- Volume 40, Issue 6, 1994

Adenovirus isolates from 52 patients with ocular infection over a 3-year period were typed by restriction endonuclease analysis in a clinical laboratory. The results indicated that adenovirus type 8 was the most common cause of adenovirus eye infection during this period, being responsible for 42 (81 %) of the 52 cases. Of 42 adenovirus type 8 isolates, 22 showed variant patterns by restriction endonuclease analysis and required multiple enzyme digests for identification. These isolates were readily identified by neutralisation tests

The nucleolar accumulation of Semliki Forest Virus (SFV) C protein was examined as a function of intact microtubules, intact microfilaments and accessible intermediate filaments. The cytoskeletal components do not seem to play a role in directing C protein to the nucleolus but nucleolar accumulation is energy-dependent and saturable. This suggests the involvement of some receptor- (or chaperon-) interaction.

The role of neuraminidase in haemagglutination and adherence to colon WiDr cells by eight strains of Bacteroides fragilis and four strains of oral black-pigmented gram-negative anaerobes was studied. Neuraminidase treatment resulted in a very small increase of haemagglutination by some of the strains but had no effect on adherence to WiDr cells by all bacterial strains tested except one strain of Prevotella intermedia (HG 110). Inhibition of neuraminidase had no effect on haemagglutination or adherence, nor was any correlation found between haemagglutinating ability and neuraminidase activity in the B. fragilis strain. The results indicated that haemagglutination and adherence of B. fragilis to WiDr cells were not mediated by neuraminidase.

Subcutaneous injection of fimbriae from Porphyromonas gingivalis strain 381 in Freund’s incomplete adjuvant (FIA) resulted in an excellent serum anti-fimbrial immuno-globulin G (IgG) response in guinea-pigs and BALB/c mice. Administration of P. gingivalis fimbriae also elicited distinct cellular immune responses to the fimbriae in terms of ear lobe reaction in BALB/c but not in BALB/c nu/nu mice, and of skin reaction in guinea-pigs. When the guinea-pigs were given a semi-synthetic adjuvant GM-53—sodium β-N-acetylglycosaminyl-(1→4)-N-acety lmuramyl-L-alanyl-D-isoglutaminyl-(L)-stearoyl-(D)-meso-2, 6-diaminopimelic acid-(D)-amide-D-alanine—and fimbriae in FIA by subcutaneous injection, more enhanced production of serum anti-fimbrial IgG and stronger cellular immune responses were induced in the guinea-pigs than in those given fimbriae alone. Synthetic peptide FP381(202-221), which corresponds to the amino-acid residue numbers 202-221 based on the amino-acid sequence of fimbrilin from P. gingivalis strain 381, elicited humoral and cellular immune responses in guinea-pigs immunised with the fimbriae or FP381(202-221). Furthermore, subcutaneous administration of synthetic peptide FP381(61–80) with GM-53 induced lesser degrees of humoral and cellular immune responses in guinea-pigs than did FP381(202-221). However, when the fimbriae or FP381(61–80) were administered with bovine serum albumin (BSA), markedly elevated levels of specific anti-BSA antibody were seen in the serum of BALB/c mice. These results clearly indicated that fimbriae from P. gingivalis 381 and their oligopeptide segments induced humoral and cellular immune responses and exhibited immuno-adjuvant activities in guinea-pigs and BALB/c mice.

Three consecutive isolates of Enterobacter aerogenes were obtained from the blood cultures of a hospitalised patient who was receiving antibiotic therapy. The initial isolate possessed an inducible cephalosporinase and was susceptible to third-generation cephalo-sporins. After ceftazidime treatment, a second isolate resistant to this antibiotic and characterised by stable overproduction of the chromosomal β-lactamase was obtained, and therapy was altered to a new combination which included imipenem. During this course of treatment, a strain of E. aerogenes was isolated that was resistant to virtually all β-lactam agents including imipenem. Comparison of biotypes and ribotyping profiles indicated that the three isolates were probably derived from a single strain which had undergone several mutations during antibiotic exposure. Examination of outer-membrane protein (OMP) preparations and lipopolysaccharide (LPS) profiles showed that the imipenem-resistant isolate lacked a major OMP and high molecular mass LPS. Furthermore, this isolate displayed reduced permeability to cephaloridine compared with the initial isolate. The introduction of a plasmid carrying a wild-type ampD allele prevented cephalosporinase production and restored β-lactam susceptibility in the imipenem-resistant isolate. It was concluded that stable derepression of class-I β-lactamase production and reduced permeability are both required for expression of imipenem resistance in E. aerogenes, and that previous exposure to cephalosporins may encourage the emergence of such strains.

Delayed post-operative endophthalmitis is a complication of modern cataract extraction and posterior chamber lens implantation. Propionibacterium acnes has been isolated in a few such cases but the majority are culture-negative, compounding surgical and medical management decisions. A method of detecting bacterial, and specifically P. acnes, DNA by the polymerase chain reaction (PCR) directed at 16S rDNA is reported. Nested PCR with universal eubacterial primers complimentary to regions of 16S rDNA conserved sequences detected 50 fg of bacterial DNA spike in normal vitreous. Nested PCR with P. acnes primers detected 10 fg of DNA. Vitreous samples from 29 patients undergoing vitrectomy for reasons unrelated to infection and 23 samples from 19 patients with delayed post-operative endophthalmitis were analysed. Four (14%) of 29 normal individuals and 17 (74 %) of 23 delayed cases gave positive results with universal eubacterial primers. None of 29 and eight of 23 samples gave positive results with P. acnes primers. The 14 % positive rate with universal primers in non-infected cases may limit their use in delayed post-operative endophthalmitis. PCR detection of bacterial DNA with specific primers from vitreous samples may prove a useful means of diagnosing delayed post-operative endophthalmitis and facilitating management decisions when conventional bacterial culture is negative.

Variant colony formation by Candida albicans has been described and the phenomenon of phenotypic switching has been studied extensively. Whereas the micro-structure of non-variant colonies has been investigated by scanning electronmicroscopy (SEM), the relationship between switched variant colonies and microstructure has not been described. The object of this study was to investigate and compare by SEM the microstructure of the normal colony type and five common variant colony types of C. albicans and to determine whether a pattern of dimorphic growth could account for the characteristic colony morphologies. A general relationship between colony type and structure was observed ; smooth colonies consisted entirely of blastospores whereas regular, irregular-wrinkled and semi-rough colonies consisted of different proportions of true hyphae and blastospores. Regular extreme-jagged shaped colonies consisted of an almost pure culture of pseudo-hyphae, and colonies producing aerial hyphae were composed of pseudo- and true hyphae, as well as blastospores. These results show a clear relationship between colony morphology and development of particular cell types.

Enrichment culture (EC) in modified buffered peptone water followed by immunomagnetic separation (IMS) with magnetic beads coated with an antibody against Escherichia coli 0157 (Dynabeads anti-is. coli O157; Dynal, Oslo) was compared with direct culture on cefixime rhamnose sorbitol MacConkey agar (CR-SMAC) and cefixime tellurite sorbitol MacConkey agar (CT-SMAC) for the isolation of E. coli O157 from bovine faeces. When used to examine bovine faecal suspensions inoculated with 12 different strains of E. coli O157, EC-IMS was c. 100-fold more sensitive for detection of the organism than direct culture on either medium. During monitoring of a dairy herd, E. coli O157 was isolated from 84 (8·2 %) of 1024 rectal swabs taken from cattle over a 4-month period; 23 (27·4 % of the 84 strains were isolated by both direct culture and IMS (15 of the 23 were isolated on both media, five on CT-SMAC only and three on CR-SMAC only), whereas 61 (72·6%) strains were isolated by IMS only. IMS is a sensitive and simple technique for the isolation of E. coli O157 from bovine faecal samples and should prove useful in elucidating further the epidemiology of this organism.

Cytotoxic necrotising factors type 1 (CNF1) and type 2 (CNF2) are produced by many Escherichia coli strains isolated from man and animals with intestinal or extra-intestinal colibacillosis. In most laboratories, CNF-producing strains are detected by a cell cytotoxicity assay and confirmed with a neutralisation assay or a mouse footpad assay. In this study, we sought to determine whether DNA probes could detect clinical isolates of E. coli producing CNF2 or CNF1, or both, without the need for cell cultures or animal assays. Two internal fragments of the gene encoding CNF2 were used as DNA probes: a 875-bp XhoI-PstI DNA fragment and an adjacent 335-bp PstI-ClaI fragment. A positive response with both DNA probes was associated with CNF2-producing strains, whereas a positive response with only the 335-bp probe was associated with CNF1-producing strains. Results of colony hybridisation experiments with 185 clinical isolates of E. coli demonstrated that these DNA probes detected CNF2-producing strains with a sensitivity and specificity of 100% and CNF1-producing strains with a sensitivity and specificity of 99 %. These two DNA probes should greatly facilitate epidemiological studies to assess the importance of CNF-producing strains as agents of diarrhoea and septicaemia.

The pre-formed urease activity of three NCTC reference strains and five clinical isolates of Helicobacter pylori was determined at room temperature (21°C) and 37°C by a viable cell count technique with a conventional urea slope test (Christensen’s agar) as well as the commercial CLO-test. The urease activity of two gastroduodenal commensals, Proteus mirabilis and Klebsiella pneumoniae, was also tested. H. pylori strains produced positive reactions with viable cell counts of 106–108 cfu within 30 min and with counts of 103–106 cfu within 2 h. For some strains, smaller numbers of organisms were needed with the CLO-test than with the conventional test, and incubation of the CLO-test strips at 37°C slightly decreased the number of organisms required for positive results. P. mirabilis produced a positive result on urea slopes with an initial inoculum of 107–108 cfu at 2 h, but no positive reaction occurred for K. pneumoniae at 12 h, even with an initial inoculum of 1011 cfu. However, both P. mirabilis and K. pneumoniae gave a positive result after incubation for 24 h with initial inocula of < 101 cfu and 103–104 cfu respectively. Incubation at 37°C significantly reduced the inoculum size of these organisms required for a positive result after incubation for 4 h when tested with the slopes, but not with the CLO-test. These findings indicate that H. pylori possesses much greater pre-formed urease activity than P. mirabilis and K. pneumoniae. False negative results for clinical detection of H. pylori in gastroduodenal biopsies may be due to small numbers of organisms, especially after treatment with antimicrobial agents, and false positive results may arise from gastroduodenal commensals or contaminants.