, Streptococcus sanguis, Streptococcus salivarius and Streptococcus intermedius were isolated and identified in our laboratory by conventional methods (10). In some cases, the API 20 Strep profile (Ayerst Italiana) was used to identify oral streptococci. NVS strains from patients with endocarditis were kindly provided by I. Van de Rijn, Winston-Salem, N.C.
FIG. 1. Culture of a molar tooth plaque sample on an M. luteus double-layer agar plate. Pinpoint colonies surrounded by a dark halo are strains of thiol-dependent streptococci that produce bacteriolytic activity on M. luteus cells.
taken from supragingival plaque, saliva, tongue, and cheek mucosae, with mean values ranging from 1.8 to as low as 0.1%. With regard to the effect of growth conditions, it was found that lytic colonies occurred two to three times more frequently when samples were incubated under anaerobic conditions than when they were incubated under aerobic conditions, and that CA medium was more suitable than mitis salivarius agar plates for detecting bacteriolytic strains (Table 1). Characterization of bacteriolytic strains from the oral cavity. Strains showing bacteriolytic activity were isolated in pure culture in order to study their morphologies, growth rates, and metabolic requirements. Table 2 gives the properties of 34 strains isolated from 20 subjects. They all appeared as a group of extremely fastidious microorganisms that grew more poorly in pure culture on medium that is conventionally used for oral streptococci. Several substrates, such as 5% sheep blood, hemin, vitamin K, and
RESULTS Growth of oral bacteria on M. luteus double-layer agar plates. When the specimens taken from the various oral sites were grown on M. luteus agar medium either aerobically or anaerobically, a variable number of microbial colonies surrounded by a clear halo of bacteriolysis was observed (Fig. 1). Bacteriolytic colonies were found in cultures of samples from all 20 subjects. The incidence ranged from less than 1 to more than 10% with respect to the other nonlytic colonies that grew on M. luteus double-layer agar plates. The incidence of bacteriolytic colonies in various areas of the oral cavity was analyzed by using different media under either aerobic or anaerobic conditions. The highest incidence was observed on the occlusal surfaces of molar teeth, where means of between 1.5 and 10.7% of the total flora that grew on the plates were found (Table 1). Far lower incidences of bacteriolytic colonies were found for samples
TABLE 2. Characterization of biochemical, cultural, and morphological properties of bacteriolytic streptococci from the oral cavity Growth on: No.No. of of strains CBA BHIA
BHIA + IsoVitaleX
THIO Aero-
Anaero-
-
-
-
+ + +
~~~~~~~BHIA + cys-
biosis
teine
+
+
+ +
+ +
+ + +
biosis C02
3 13 18
Bacteriolytic Gram-
stainingb + + +
Biochemical
activity on: characterization Bio Satel- -Bio-_____ litism Todd-Hewitt THIO type' agar + cysteine PYRA PAL a-Gal P-Gal Il-Gur Tre ________________
+ + +
+ + +
-
+ + +
-
+
+
-
+
I
-
-
+
-
II
III a Abbreviations: CBA, Columbia blood agar; BHIA, brain heart infusion agar; THIO, thioglycolate medium; PYRA, pyrrolidonylarylamidase; PAL, alkaline phosphatase; cx-Gal, ax-galactosidase; P-Gal, ,i-galactosidase; ,i-Gur, P-glucuronidase; Tre, trehalose; -, +, and + indicate negative, partially positive, and positive reactions, respectively. b Gram staining often revealed gram-variable strains when grown on either Columbia blood agar or brain heart infusion agar. c Biotypes were identified according to Bouvet et al. (6).
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cystine, which are required by many microaerophilic or anaerobic oral bacteria, were unable to stimulate growth. Growth occurred only in medium supplemented with IsoVitaleX. When we evaluated the effect of the various IsoVitaleX components on cell growth, only cysteine turned out to have a stimulatory effect equal to that of complete IsoVitaleX. Cysteine showed maximal activity at 100 to 200 ktg/ml and could be substituted by 20 ,ug of vitamin B6 (as pyridoxal phosphate) per ml. The finding of a suitable medium for growth allowed us to characterize bacteriolytic strains for both their morphologies and metabolic requirements. They appeared coccal in shape with short chains; when grown on either CA or Todd-Hewitt medium supplemented with cysteine, they were variable by Gram staining, whereas when grown on thioglycolate medium with cysteine, they were all markedly gram positive. The phenomenon of satellitism, which is a characteristic of most thiol-dependent streptococci, was present when they were cultured on CA near the colonies of Staphylococcus epidermidis. All of the strains tested were nonhemolytic on CA-cysteine with 5% sheep blood, and in all cases, growth under aerobic conditions was poor, whereas under anaerobic conditions growth was generally excellent. A good rate of growth could also be obtained in a 5% C02 atmosphere, especially after several passages in pure culture. By using the API 20 Strep system, we found that most strains did not fall into any known profile and could not be identified at the species level. They were thus defined as NVS. Biochemical tests for the identification of the biotypes of the NVS isolated were performed by the method of Bouvet et al. (6). As shown in Table 2, 3 strains were found to belong to biotype I, 13 to biotype Il, and 18 to biotype III. The bacteriolytic activities of the isolated thiol-dependent strains were present in most media in which the lytic strains were grown, such as CA, brain heart infusion agar, and Todd-Hewitt medium, all of which were supplemented with cysteine; but it was completely absent when they were cultured on thioglycolate medium supplemented with cysteine. M. luteus AH-47 was shown to be extremely susceptible
to the lytic activity of NVS, as it was to the lytic activity of lysozyme (MIC, 0.05 ,ug/ml). M. lysodeikticus (Sigma) was more resistant to lysozyme and was equally more resistant to the lytic activity of NVS. Other wild-type strains of the micrococci, such as M. lylae and M. roseus as well as a strain of Staphylococcus aureus and a strain of Enterococcusfaecium, for which MICs of lysozyme were greater than 2 ,ug/ml, were poor substrates for the lytic activity of NVS, particularly for those belonging to biotype I (data not
shown). In order to establish the frequency of production of lytic enzymes among the thiol-dependent strains present in the oral cavity, we isolated several hundred viridans group streptococci on CA medium supplemented with 5% sheep blood and cysteine. Among the more than 1,000 colonies that we examined, 104 colonies were found to be thiol dependent. Morphological analysis, culture requirements, conventional biochemical tests, and the use of the API 20 Strep profile allowed us to assigne the colonies to the NVS group. Ail of the colonies secreted enzymes with clear bacteriolytic activity on M. luteus double-layer agar plates (data not shown). Bacteriolytic activities of some non-thiol-dependent reference viridans group streptococci and some thiol-dependent streptococci isolated from humans with infections. Finally, we examined the bacteriolytic activities of thiol-dependent strains isolated from humans with infectious endocarditis
1625
BACTERIOLYTIC ACTIVITY OF NVS
TABLE 3. Bacteriolytic activities of some reference strains of thiol-dependent streptococci from pathologic origin and of reference viridans group streptococcal species
Identification
of No. strains tested
No. of strains bacterishowing
(species or group)
NVS from humans with endocarditisa S. morbillorum S. mitis S. mutans S. sanguis S. salivarlus S. intermedius Peptostreptococci Peptococci
3 5 8 il 6 4 3 7 4
3 O 0 0 0 0 o 0 0
a For strains from humans with
olytic activity
endocarditis, see reference 37.
and of some other reference streptococci normally found in the human oral cavity which are known to require neither thiols nor vitamin B6 for growth. We found that all the thiol-dependent strains from humans with endocarditis were bacteriolytic, while all the non-thiol-dependent reference strains of human oral streptococci were not (Table 3). DISCUSSION Among the saprophytic oral flora, a group of streptococci that produces enzymes with bacteriolytic activity on M. luteus has been identified. All the strains demonstrating this property were found to be thiol dependent and were defined as NVS. Conversely, ail of the 104 oral streptococcal isolates demonstrating the characteristics of the NVS secreted enzymes with bacteriolytic activity. Furthermore, when we examined the reference strains from infectious sources, all the NVS from humans with endocarditis were bacteriolytic. Thus, it seems that the excretion of enzymes with bacteriolytic activity by oral streptococci is always coupled with thiol dependency. The production of a bacteriolytic enzyme by oral streptococci has been described recently by Baba (1) and Baba et al. (2). They studied the bacteriolytic activities of streptococci in an extremely species-specific system, in which a strain of S. mutans was able to lyse living cells of S. sanguis ATCC 10558, but not those of S. sanguis ATCC 10556 or ATCC 10557. Thus, they concluded that the S. mutans strain showed a bacteriocin-like activity on S. sanguis rather than a bacteriolytic activity such as that of lysozyme; in fact, in our experiments, among 11 reference strains of S. mutans, none showed any sort of bacteriolytic activity on M. luteus (Table 3). Therefore, the production by oral streptococci of an enzyme with bacteriolytic activity on M. luteus seems to be a property that mimics that of lysozyme more than a strictly species-specific interaction. In fact, in our experience, oral NVS were able to lyse killed cells of M. luteus AH-47 highly efficiently, but they lysed killed cells of M. lysodeikticus (Sigma) less efficiently and other wild-type lysozyme-resistant strains of micrococci and some strains of staphylococci and enterococci quite poorly. NVS are a group of fastidious microorganisms whose presence in the human flora has been recognized since the early 1960s (11). Interest in the study of these bacteria has increased in the past 10 years, since they were found to be involved in some very important and severe human infections. In fact, between 5 and 10% of cases of streptococcal endocarditis are caused by NVS (25), and as for the other
1626
POMPEI ET AL.
viridans group streptococci, their main location in humans is believed to be the mouth (12). Because of their peculiar growth requirements, NVS are often overlooked in blood cultures, causing a considerable number of false-negative hemocultures in microbiological laboratories that do not use thiol-enriched media (24). The diagnostic problem in the recognition of NVS in the blood of patients with endocarditis is of considerable importance because of the higher resistance of these streptococci than of other viridans group streptococci to penicillin (13, 30). This fact has led most investigators to stress the need for the use of thiol-enriched medium in microbiological laboratories when culturing blood samples from patients suffering from endocarditis (16, 24). In addition, it has been claimed that fastidious microorganisms resembling NVS are involved not only in endocarditis but also in other infections, both in humans and in animals. Conjunctivitis (3), periorbital abscesses (21), and corneal ulcers in horses (7) have been found to be caused by a variety of NVS isolates. The actual taxonomic position of NVS is still uncertain. Strains of NVS from patients with endocarditis have been assigned to different species by various investigators (5). Cooksey et al. (8) and Van de Rijn and George (37) stated that different methods of identification led to a variable classification of NVS strains. Recently, in a series of papers (36-38), Van de Rijn and co-workers have tried to resolve the problem of the taxonomy of NVS. Even though they found some similarities between NVS and S. mitis, such as the production of a chromophore by both groups in a chemically defined medium, immunochemical studies allowed them to separate NVS into three serotypes, whereas no similar serotypes could be demonstrated either in the S. mitis species or in other oral streptococcal species (36-38). Finally, in an analysis of biochemical properties and penicillin-binding proteins, Bouvet et al. (6) have shown that NVS are actually substantially different from S. mitis and S. sanguis and also from other viridans group streptococci. On the basis of these findings, they proposed that NVS could be included in at least two new species of streptococci (4). The production of bacteriolytic activity by oral NVS is an additional original property that supports the findings of Van de Rijn and colleagues (36-38) and Bouvet et al. (4) that these strains could represent a group with characteristics different from those of all other viridans group streptococci. As mentioned above, lysozyme has been shown to produce a variety of effects on oral bacteria, such as a decrease in glucose incorporation (32), an interaction with the adherence mechanisms of streptococci to the tooth surface (31), and the alteration of bacterial aggregation (15); in addition, lysozyme-like enzymes of staphylococci have been demonstrated to play an important role in the pathogenicity of these microorganisms and in their resistance to phagocytosis by polymorphonuclear leukocytes (33). Considering that NVS were demonstrated in this study to produce constantly an enzyme with lysozyme-like activity and that they were isolated mostly from the occlusal surface of molar teeth, which is also the area where cariogenic S. mutans strains are more often isolated (14), a possible interaction between NVS and S. mutans, with the involvement of the lytic enzyme(s), is under investigation. ACKNOWLEDGMENTS We are indebted to I. Van de Rijn, M. G. Menozzi- and G. Rossolini for supplying some reference streptococcal strains. We thank G. Foddis for excellent technical assistance. We are particularly grateful to G. Satta for helpful suggestions and discussion.
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