An Approach to the Taxonomy of Gram-positive Anaerobic Cocci. 4. The Cell Wall Composition

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1 An Approach to the Taxonomy of Gram-positive Anaerobic Cocci. 4. The Cell Wall Composition Jaber S. Orwa,* Mutwakil G. Ahmed,** Timothy J. Coleman,*** Abstract: The classification of Gram-positive anaerobic cocci (GPAC) has always been very unsatisfactory. Attempts to clarify the taxonomy of GPAC have involved several methods including the analysis of the composition of bacterial cell wall. In an attempt to obtain information on the cell walls of 24 strains of GPAC isolated from clinical materials at St. Lukes, Guilford, and St. Thomas Hospital, London, the amino acid and amino sugar composition was quantitatively determined by an amino acid analyzer, using the method of Park and Hancock. The present study has revealed that the evaluation of the cell wall components is an accurate and reproducible method for classification of GPAC. The investigation showed that there are major differences in the amino acid composition of the cell walls of the members of GPAC. On balance, the separation of most GPAC using this system in general is consistent with the principles of Holdman et al (1977) and added valid characteristics for certain organisms such as differentiation of the species of Pst. micros from Pc. magnus, and recognition of the species of Pc. variablis and Pc. anaerobius as a variant species of Pc. magnus. The most unexpected finding was the designated reference strains of Pc. asaccharolytius varied significantly in their cell wall components. Therefore, additional strains must be studied in order to clarify the characterization and hence the nomenclature of such organisms. Introduction: The first report of micro-organisms arranged in packets was in However, the first isolation of the gram-positive anaerobic cocci (GPAC) was, in 1893, from a case of suppurative bartholinitis. 2 Gram-positive anaerobic cocci (GPAC) are a heterogeneous group of organisms defined by their morphological appearance and their inability to grow in the presence of oxygen. GPAC are part of the normal flora of the mouth, upper respiratory and gastrointensinal tracts, female genitourinary system and skin. 3-7 GPAC are commonly present in human clinical specimens; data from four surveys of anaerobic infections are consistent that they account for about 25 to 30% of all anaerobic isolates. They are not involved in any single specific disease process; rather they may be present in a great variety of infections involving all areas of the human body. These infections may range in severity from mild skin abscesses, which disappear spontaneously after incision and drainage, to more serious and life-threatening infections such as brain abscess, bacteremia, necrotizing pneumonia, and septic abortion. Infection by GPAC usually involves invasion of devitalized tissue by organisms that are part of the normal flora of the affected tissue or of the surrounding area GPAC have been poorly studied for several reasons; which include an inadequate classification, difficulties with laboratory identification, and the mixed nature of the infections from which they are usually isolated. GPAC have been fair game for amateur taxonomists. The classification has always been very unsatisfactory; but a few chemical tests can be helpful. Hare and coworkers put forward the original scheme to classify the GPAC into nine groups according to fermentation of carbohydrates (glucose, fructose, maltose and sucrose) and organic acids. All strains of the nine groups are Grampositive (except group V which is gram negative), cocci arranging either in clusters or in chains, with the exception of group VI and VII showing no particular arrangement under the microscope. On the basis of fermentation of carbohydrates and organic acids group I is characterized by its ability to ferment glucose, fructose and maltose. Group III and VI ferment all carbohydrates tested, the former group differs *) Department of Microbiology, El-Najah Polyclinic, Sebha Libya. **) Department of Medicine, Faculty of Medicine, Sebha University, Sebha Libya. ***) Department of Microbiology, Faculty of Medicine, University of Surrey, U.K. 44 Sebha Medical Journal, Vol. 7(2), 2008.

2 from the latter in the fermentation of pyruvate. Group IV is readily separated from other groups, since fructose is the only sugar utilized. Both group II and IX do not ferment carbohydrates, but the former is different from the latter in the fermentation of pyruvate and citrate (group II produces gas from pyruvate and citrate). The remaining groups VII and VIII are separated from the others on the basis of the fermentation of glucose and fructose only, and the only difference between these two groups is the production of gas from carbohydrates (glucose and fructose) and pyruvate by group VIII, (Table 1) However, this conventional carbohydrate have proven unsuccessful in distinguishing between the various anaerobic cocci. 15 Attempts to clarify the taxonomy and to improve the identification of GPAC have involved various methods that included the analysis of the composition of the bacterial cell wall. The most empirical staining method for differentiation of bacteria is the Gram stain. Gram reaction is correlated with a variety of important cellular characteristic, of these the structural and the specific chemical properties of the cell wall which are markedly different between Gram positive and Gram negative bacteria. The bacterial cell walls contain several classes of heteropolymers. The most ubiquitous and important among these in respect of cell wall function is the peptidoglycan. This polymer is stable, unaffected by variations in culture media or condition of growth. 12,13 It has been described as a 'bag-shaped molecule' since it forms the microfibillar mesh that has some elasticity and considerable tensile strength which confers rigidity and morphological integrity of the bacterial cell wall. 14 Bacteria with such rigid cell wall withstand osmotic up to 20 atmosphere. On the other hand peptidoglycan in some bacteria is a favourable target for attack by certain antibiotics, such as penicillin and cephalosporin, providing the basic for the action of many specific chemotherapeutic agents. 15 Gram-positive bacteria are composed mainly of peptidoglycan (30-70% of the total cell wall). In contrast Gram-negative bacteria contain little peptidoglycan (less than 10% of the total cell wall). The composition and the structure of the cell wall is consistent species specific characteristic of bacteria and there is considerable variation between Gram-positive species. 16 In general the main chemical component of the rigid structure are amino acids and amino sugars; amino sugars account for as little as 9-11% of the cell wall. 17 Goodfellow (1977) suggested that chemotaxonomic markers should be important in the classification of micro-organisms. 18 Lechevalier accepted the chemical composition of the cell wall as a criterion for classification of aerobic actinomycetes. 19 The quantitative analysis of the cell wall fractions has been widely used as one of the important keys for the classification of a great variety of Grampositive bacteria and enabled several researchers to emphasize the relationships between the patterns of cell wall amino acid composition and the taxonomic grouping Salton suggested that the complete separation of the cell wall amino acids and amino sugars would be the best way of detecting any relationships between the various species and may constitute a decisive contribution to reclassification of bacteria that were formerly classified improperly. 12 There are few reports concerning the study of cell walls of GPAC. The species of Pc. indolicus, Pc. niger, Pst. micros, Pst. parvulus, Pst. productus and Gffkya anaerobius remained untested. Therefore, it now seems worth while to focus on the correlation of the amino acid and amino sugar components of the GPAC. Materials and Methods: The sources of cultures were a total of 116 strains of GPAC isolated from clinical materials in St. Luke's Hospital, Guilford and St. Thomas's Hospital, London. All specimens were routinely examined for anaerobic bacteria. The reference strains 24 were obtained from the American Type Culture Collection (ATCC), Virginia Polytechnic Institute and State University (VPI) and the National Collection of Industrial Bacteria (NCIB), (Table 2). The specimens were streaked onto freshly prepared blood agar plates using a swab or a sterile platinum loop. The remainder of the specimen was incubated in chopped meat medium. The inoculated blood agar plates were placed in anaerobic jars. An inoculated blood agar with a culture of clostridium tetani was incubated in the anaerobic jar to act as a control for adequate anaerobiosis. 15 A modified sachet containing platinum catalyst was reactivated each time the jar was used by heating to 160 for 90 minutes. 45 Sebha Medical Journal, Vol. 7(2), 2008.

3 Glucose Fructose Maltose Sucrose Pyruvate Citrate Lactate An Approach to the Taxonomy of Gram-positive Anaerobic Jaber S. Orwa, et al. The anaerobic jars were prepared by the use of the evacuation-replacement method. 16,17 In this method the anaerobic jar was filled with freshened catalyst. A tube of freshly prepared methylene blue indicator was placed in the loaded jar. The jar was sealed and evacuated to an internal pressure of about -440 mmhg by water pump. After three series of partial evacuation the anaerobic jar was filled with a gas mixture containing 90% hydrogen and 10% carbon dioxide and left for 25 minutes. Since the catalysis creates a reduced pressure within the jar due to decrease in the amount of gases, the valve to the mixed gas supply was reopened and the jar was filled with mixed gas and incubated. The colourless appearances of the methylene blue in the indicator capsule and the presence of swarming growth in the Clostridium tetani plate, were an indication of good anaerobiosis in the jar. After 6 days' incubation, the various types of colonies were examined and marked. On the basis of primary screening using the Gram stain, the colonies of GPAC were picked, inoculated into chopped meat media (C.M.) and incubated for 24 hours. The cultures of GPAC were transferred to the microbiological laboratory in Surrey University for further study. All strains were plated in parallel on three Brain Heart Infusion Agar (BHIA); one of which was incubated aerobically, the second in candle jar, and the third in an anaerobic atmosphere. All plates in different atmospheres were incubated at 37 C for 6 days. After this period of incubation, the strains were accepted as GPAC if they had grown only anerobically. One pure colony was picked and used for subsequent inoculation of C.M. medium and for Gram stain. The inoculated C.M. medium was incubated in an anaerobic cabinet overnight, stoppered firmly, and the culture was preserved at 4 C until needed. Table 1: Fermentation characteristics of anaerobic gram-positive cocci. Group Morphology Fermentation of carbohydrate Fermentation of organic acid (Str. Putridus) Gram-positive chains, cocci small AG AG AG - G - - II Gram-positive, clusters G G - III Gram-positive, clusters, large cocci AG AG AG AG G - - IV Gram-positive, clusters G+ - - V Gram-positive, clusters G - - (Veillonellase?) VI Gram-positive chains and clusters A A A A VII Gram-positive chains and clusters A A VIII Gram-positive clusters AG AG IX Gram-positive clusters A = acid; G = Gas; + = moderate amount. The identification of the isolates was based on the method described by Holdeman et al, in the Virginia Polytechnic Institute Laboratory Manual of Anaerobes. 18 Accordingly, GPAC were identified on the basis of morphology, biochemical examination and the detection of the end products from the fermentation of carbohydrates by means of Gas Liquid Chromotography (GLC). Carbohydrate fermentation tests included glucose, galatose, rhamnose and mannose. The basal medium was peptone yeast extract containing 0.5 g for both peptone and tripticose; 1.0 g yeast extract; and 0.4 ml salt solution per 100 ml of distilled water. 18 The basal medium was supplemented, per 100 ml. with 0.5 ug of vitamin K 1 ; 0.5 mg of haemin; 0.05 g of cysteine hydrochloride as reducing agent; g of resazurin as an indicator of anaerobiosis; and 0.02 ml of Tween-80. All sugars were added to the basal medium in a final concentration of 1%. A total of 47 cultures of GPAC were tested for their fermentation of glucose, galactose, 46 Sebha Medical Journal, Vol. 7(2), 2008.

4 rhamnose and mannose in peptone yeast extract. The products of carbohydrate fermentation volatile and nonvolatile fatty acids were detected by gas liquid chromatography. Organisms: Out of a collection of 116 Gram-positive anaerobic cocci (GPAC) from a variety of clinical sources; at St. Lukes Hospital, Guilford and St. Thomas Hospital, London; twenty-four strains were selected for the bacterial cell wall study to include representative from each of identified species. The main emphasis was to select isolates most typical from a range of well characterized species. Media: Schaedler broth medium supplemented with haemin and Vit. K 1 was prepared in an anaerobic cabinet. The test organisms were grown anaerobically for 24 h at 37 C on the Blood Heart Infusion Agar (BHIA). Subsequently one colony was grown in 5 ml of Schaedler broth anaerobically, for 5 h at 37 C and 1 ml of the incubated medium was used to inoculate 250 ml of Schaedler broth. The inoculated medium was incubated in an anaerobic atmosphere for 18 h. Preparation of the cells of GPAC:- At the end of incubation the growing culture was checked for purity by Gram stain. Formalin 0.5% was added to the culture for 24 h in order to kill the bacterial cells. 12 The suspension was harvested by centrifugation of 6500 rpm at 4 C for 20 min. in a refrigerated centrifuge. The supernatant fluid was removed and the packed cells were washed three times with physiological saline and stored in small bottles at -20 C until needed. Determination of the dry weight: Ten milliliters of the washed cell wall suspension were pipetted into a weighed dry centrifuge tube and centrifuged to deposit the cell. The supernatant was poured off and the cell pellet was washed with cold water and recentrifuged. The centrifuge tube containing the pellet was dried in a vacuum oven at 80 C for 6 h and stored in a desiccator, the weight being determined on two occasions. The weight was recorded if the weightings were identified. Dry weight was expressed in milligrams per milliliter. Preparation of the cell walls: In order to remove all low molecular weight compounds such as the cytoplasmic pool of amino acids, nucleic acids, and techoic acid from the cell walls, a volume containing 10 mg of the cell wall suspension was centrifuged and the sediment was suspended in 20 ml of 10% trichloroacetic acid, heated in a water bath for 20 min, and centrifuged. The sediment was washed with deionized water and recentrifuged. In order to digest the cellular protein the sediment was suspended in 5 ml trypsinphosphate buffer, and incubated at 37 C in a shaker for 4 h and centrifuged. The sediment was washed twice with distilled water. The prepared cell walls (peptidoglycan) were kept at -20 C until needed. Hydrolysis of the prepared cell walls: A sample of 5 mg of the cell wall was introduced into a dry heavy walled pyrex test tube and 1 ml of 6 N-HcL was added. The test tube was constricted with a small, sharp flame. After cooling the contents were cooled with ice, the test tube was evacuated with an oil pump and the tube was finally sealed with a sharp flame under vacuum. The sealed test tube containing the mixture to be hydrolyzed was kept at 105 C in an oven for 18 h. After the hydrolysis was completed, the test tube was allowed to cool and opened with a small, sharp flame directed just below the sealed tip. The upper part of the test tube was cut off and the contents were transferred into a small flask. The hydrolysate was mixed with deionized water and evaporated to dryness three times in order to eliminate hydrochloric acid. The residue was dissolved in 4 ml citrate buffer of ph 2.2 and stored at -20 C. Quantitative amino acid and amino-sugar analysis: An equivalent of 1.25 mg (1 ml) of Hclhydrolyzed cell wall preparation was analyzed in an amino acid analyzer. Results: Table 2-4 show summary of the amino acid and amino sugar composition in the cell walls of GPAC. 47 Sebha Medical Journal, Vol. 7(2), 2008.

5 Discussion: In an attempt to obtain information on the cell walls of GPAC, the amino acid and amino sugar composition was quantitatively determined by an amino acid analyzer. The results of the analysis of 24 species are summarized in Table 2, where the molar ratios are compared with glutamic acid. The results reported for Pc. prevotii, Pc. indolicus, Pc. magnus, Str. constellatus and Str. morbillorum were duplicated in two batches of Schaedler broth. The two patches gave quantitatively similar results in composition indicating good reporducibilities of the molar ratios of amino acids and amino sugars. This point is further underlined by close agreement between the present results and those of Schliefer and Nimermann. 16 The finding in the case of Pc. asaccharolyticus (ATCC 14963); Pc. variabilis (ATCC 14955) and Pc. saccharolyticus (ATCC 14953) for example were identical with those of Schleifer and Nimermann 16 with the same strains; and there is the same of correspondence in the case of Pc. prevotii (ATCC 14952) although in this instance a different strain (Pc. prevotii 9321) was used. However, it should be noted that Bahon and co-workers (1966) 24 reported that the cell walls of Str. intermedius contained glutamic acid, alanine, and lysine in the molar ratio 1:4:1.1, whilst Latham et al (1979) 25 found that cultures of this species contain the same acids in the molar ration 1:3.5:1; in addition the presence of serine in a molar ratio of 0.5 per glutamic acid was variable. The cell walls of Str. constellatus and Str. morbillorum have been claimed to have the same molar ratio as that of Str. intermedius. The present data showed that all the streptococci except Str. constellatus (ATCC 27573) contained glycine and serine in the cell wall in addition to glutamic acid, alanine and lysine. However, Str. morbillorum differs from other streptococci in that the cell walls contain aspartic acid and threonine. All of the preparations of the cell walls of GPAC contain alanine, glutamic acid, muramic acid and glycosamine. This suggests a common primary structure in the cell walls of GPAC. However, Pc. glycinophilus, Pc. saccharolyticus, Megasphaera elsdenii, and species of streptococcus possess a substantial amount of alanine in sharp contrast to the proportions in the cell walls of the other GPAC. The ratios between hexoamines (muramic acid and glucosamine) varied; probably because muramic acid was labile in the process of Hcl hydrolysis. Another possible explanation of the lower ratio could be the colour contrast of ninhydrin used for calculation. 21 Apart from glutamic acid and alanine, the amino acids next in abundance in the walls of GPAC are glycine, lysine, aspartic acid, serine, ornithine, diaminopimelic acid and threonine. The cell walls of the majority of strains contain lysine as a major component. Only the cell walls of Pc. asaccharolyticus (ATCC 14963), Pc. aerogenes (NCIB 10064), Pc. niger, Pc. indolicus, Pst. micros, Pst. productus, and M. elsdenii which contain no lysine are different. Aspartic acid, serine, threonine and ornithine occurred less frequently in the cell wall preparations of GPAC. Aspartic acid was detected in significant amounts with a range of molar ratio to glutamic acid in 14 out of 24 GPAC strains whereas serine was found in nine strains. The following two strains of GPAC contain diaminopimelic acid (DAP) as a mojar constituent; Pst. productus (ATCC 27340) and Megasphaera elsdenii. Among the tested strains, the cell walls of Pc. asaccharolyticus (NCIB 10064); Pc. aerogenes (ATCC 27340); Pc. indolicus; Pst. micros, and Vellonella parvulus contained ornithine as a major constituent, whereas threonine was detected in the species of Pc. asaccharolyticus (ATCC 29743) and Str. morbillorum. The hydrolysate of the cell wall preparation of the species of Pc. prevotii and Gaffkya anaerobia have an identical composition of glutamic acid/ alanine/ glycine/ lysine/ muramic acid/ glucosamine in molar ratio (1: 0.5: 0.7: 0.9: 0.4: 0.3). In most respects the composition of the species of Pc. saccharolyticus was similar to that Pc. glycinophilus, Str. constellatus (ATCC 27823) and Str. intermedius. The molar ratios for amino acid wall composition of the species of Pst. anaerobius resemble those of Pst. parvulus and Sarcina ventriculi, but the strains of Pst. anaerobius contain high molar ratio of aspartic acid and lower lysine than that of the strains of Pst. parvulus, whilst the strain Sarcina ventriculi contains lower molar ratio of alanine and aspartic acid and higher ratio of glycine than that of Pst. anaerobius. It should be emphasized that these results refer only to the major amino acid components and do not exclude the possibility that the cell walls of some GPAC possess components 48 Sebha Medical Journal, Vol. 7(2), 2008.

6 containing additional amino acids in minor amounts. Sometimes certain amino acids identified pose a problem of interpretation since it is difficult to decide whether such amino acids are part of a cell wall structure or whether they are derived from cytoplasmic remnants or represent the remains of surface protein layers. However, several workers emphasized that aromatic amino acids (phenylalanine, tyrosine, and tryptophan), sulphur containing amino acid (cysteine, methionine), branched amino acids (leucine, isoleucine, and valine), histidine, arginine and proline are non-wall amino acids In conclusion, the present study has shown that the evaluation of the cell wall components is an accurate, reproducible method for classification of GPAC. The study revealed that there are major differences in the amino acid composition of the cell walls of the members of GPAC. Although, the detailed structure of the peptidoglycan was not studied, the molar ratio of the amino acid composition of this structure varied significantly according to species. These differences reinforce and amplify the usefulness of the amino acid utilization for the classification of these organisms. On balance, the separation of most GPAC using this system generally agrees with the principle of Holdmann et al (1977), and added valid characteristics for certain organisms such as the differentiation of the species Pst. micros from Pc. magnus, and recognition of the species of Pc. variabilis and Pc. anaerobius as a variant species of Pc. magnus. 26 The most unexpected finding was that designated reference strains of Pc. asaccharolyticus varied significantly in the pattern of cell wall composition. Therefore, additional strains must be studied in order to clarify the characterization and hence the nomenclature of such organisms. Acknowledgement: This work was financed by the Ministry of Health and Agriculture, Libya. The authors are grateful to Dr. B. J. Lloyd and M. O. Moss who provided useful discussions and helpful ideas. We are greatly indebted to the technical assistance of staff of the Department of Microbiology at the University of Surrey for their expertise and efficiency on the experimental apparatus. The authors express thanks and appreciation to the members of the Department of Microbiology in St. Lukes Hospital, Guilford and St. Thomas's Hospital, London. Technical assistance of Mr. Hussein El Jamal at African Computer Center, Sebha, and Mohamed Elfar, Enajah Polytechnic, Sebha, Libya, is highly appreciated. 49 Sebha Medical Journal, Vol. 7(2), 2008.

7 Table 2: Summary of the amino acid and amino sugar composition in the cell walls of GPAC. Molar ratios (glutamic acid = 1) Species Glu Glu Ala Gly lys DAP Ser Asp Thr Orn Mur NH 2 Pc. asaccharolyticus* ATCC Pc. asaccharolyticus ATCC Pc. aerogenes NCIB 10064* Pc. niger* ATCC Pc. prevotii ATCC Pc. indolicus* ATCC Pc. magnus * ATCC Pc. anaerobius ATCC T 0.4 Pc. varabilis ATCC Pc. glycinophilus * ATCC Pc. saccharolyticus ATCC Pst. anaerobius * ATCC Pst. micros * ATCC Pst. parvulus* VPI Pst. productus* ATCC G. anaerobius * VPI Sarcina ventriculi VPI Str. constellatus ATCC Str. constellatus EATCC Str. intermedius ATCC Str. morbillorum ATCC Str. morbillorum ATCC M. elsdenii ATCC V. parvulus* ATCC *) The species marked with an asterisk have not had their cell walls investigated previously. T = Trace amounts. GPAC = gram positive anaerobic cocci. 50 Sebha Medical Journal, Vol. 7(2), 2008.

8 Tested strains An Approach to the Taxonomy of Gram-positive Anaerobic Jaber S. Orwa, et al. Table 3: The production of volatile fatty acids from glucose, galactose, rhamnose and mannose by 47 species of GPAC Speicies Glucose Galactose Rhamnose Mannose A Pc ib B iv v ic c A Pc ib B iv v ic c A Pc ib B iv v ic c A Pc ib B iv v ic c G. anaerobia M. elsdenii Pc. asaccharolyticus Pc. magnus Pc. prevotii Pc. saccharolyticus Pst. anaerobius Pst. micros Pst. productus Str. intermedius Symbols: A = acitic acid; Pc = propionic acid; ib = isobutyric acid; B = butyric acid; iv = isovaleric acid; v = valeric acid; ic = isocaproic acid; c = caproic acid; I = number of srtrains positive. GPAC = gram positive anaerobic cocci. 51 Sebha Medical Journal, Vol. 7(2), 2008.

9 Tested strains An Approach to the Taxonomy of Gram-positive Anaerobic Jaber S. Orwa, et al. Table 4: The production of nonvolatile fatty acids from glucose, galactose, rhamnose and mannose by 47 species of GPAC Speicies Glucose Galactose Rhamnose Mannose Py La S Py La S Py La S Py La S G. anaerobia 2 0/2 2/2 2/2 0/2 1/2 1/2 0/2 1/2 1/2 0/2 1/2 1/2 M. elsdenii 1 0/1 1/1 1/1 0/1 1/1 1/1 0/1 1/1 1/1 1/1 1/1 1/1 Pc. asaccharolyticus 4 0/4 3/4 3/4 0/4 2/4 3/4 0/4 4/4 4/4 0/4 4/4 4/4 Pc. magnus 8 0/8 8/8 7/8 0/8 7/8 4/8 0/8 8/8 8/8 0/5 5/8 8/5 Pc. prevotii 6 1/6 6/6 6/6 0/6 6/6 6/6 0/6 6/6 6/6 0/6 6/6 6/6 Pc. saccharolyticus 2 0/2 2/2 1/2 0/2 2/2 2/2 0/2 2/2 1/2 0/2 2/2 2/2 Pst. anaerobius 10 2/10 10/10 8/10 0/10 9/10 9/10 0/10 10/10 10/10 0/10 10/10 9/10 Pst. micros 4 0/4 4/4 4/4 0/4 4/4 4/4 0/4 0/4 4/4 0/4 4/4 4/4 Pst. productus 5 0/5 5/5 3/5 0/5 5/5 5/5 0/5 5/5 5/5 0/5 4/5 4/5 Str. intermedius 5 0/5 5/5 4/5 0/5 5/5 4/5 0/5 5/5 5/5 0/5 5/5 2/5 Symbols: Py = pyruvic acid; La = lactic acid; S = succinic acid. 1 = number of strains positive/number of strains tested. GPAC = gram positive anaerobic cocci. 52 Sebha Medical Journal, Vol. 7(2), 2008.

10 References: 1. Goodsir J. History of a case in which a fluid periodically ejected from the stomach contained vegetable organisms of an undescribed form. With a chemical analysis of the fluid by George Wilson. Edinburgh Med. Sur. J. 1842; 57: Veilton M.A. On the anaerobic micrococcus found in putrid suppuration. C. Soc. Biol. 1893; 45: Bartlett J.G. Anaerobic cocci. In: Mandell G.L., Douglos R.G., Bennett J.E., editors. The principles and practice of infectious diseases. 3 rd ed. New Yoork, N.Y., Churchill Livingstone, Inc., 1990; Ezak T., Oyaizu H., Yabuuchi E. The anaerobic gram-positive cocci. In: Bolows A., Truper H., Drartin M., Harder W., Schleifer K., editors. The prokaryotes. 2 nd ed. Berlin, Germany, Speinger-Verlag, KG. 1992; Hillier S., Krohn L., Robe S., et al. The normal vaginal flora. Clin. Infect. Dis. 1993; 16: Neut C., Lesieur V., Romond C., et al. Analysis of gram-positive anaerobic cocci in oral, fecal and vaginal flora. Eur. J. Clin. Microbiol. Infect. Dis. 1985; 4: Summanen P., Baron E., Citon D., et al. Wodsworth anaerobic bacteriology manual. 5 th ed. Belmont, Calif. Star publishing Co., Brook I. Recovery of anaerobic bacteria from clinical specimens in 12 years at two military hospitals. J. Clin. Microbiol. 1988; 26: Holland J., Hill E., and Altemeier W. Numbers and types of anaerobic bacteria isolated from clinical specimens since J. Clin. Microbiol. 1977; 5: Murdoch D., Tabaqcholi S., and Mitchelmore I. The clinical importance of gram-positive anaerobic cocci isolated at St. Bartholomew's Hospital, London, in J. Med. Microbiol. 1994; 41: Wien M., Baldwin A., Eldon C., et al. The anaerobic culture of clinical specimens: a 14-month study. J. Med. Microbiol. 1977; 10: Cummins C.S., and Harrio H. The chemical composition of the cell wall in some Gram positive bacteria and its possible value as a taxonomic character. J. Gen. Microbial. 1956; 14: Achockman G.D., Klob J.J., and Toennies G. Relations between bacterial cell wall synthesis. J. Biol. Chem. 1958; 230: Mitchell P., and Moyle J. Osmotic functions and structure in bacteria. Symp. Soc. Gen. Microbial. 1956; 6: Park J.T., and Strominger J.L. Mode of action of penicillin. Biochemical basis for the mechanism of action of penicillin and for its selective toxicity. Science. 1957; 125: Schleifer K.H., and Nimmermann E. Peptidoglycan types of strains of the genus peptococcus. Arch for Microbiol. 1973; 39: Salton M.R. Mocrobial cell wall. Ciba lectures in microbial biochemistry. New York. John Willey Goodfellow M. Characterization of Mycobacterium, Nocordia, Corynebacteriun and related taxa. Ann. Soc. Belg. Med. Trop. 1973; 53: Lechevalier M.P., and Lechevalier H. Chemical composition as a criterion in the classification of anaerobic actinomycetes. Int. J. Syst. Bacterial. 1970; 20: Cummins C.S., and Harris H. Studies on the cell wall composition and taxonomy of Actinomycetes and related groups. J. Gen. Microbial. 1958; 18: Salton M.R. The bacterial cell wall. London Elsevier Publishing Company Park J.T., and Hancock C. A fractionation procedure for studies of the synthesis of cell wall mucopeptide and other polymers in cell of Staphylococcus aureus. J. Gen. Micriobial. 1960; 22: Schleifer K.H., and Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacterial. Rev. 1972; 36: Bahan A.N., Kung P.C., and Hayashi J.A. Chemical composition and serological analysis of the cell wall of Peptostreptococcus. J. Bacterial. 1966; 91: Lathan M.J., and Eliasabeth S.M. Anaerobic cocci from the bovine alimentary tract, the amino acids of their cell wall peptidoglycan and those of various species of anaerobic streptococcus. J. Appl. Bacterial. 1979; 47: Holdeman L.V. Cato E.P. and Moore W.E. Anaerobe laboratory manual, 4 th ed. Virginia Polytechnic Institute and State University, Blacksburg Sebha Medical Journal, Vol. 7(2), 2008.

11 54 Sebha Medical Journal, Vol. 7(2), 2008.