The characterization and occurrence of clinically important gram-negative anaerobic bacilli

• Main Author: Duerden, B. I.
• Published: University of Edinburgh 1979
• Subjects: 616.9
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649752

Awareness of the Bacteroidaceae as important members of the normal human flora and as pathogens has increased dramatically in recent years. Classification, however, has been confused and the identification of isolates difficult. In particular, the role of pigment production in the classification of Bacteroides spp. was debated. The aims of this investigation were (i) to study the classification of Bacteroidaceae with specific reference to pigment production by B. melaninogenicus; (ii) to examine conventional bacteriological tests for the characterization and identification of clinically important gram-negative anaerobic bacilli; and (iii) to apply these methods to the study of Bacterosdes spp. isolated from the normal human flora and from infections. In studies on pigment production, B. melaninogenicus strains produced a characteristic pigment when grown on media containing blood. The pigment was extracted by ultrasonic disintegration of washed cells of strains of B. melaninogenicus grown in blood broth and on blood agar. It was intra-cellular or cell-associated, soluble in water and had the spectrophotometric characteristics of a derivative of haemoglobin. No such pigment was extracted from strains of B.fragilis, F.necrophorum and Cl. clostridiiforme. The pigment was unrelated to the dense black colloidal precipitate of ferrous sulphide that resulted from the production of H2S by Bacteroides spp. and facultative species in the presence of ferrous ions. However, the pigment-producing strains were not a homogeneous species and were divided into three subgroups: B. melaninogenicus ss. melaninogenicus, ss. intermedius and ss. asaccharolyticus. A scheme for the identification of unknown isolates of Bacteroides spp, was devised following studies in which 165 reference strains and laboratory isolates were subjected to a series of simple laboratory tests that included conventional biochemical and fermentation tests, tests for resistance to antibiotics, and tolerance of dyes and bile salts. These tests allowed a clear separation of strains into three main groups - B.fragilis, B.melaninogenicus and Pusobacterium spp. - and certain tests were useful for identifying the subspecies of B. fragilis and B. melaninogenicus. The classification of B. melaninogenicus and related species was further studied in a series of tests with 175 strains of B. melaninogenicus, 17 strains of B. oralis and 6 strains of B. ochraceus. The pigmented asaccharolytic strains formed a distinct group and have been assigned a separate species - B. asaccharolyticus. B.melaninogenicus ss. intermedius strains formed a homogeneous group. B. ochraeceus was distinguished from other Bacteroides spp. by its ability to grow in air plus 10% C02 and its resistance to metronidazole; it is suggested that it should be removed from the genus Bacteroides. B.melaninogenicus as. melaninogenicus and B. oralis gave similar patterns of results and were often indistinguishable except for the production of pigment by B. melaninogenicus strains. The following system of classification was derived after studies with additional reference and referred strains. The Bacteroidaceae were divided into 4 main groups - B.fragilis group, B. melaninogenicus/oralis/ruminicola group, asaccharolytic group and Fusobacterium group. The B.fragilis group comprised the 5 subspecies of B.fragilisi that have been reinstated to species rank - B. fragilis, B. vulgatus, B. distasonis, B.thetaiotaomicron and B.ovatus - and several related species - B. splanchnicus, B. eggerthii, B. uniformis and B. variabilis. The B. melaninogenicus/oralis/ruminicola group contained the 2 saccharolytic subspecies of B. melaninogenicus, as. melaninogenicus and as. intermedius, a weakly fermentative subspecies - as. levii, and 4 non-pigmented specie's - species B.bivius, B.disiens and B.ruminicola. Bs oralis and B.melaninogenicus as. melaninogenicus strains share many characteristics and it is suggested that pigment production might not .be a valid criterion for their separation. The asaccharolytic group included'B.asaccharolyticus, B.corrodens and non-pigmented strains that were not further identified, and the:Fusobacterium group was represented by reference strains of F.polVmorphum, F.varium, F.necrogenes, F.necrophorum and L.buccalis. These species were identified by a combined set of 'tolerance tests with taurocholate, deoxycholate, Victoria blue 4R and ethyl violet, antibiotic disk resistance tests with neomycin 1000pg, kanamycin 1000pg, penicillin 2 units and rifampicin l5pg, pigment production and biochemical tests for indole production, gelatin digestion, aesculin hydrolysis and the fermentation of glucose, lactose, sucrose, rhamnose, trehalose, mannitol and xylose. Strains were allocated to the appropriate group by the results of the tolerance and resistance tests and to species/subspecies level by the results of biochemical and fermentation tests.The scheme was evaluated satisfactorily in studies with Bacteroides strains isolated from the normal human flora and clinical infections. Specimens of faeces, vaginal secretions and sub-gingival plaque were obtained from 20 normal healthy adults. A heavy growth of Bacteroides spp. was' obtained from all specimens of faeces and 10 colonies were selected from each subject for identification. Most (84%) isolates belonged to the B.fragilis group. The commonest species/subspecies were B.fragilis as. vulgatus and as. thetaiotaomicron (22% of B.fragilis-group isolates each 5, Ss distasonis (18%) and the B.eggerthii/variabilis group (14%). B.fragilis ss. fragilis accounted for only 9% of B.fragilis-group isolates. Bacteroides spp. were recovered from 65% of vaginal specimens. Most (78%) isolates belonged to the B. melaninogenicus/oralis/ruminicola group and the commonest species/subspecies were B. bivius/disiens (42% of the group isolates) B. melaninogenicus as. melaninogenicus (16%) and as. intermedius (22%). Only 6 B.fragilis strains were identified and 5 were from a single subject. A heavy growth of Bacteroidaceae was obtained from all specimens of sub-gingival plaque; 68% of isolates were members of the B. melaninogenicus/ oralis/ruminicola group. B.oralis (42% of the group isolates), B.melaninogenicus as. melaninogenicus (26%) and ss I'ntermedius (17%) were the commonest species. Fusobacterium spp. and L.buccalis were common isolates from sub-gingival plaque and accounted for 36 isolates,In studies of the role of Bacteroides spp. in infections, 399 significant isolates were obtained from 356 specimens from 332 patients. A variety of species were identified; the B.fragilis group accounted for 261 isolates and there were 55 isolates of B.asaccharolyticus. Many (68%) were from infections related to the gastro-intestinal tract but others were from gynaecological, soft tissue and a variety of other infections. B.fragilis ss. fragilis accounted for 51% of-all isolates and 78% of B.fragilis-group isolates, which indicates that this subspecies has particular pathogenic potential, not only in infections-derived from the gastro-intestinal tract. The Bacteroides app. were isolated in pure culture from only 26% of the infections, 73% were mixed infections with Bacteroides spp. and facultative organisms that may act synergistically.Bacteroides spp. can be identified by a simple set of conventional bacteriological tests that can be performed by any diagnostic laboratory. These studies have shown that different species are predominant in the normal flora of the mouth, faeces and vagina and that a number of species, particularly B.fragilis as. fragilis, form only a minor part of the normal flora but are the commonest pathogens.