Oligopeptidase Activity of Gram-Positive Anaerobic Cocci Used for Rapid Identification

J. Gen. Appl. Microbiol., 31, 255-265 (1985)

OLIGOPEPTIDASE ACTIVITY OF GRAM-POSITIVE ANAEROBIC COCCI USED FOR RAPID IDENTIFICATION

TAKAYUKI EZAKI AND EIKO YABUUCHI

Department of Microbiology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan

(Received June 3, 1985)

The amidase and oligopeptidase activities of peptostreptococci, peptococci and anaerobic strains of streptococci were studied. Most of the asaccharolytic species of the Genus Peptostreptococcus, which include Pepto- streptococcus micros, Peptostreptococcus magnus, Peptostreptococcus asaccharolyticus, Peptostreptococcus indolicus, and Peptostreptococcus prevotii, had very strong peptidase activities. Peptostreptococcus productus and Peptostreptococcus anaerobius, which are strongly or weakly saccharolytic species, had very weak peptidase activities. Most of the anaerobic strains of streptococci were strongly saccharolytic but their peptidase activities were divided into two groups: a high activity group and a low activity group. The former group includes Streptococcus intermedius, Streptococcus pleomorphus, Streptococcus constellatus, and Streptococcus morbillorum. The latter group includes Streptococcus hansenii and Streptococcus parvulus. Anaerobic cocci were grouped into 7 phenons according to the analysis of their lower fatty acids as metabolic end products, and the indole and nitrate reduction tests. Several amidase and oligopeptidase tests, which are useful for differentiation at the species level in each phenon, were selected to prepare a new scheme for a rapid identification system of anaerobic cocci.

Most members of the genus Peptostreptococcus attack carbohydrates hardly at all but they decompose proteins and oligopeptides and use the products as their major energy source. Some of these traits of peptostreptococci have already been described by an early worker (1). However, the description was limited to amino acid utilization of several Peptostreptococcus and Peptococcus species and the taxonomy of the gram-positive anaerobic cocci has recently been greatly changed (2-4) according to the guanine plus cytosine contents and the homology value of the deoxyribonucleic acids in the organisms. The distribution of the 255 256 EZAKI and YABUUCHI VOL. 31 peptidase activities among gram-positive anaerobic cocci, including new members of the genus Peptostreptococcus, should therefore be reexamined. Biochemical tests for differentiating members of the genus Peptostreptococcus have been fairly limited. The distribution of amidase and oligopeptidase activities among the gram-positive anaerobic cocci seems to be characteristic, although these enzyme activities have not yet been used in the taxonomy of these organisms. We performed 53 peptidase tests on 105 strains in order to obtain information useful in characterizing and differentiating members of the genus Peptostreptococcus and other species of gram-positive anaerobic cocci. Recently, chromogenic substrates have been introduced for rapid identification of pathogenic organisms. From this, some information on the amidase of anaerobic cocci has become available but the information is limited to only a few species. We tested not only 20 amino acids but also 33 oligopeptidase activities of all members of the genera Peptococcus and Peptostreptococcus, and the anaerobic strains of Streptococcus and Staphylococcus.

MATERIALS AND METHODS

Bacterial strains. The nomenclatural type strains for the 8 Peptostreptococcus

Table 1. Type strains and number of clinical strains used in this study. 1985 Oligopeptidase Activity of Cocci 257

Table 2. Grouping of anaerobic gram-positive cocci by phenons.

species, 2 Peptococcus species, l Staphylococcus species, and 6 anaerobic or microaerophilic Streptococcus species were used together with 88 clinical isolated previously identified as members of these 4 genera (Table 1). The method of species level identification of these anaerobic cocci has been described previously (4). All the 17 species were divided into 7 phenons (1-3, 4a, 4b, 5a, 5b) according to the analysis of metabolic end product and the indole and nitrate tests (Table 2). Peptidase activities. All 105 strains were anaerobically cultured in Gifu anaerobic medium (GAM) broth (Nissui, Japan) for 18 hr at 37°C. Cells were harvested by centrifugation, washed twice with saline and suspended in distilled water at a concentration of Mcferland turbidity standard Nos. 4 to 5. One drop of the cell suspension was applied to dried chromogenic substrate of API (Appareils et Procedes d'Identification, Montalieu Vercieu, France), peptidase galleries No. 1 to No. 6 and API ZYM. After 4 hr incubation at 37°C, the peptidase activities of anaerobic cocci were determined by colorimetric procedures according to the manufacture's instructions. 258 EZAKI and YABUUCHI VOL. 31

RESULTS AND DISCUSSION

Our attempt to differentiate each species of anaerobic cocci with the peptidase test alone was not successful. We then grouped the species into several phenons and tried to differentiate each species belonging to a phenon by peptidase tests (Table 2). The species in each phenon is characterized as follows.

Caproic acid producing cocci (phenon 1 and 2) Peptocoecus niger does not have any detectable amount of peptidase activity while Peptostreptococcus anaerobius has several peptidase activities as shown in Fig. 1. Glutamine, proline, arginine-arginine, glutamine-histidine, and serine- tyrosine, arylamidase tests effectively differentiated these two organisms. Two organisms in this group are easily differentiated by analysis of their metabolic 1985 Oligopeptidase Activity of Cocci 259

i .b. iwJ. Fig. 1. Oligopeptide-arylamidase (-AMD) activities of gram-positive anaerobic Cocci. Peptidase activities; !, 10 nmol or more substrates hydrolyzed. 0, 5-10 nmol substrates hydrolyzed : blank, substrates lower than 5 nmol hydrolysed. ( ), number of strains examined, including the type strain for the species. products. Peptococcus niger produces n-caproic acid while Peptostreptococcus anaerobius produces iso-caproic acid from anaerobic peptone-yeast-glucose medium (PYG). So peptidase test to differentiate these two organisms have little value.

Anaerobic or microaerophilic species of the genus Streptococcus (phenon 3) Some organisms in this group were once placed in the family Peptocoecaceae but can grow under aerobic environment or in incubation with 10% CO2. Most clinical strains require a strictly anaerobic condition to grow at the primary isola- tion and are thus often misidentified as peptococci or peptostreptococci. No 260 EZAKI and YABUUCHI VOL. 31

Table 3. Tests for the differentiation of butyrate and in dole positive peptostreptococci (phenon 4-a).

aerobic strains of S. parvulus or S. hansenii capable of growing in an aerobic environment have been reported. This means that the genus Streptococcus contains strictly anaerobic species. These anaerobic species of streptococci can be differentiated from strains of Peptococcus and Peptostreptococcus species by gas chromatographic analysis of the metabolic products. An organism whose major metabolic end products in PYG broth is only lactate, must be identified as a strain of Streptococcus species even if it is strictly anaerobic, since the present genera of the family Peptococcaceae do not contain such a species. Oligopeptidase profiles successfully characterized most species of organisms in this group (Fig. 1). The two anaerobic species, S. hansenii and S. parvulus failed to exhibit any peptidase activity and could not be differentiated from each other by peptidase tests.

Butyrate producing peptostreptococci (phenon 4) Biochemical tests to differentiate organisms in this group are listed in Tables 3, 4. Indole positive organisms (phenon 4-a). As shown in Table 3, unclassified homology group A organisms may be identified as Peptostreptococcus asaccharolyticus by a combination of several conventional tests alone. However, the strains of this group did not have any detectable enzyme activity, while Peptostreptococcus asaccharol yticus had very strong peptidase activities. 1985 Oligopeptidase Activity of Cocci 261

Table 4. Tests for the differentiation of butyrate positive and indole negative peptostreptococci (phenon 4-b).

Group A organisms produce abundant hydrogen gas, the strongest gas production of any anaerobic cocci. Weak acid production from glucose and positive alkaline phosphatase activity are generally useful to separate this organism from P. asaccharolyticus. The peptidase activities of this unclassified group A are clearly different from P. asaccharolyticus. The application of some peptidase tests listed in Table 2 for the identification of anaerobic cocci may be helpful to discriminate group A from P. asaccharolyticus. Peptostreptococcus indolicus, which is rarely isolated from human clinical specimens, is easily differentiated from other members of phenon 3 by conventional biochemical tests because it produces coagulase and reduces nitrate. Peptidase profiles of this organism has little value in differentiating it from P. asaccharolyticus but are useful i n differentiating it from group A. Indole negative species (phenon 4-b) (Table 4). Two clinically important species, Peptostreptococcus prevotii and Peptostreptococcus tetradius, can be differentiated either by conventional tests or by enzyme activities. Peptococcus heliotrinreducens may slip into this group because this species produces a trace amount of butyric acid. P. heliotrinreducens is also easily separated from Pepto- streptococcus tetradius and from Peptostreptococcus prevotii by either conventional 262 EZAKI and YABUUCHI VOL. 31

Table 5. Tests for differentiation of butyrate and nitrate negative peptostreptococci (phenon 5-a).

or peptidase tests in T able 4.

Butyrate negative organisms (phenon 5) Nitrate negative species (phenon 5-a) (Table 5). Among phenon 5-a species, Peptostreptococcus magnus is a medically important species because it is frequently isolated from various human clinical specimens. A DNA-DNA homology study of this organism and Peptostreptococcus micros revealed that the species are genet- ically distinct (4), but separation of P. magnus from P. micros by the proposed biochemical tests is indefinite. Alkaline phosphatase activities and cellular protein profiles of the two species have already been reported as useful traits for differentiating them (3, 5). The cellular protein profiles may be taxonomically useful but at present it is not applicable as a routine laboratory test. In our study, the alkaline phosphatase test is not always reliable for differentiating two species because, 1985 Oligopeptidase Activity of Cocci 263

Table 6. Tests for differentiation of nitrate reducing anaerobic cocci (phenon 5-b).

while the alkaline phosphatase activity in P. micros is strong, in considerable num- bers of P. magnus strains the enzyme activity is also detectable (4). The peptidase activity of the two species is commonly very strong, as shown in Fig. 1. Some enzyme activities listed in Table 5 may be applicable for the identification of these species. Anaerobic species of streptococci (S. parvulus, S. hansenii and S. pleomorphus) and so-called microaerophilic streptococci (S. intermedius, S. morbillorum and S. constellatus) may at times be misidentified as members of phenon 5-a. In such a case, they must be differentiate from P. productus because both P. productus and these streptococci are strongly saccharolytic. P. productus is easily distinguished from S, intermedius, S. pleomorphus, and S. constellatus, which have strong peptidase activities, because of its weak peptidase activity. Other streptococci, which have very low or undetectable peptidase activities, can be differentiated from P. productus by conventional biochemical method. Nitrate positive species (phenon S-b). Peptococcus saccharolyticus has been transferred to the genus Staphylococcus (6) and a new combination, Staphylococcus saccharolyticus was proposed. However, most strains of the species do not grow under aerobic conditions. So it should be included in a differentiation scheme for anaerobic cocci. Classical biochemical tests may suffice to identify this species. 2e4 EZAKI and YABUUCHI VOL. 3 ]

No peptidase activity was detected in this organism and the species is easily differentiated from the other anaerobic cocci as shown in Fig. 1 and Table 6. Peptococcus heliotrinreducens was isolated from sheep rumen and has not yet been isolated from human clinical specimens. This species is asaccharolytic and shows strong enzyme effects on a variety of amino acids and oligopeptides. The peptidase profile resembles that of P. micros and P. magnus. The pyrrolizidine reducing character of this organism (Fig. 1) also resembles P. micros and P. magnus but this species is easily differentiated from these by nitrate test. In anaerobic PYG medium, the P. heliotrinreducens strain produces a trace amount of butyrate. If butyrate is detected, the strain should be differentiated from Peptostreptococcus prevotii and Peptostreptococcus tetradius based on biochemical tests as shown in Table 4. From the above results we prepared a scheme to identify anaerobic cocci within 4 hr as indicated in Fig. 2. When an organism is incubated in a pre-reduced liquid medium, the supernatant can be used for the analysis of the metabolic end products and the sedimented cells can be used for the peptidase tests and both results can be obtained within a few hours. Recently, several rapid identification systems for anaerobic cocci are commer- cially available but the species included in their systems are limited and species level identification is not yet satisfactory. The scheme given in Fig. 2 is applicable for all members of the genus Peptococcus, Peptostreptococcus and anaerobic strains of streptococci. 1985 Oligopeptidase Activity of Cocci 265

We thank Jean-Pierre Gayral (Laboratories de Recherche API, La Balme-Les-Grottes, 38390 Montalieu-Vercieu, France) for supplying the API peptidase galleries.

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