- Volume 38, Issue 3, 1993

A DNA amplification method was used to detect Mycoplasma pneumoniae in clinical samples. M. pneumoniae 16S ribosomal RNA gene sequences were selected as the amplification target region. The polymerase chain reaction (PCR) with purified DNA fragments as templates yielded an expected 88-bp fragment from M. pneumoniae but not from other Mycoplasma spp. nor from any of the other bacteria assayed. With this method, the 88-bp product specific for M. pneumoniae could be obtained from a minimum of 0.05 pg of M. pneumoniae DNA. Subsequently this PCR technique was used for the detection of M. pneumoniae in throat-swab samples. Twenty-two of 30 culture-positive clinical samples gave positive results in the PCR test. Thirty-two culture-negative clinical samples and 33 samples from healthy volunteers, of which only one was culture-positive, gave negative results in the same PCR test. This PCR method is useful for the direct detection of M. pneumoniae in clinical samples.

The role of a 54-kb plasmid in the virulence of Salmonella Enteritidis strains of phage type 4 was examined in mice, young chickens and adult laying-hens. Although the plasmid was essential for full expression of virulence in mice, differences in expression of virulence by this 54-kb plasmid were noted among strains; thus, introduction of the plasmid to a naturally occurring strain that lacked it did not make that strain virulent. In newly hatched chickens, virulence of an Enteritidis strain of phage type 4 by oral or parenteral routes was unrelated to possession of this plasmid which, similarly, played no role in infection in egg-laying hens. When a strain of Enteritidis phage type 4 and a plasmid-cured strain derived from it were given orally to chickens, both strains were equally invasive and their patterns of localisation in spleen, liver and ovaries were similar and they were isolated at similar frequencies from cultured, laid eggs.

Monoclonal antibodies (MAbs) were developed to Bacillus piliformis isolate-specific flagellar epitopes and used to group B. piliformis isolates on the basis of epitope expression. BALB/c mice immunised with flagella purified from various B. piliformis isolates served as the source of immune spleen cells for fusion with SP2/0Ag14 myeloma cells. Evaluation of hybridoma culture medium by ELISA against various bacterial species and B. piliformis isolates indicated that 482 of 2127 hybridomas secreted antibodies specific for B. piliformis. Specificity of MAbs for flagellar epitopes was demonstrated by indirect fluorescent antibody assays and Western blot analyses. Probing of 10 B. piliformis isolates with MAbs indicated that four B. piliformis isolates each possessed a distinct and isolate-specific flagellar epitope; five other isolates shared a common flagellar epitope. One isolate did not react with any of the MAbs specific for flagellar epitopes. Thus, B. piliformis isolates could be grouped into six antigenically distinct groups based upon flagellar epitope expression. Additionally, a MAb reactive with a cell-associated component recognised all but one isolate. This serological grouping of B. piliformis isolates agrees with the grouping of isolates based upon genetic and physiological characteristics, and supports the assertion that there are different strains among B. piliformis isolates.

A commercial DNA probe kit (Gen-probe) for the detection of rRNA from legionellae was evaluated for its accuracy in diagnosing Legionnaires’ disease in 167 patients with pneumonia. The test was performed on freshly obtained clinical respiratory tract samples. Cultures and direct immunofluorescence antibody (DFA) staining of the samples and serological tests were performed simultaneously for all patients. The probe assay result was positive in six patients; five of them had other laboratory evidence of disease (positive cultures or positive serological results or both). Depending on the diagnostic criteria, the probe test had a sensitivity of 31–67%, a specificity of 99% and positive predictive values of 67–83%. The diagnostic performance of the DNA probe assay in this study was superior to that of the DFA test. The results indicate that the examination of respiratory tract secretions by the Gen-probe kit is a suitable screening test for the diagnosis of Legionnaires’ disease.

The bacteriology of brain abscess is complex-both aerobic and anaerobic organisms are involved, and their incidence varies from centre to centre. In this study of 50 consecutive cases of brain abscess, the value of both modern imaging techniques and the time-honoured Gram’s stain was demonstrated. The Gram’s stain showed organisms in 41 cases (82%) and culture was positive in 44 cases (88%). Thirty cultures yielded pure aerobic growth: Staphylococcus aureus. β-haemolytic streptococci and Proteus spp. were pre-dominant. Five cases gave mixed aerobic cultures, and in seven cases anaerobes were isolated. Of these, three showed a mixed aerobic and anaerobic flora. Mycobacterium tuberculosis was cultured from one of the samples, which had shown acid-fast bacilli on direct smear. M. fortuitum was cultured from one sample, although no organisms were seen in the gram-stained preparation.

Isolates of group B streptococci (GBS) from neonates with early-onset septicaemia are associated with particular restriction endonuclease digestion patterns (RDP type Ia-3 and III-3) of chromosomal DNA. Opsonophagocytosis of serotype Ia and serotype III GBS isolates was studied by the luminol-enhanced phagocytic chemiluminescence (CL) assay. Pools of serum containing GBS type-specific antibody levels equivalent to or just above levels typically found in sera from mothers of infected infants were used. CL intensities induced by GBS isolates of RDP types Ia-2, Ia-3 and III-3 were lower than those of the other RDP types of the same serotype. Opsonophagocytosis was more efficient with serum containing higher concentrations of type-specific antibodies but for RDP type III-3 strains these differences were much less marked than for other RDP types. CL intensity did not correlate with cell surface charge, hydrophobicity or sialic acid content of GBS. Results demonstrate that certain GBS RDP types are more resistant to opsonophagocytosis and suggest that potentially virulent strains with genetic homogeneity may exist.

Three traditional assays were used to determine the minimal bactericidal concentration (MBC) and minimal inhibitory concentration (MIC) for Streptococcus pyogenes (group A streptococci) in two phases of growth and the time taken to kill the organisms. Three other methods were used for the determination of penicillin tolerance: a cell-lysis assay. the β-lactamase disk method and the replication method. Twenty strains, comprising penicillin-tolerant clinical isolates and two laboratory mutants, were used to evaluate the six tests. Results indicated that two groups of S. pyogenes can be distinguished—four highly tolerant and three moderately tolerant strains. The moderately tolerant strains were not recognised when rapidly growing instead of stationary cultures were used for the MBC and MIC determinations. The MBC/MIC ratio for tolerant strains was > 100. Tolerance percentage ranged from 0.30 to 1.07 and 0.29 to 3.96 for cultures in the mid-logarithmic and stationary phases of growth, respectively. The cell-lysis assay, the β-lactamase disk method and the replication method may be used to screen for tolerance. Detection of high or moderate tolerance by determining the MBC/MIC ratio for mid-logarithmic or stationary cultures is recommended.

Methicillin-resistant strains of Staphylococcus aureus isolated during 1985 and 1987 at a Nigerian hospital were compared by resistance profiles, plasmid analysis, and pulsed-field gel electrophoresis of chromosomal DNA. The results indicated that the isolates from the two periods were unrelated with regard to all three aspects. None of the isolates was similar to the classical MRSA nor to the epidemic MRSA of Australia or the UK. The MRSA isolated in 1985 had a similar plasmid to MRSA isolates from Singapore, but differed from them when compared by pulsed-field gel electrophoresis.

The opsonic requirements of Helicobacter pylori were investigated in a series of experiments with human polymorphonuclear leucocytes (PMNL). Pre-incubation of H. pylori with pooled normal human serum (NHS) in concentrations of 5–20% significantly increased the uptake of radiolabelled bacteria by PMNL. Treatment of the bacteria with NHS 30% caused the release of radiolabel and this effect was abolished by heating serum to 56°C, suggesting that H. pylori is serum-sensitive and that complement is involved. Opsonisation of H. pylori with NHS concentrations of 10–30% significantly increased PMNL chemi-luminescence. Removal of specific antibody had no effect. Removal of either the classical or alternative complement pathways produced no significant change in PMNL chemilum-inescence, indicating that either pathway is sufficient for opsonisation on its own. The results confirm that complement is the most efficient opsonin for H. pylori.

Two clones of Escherichia coli O27:K1:H31 and O2:H7, isolated from patients with urinary tract infection or bacteraemia, failed to grow in a synthetic minimal medium (MM) of low osmolality. They were considered to be osmo-remedial because they grew well when sufficient amounts of NaCl, mannitol or sucrose were added to raise the osmolality of the medium to > 300 mOsm/kg. The defect could also be corrected by nicotinamide or its precursors quinolinic and aspartic acids. Each clone had a unique DNA restriction enzyme profile, fimbriae and antibiotic susceptibility patterns. The osmo-remedial variants were unstable and underwent phenotypic modulation to form mixtures with osmo-tolerant forms when grown in MM. They tended to form satellites of small colonies around large colonies of osmo-tolerant cells on MM agar plates. The penicillin method of Davis was used to separate the two forms. Nicotinamide induced the expression of ompF when the osmo-remedial strains were grown under conditions of low osmolality. It is possible that the variants are defective in the synthesis of membrane-derived oligosaccharides or outer-membrane proteins, but this has yet to be determined.

Nine strains of Escherichia coli isolated from infants with diarrhoea between 1947 and 1960 and designated “enteropathogenic” were examined for phenotypic and genetic characters associated with virulence. Each strain belonged to a different serotype. All the isolates were historically significant in that they were amongst the first strains of E. coli reported to be causally associated with infantile diarrhoea. Five strains possessed the virulence properties of class I enteropathogenic E. coli (EPEC). All these strains were isolated originally from symptomatic children during outbreaks of diarrhoea. Two isolates from sporadic cases of diarrhoea fulfilled the criteria for classification as class II EPEC. One strain was identified as enteroaggregative E. coli and the other carried no known virulence-associated properties. These findings indicate that most early isolates of E. coli which were designated “enteropathogenic” were indeed EPEC, as currently defined.

Aeromonas sp., grown in tryptone soya broth supplemented with yeast extract, 0.6%, pH 7.5, and incubated with agitation at 100 oscillations/min for 15 h at 37°C produced optimal amounts of β-haemolysin and cytotonic enterotoxin. More prolonged incubation resulted in the loss of enterotoxic activity and anion exchange chromatographic analysis indicated the presence of a moiety capable of brerking down the toxin. Anion exchange fast protein liquid chromatography resulted in a single peak of haemolytic activity and two peaks with enterotoxic activity. The cytotonic enterotoxin was purified from the fraction most active in the infant mouse assay; the second peak, which did not cross-react immunologically, may represent a second cytotonic enterotoxi. Neither peak was observed in the chromatographic fractions of filtrates from strains devoid of activity in the infant mouse assay. Purified enterotoxin, estimated to have a mol. wt of 15kDa by SDS-PAGE, caused fluid accumulation in the infant mouse assay, was non-haemolytic to rabbit erythrocytes, caused an increase in cAMP activity in tissue culture cells and did not cross-react immunologically with components of cholera toxin or the whole toxin. Purified β-haemolysin had an estimated mol. wt of 55 kDa, lysed rabbit erythrocytes and did not cause fluid accumulation in the infant mouse test.

Edited by R. R. Watson. 1991. ISBN 0-89603-206-X. Humana Press, New Jersey. Pp. 620. $99.50.

Edited by H. J. KUNG and P. K. VOGT. 1991. ISBN 3-540-53857-7. Springer-Verlag, Berlin. Pp. 179. £136.

Edited by M. Borgers, R. Hay and M. G. Rinaldi. ISBN 3–540–97504–7. Springer-Verlag, New York. Pp. 274.DM 228.00.

Edited by G. Orefici. 1992. ISBN 3–437–1 1362–3. Gustav Fischer Verlag. Pp. 569. DM 239.00.

G. P. BODEY, D. MILATOVK and I. BRAVENY. 1991. ISBN 3-528-07838-3. Verlag Vieweg, Wiesbaden. Pp. 270. DM 36.00.