- Volume 34, Issue 5, 1991

Single and mixed infections with 11 clinical isolates of Propionibacterium acnes and three facultative bacteria (Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae) were studied in a subcutaneous abscess model in mice. Abscesses were induced by pure cultures of six of 11 strains of P. acnes and by the three facultative bacteria. The abscesses produced by each of the six “virulent” P. acnes isolates mixed with S. aureus, E. coli or K. pneumoniae were larger than those induced by the single organisms in 16 of the 18 combinations. There was a significant increase in the numbers of the six P. acnes strains in 13 of the 18 bacterial mixtures and in the numbers of the facultative bacteria in 17 of the 18 combinations. These data illustrate the potential virulence of some P. acnes strains and their synergic capacity with facultative bacteria.

The lipopolysaccharides (LPS) of the 10 species of the genus Bacteroides (sensu stricto) were extracted by the proteinase K method and their resolution compared by several methods of polyacrylamide gel electrophoresis (PAGE). These included sodium dodecyl sulphate (SDS)-PAGE with and without urea, polyacrylamide gradient gels and Tricine [N-Tris (hydroxymethyl) methyl glycine]-SDS-PAGE. The original discontinuous system showed good resolution of LPS from B. thetaiotaomicron, B. caccae and B. ovatus and this was enhanced by urea; B. vulgatus showed a typical ladder pattern associated with repeating polysaccharide units of the O side chains. The LPS profiles of the other species, including B. fragilis, were poorly resolved; the majority of components migrated with the leading edge of the wave front. The resolution of the LPS of these species was marginally improved with gradient gels but the majority of components were separated only within the regions of high polyacrylamide concentration. The Tricine-SDS system was consistently superior to the other methods, with excellent resolution of the LPS profiles of all Bacteroides species.

In this study the ability of strains of Shigella dysenteriae serotype 1 to agglutinate mammalian erythrocytes is attributed to the polysaccharide fraction of bacterial-cell lipopolysaccharide (LPS). LPS obtained from a rough, mutant strain of S. dysenteriae serotype 1 lacking the O-antigen polysaccharide side-chain, did not agglutinate erythrocytes, clearly demonstrating a link between O-antigen polysaccharides and haemagglutinating activity (HA). Strains of S. dysenteriae serotype 1 adhered well to cultured Henle Intestinal 407 cells, whereas rough strains adhered poorly. Pre-treatment of bacteria with LPS-specific antisera inhibited both HA and binding to cultured human-intestinal cells. The contribution of the polysaccharide side-chain and its associated HA–which appear to facilitate binding to cultured cells–to bacterial attachment to colonocytes and to the pathogenesis of shigellosis in vivo needs to be confirmed in animal studies.

A restriction fragment length polymorphism (RFLP) typing method was developed for Neisseria meningitidis. A cloned EcoRI fragment from a Neisseria meningitidis Group B serotype 15P1.16 sulphonamide-resistant strain was used to probe Southern blots of total chromosomal DNA restriction fragments (enzyme Aval). A group of 75 apparently unrelated organisms gave rise to 26 different restriction fragment length patterns and two different groups of epidemiologically related strains had RFLP patterns that were distinct for each group. The technique was highly reproducible and discriminatory. The RFLP data were compared with the results of serotyping and subtyping and isoenzyme electrophoretotyping. The RFLP data were consistent with those from the alternative typing methods; clones defined by isoenzyme analysis were subdivided by this technique. The use of RFLP typing by cloned probes should be of considerable epidemiological value.

An enzyme-linked immunosorbent assay (ELISA) has been evaluated for coprodiagnosis of giardiasis with anti-trophozoite antibody to capture specific Giardia lamblia stool antigen (GLSA), which was then detected by specific antibody conjugated with horseradish peroxidase. GLSA was demonstrated in stool eluates from all the 24 confirmed cases of giardiasis. None of the stool eluates from apparently healthy subjects or from patients carrying intestinal parasites other than G. lamblia had GLSA. Of the 25 microscopynegative clinically suspected cases of giardiasis, 17 (68%) patients had GLSA in their stool eluates; these patients responded to anti-giardial therapy. The specific antigen was isolated and affinity-purified by the use of specific antibody; it had a Mr of 66 Kda, and its immunoreactivity was lost after treatment with heat or trypsin but unaltered by metaperiodate. ELISA seems to be a sensitive and specific method for copro-diagnosis of giardiasis, especially in highly suspected cases.

A collection of 223 strains of non-typable Haemophilus influenzae was assembled. Most strains were isolated from hospital in-patients in North East Scotland between 1984 and 1989. These isolates were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell polypeptides. Variability was assessed in terms of apparent molecular weight differences between the protein profiles. Isolates were grouped on the basis of Dice coefficients of similarity and assigned to clones. Of the 223 strains, 147 were unique; the remaining strains were assigned to multi-member clones of which 13 clones had two members, one clone had three members, three clones had four members, three clones had five members, two clones had six members and one clone had eight members. The longest time interval between isolation of clonally related strains virtually equalled the limits of the study. Isolates from a childrens hospital were significantly less diverse than those from patients in adult wards.

Human polymorphonuclear leucocytes (PMNL) underwent several changes in response to challenge with Neisseria gonorrhoeae, namely (1) an increase in oxygen uptake, (2) changes in membrane electrical properties, and (3) increased transport of chloride ions (C1−) across the membrane. Mean oxygen consumption and C1− uptake by PMNL were stimulated by both pilate (P+) and non-pilate (P−) gonococci, although the levels were much reduced in the presence of P+ organisms. P+ gonococci also initiated low levels of polarisation or depolarisation in contrast with P− cells, which caused hyperpolarisation followed by depolarisation in the PMNL. Most of the strains showed these patterns. High performance liquid chromatography of extracts of unstimulated PMNL and of PMNL challenged with gonococci confirmed production of hypochlorous acid (HOC1) in the leucocyte. Furthermore, addition of radiolabelled C1− to the PMNLs showed that some of the C1− taken up by the cells in response to gonococcal challenge was incorporated into the HOC1, suggesting a direct relationship between stimulation of C1− uptake and production of active chlorine compounds in the leucocyte.

A collection of 256 clinical strains and 40 reference strains of gram-positive anaerobic cocci (GPAC) was studied, to characterise the recognised species more fully and to define groups of strains which might correspond to previously undescribed species. The methods used were: gas-liquid chromatography (GLC) for the detection of volatile fatty acids (VFAs); determination of the pre-formed enzyme profile with a commercially available kit, ATB 32A; microscopic appearance; colonial morphology; and antibiotic sensitivity tests. Strains were placed in one of five VFA groups according to their GLC profile; 96% of strains were further assigned to 12 groups by their enzyme profile. There was >99% agreement between the two methods.Of 111 clinical strains in the VFA-negative group, 110 gave one of three distinct enzyme profiles corresponding to Peptostreptococcus magnus, P. micros and P. heliotrinreducens. The assignment of strains to groups based on their microscopic appearance and colonial morphology agreed well with groupings according to enzyme profile. Identification of butyrate-producing GPAC was unsatisfactory because it relied heavily on the enzyme profile; testing for indole production was of limited discriminative value. Most strains of P. asaccharolyticus and P. indolicus were very similar in enzyme profile, microscopic appearance and colonial morphology, but a sub-group of P. asaccharolyticus could be distinguished. A further indole-positive group corresponding to Hare group III was also noted. Strains of P. prevotii and P. tetradius were very similar, but easily distinguished from other butyrateproducing GPAC. However, 45% of the butyrate-producing cocci could not be assigned to recognised species; most of these were assigned to one of two new groups, the ADH group and the bGAL group, by their enzyme profile, microscopic appearance and smell. Four strains that produced a terminal VFA peak of isovaleric acid formed a new group designated ‘ivoricus’. Reliable features for the identification of P. anaerobius were GLC (all GPAC that produced isocaproic acid were identified as P. anaerobius), enzyme profile and sensitivity to SPS. Two clinical strains that produced caproic acid were identified as Hare group VIII; they were distinguished from Peptococcus niger by their enzyme profile and colonial morphology.A phenotypic classification based on GLC and enzyme profile is presented, with a method for the identification of most strains of GPAC within 48 h of primary isolation.