- Volume 36, Issue 4, 1992

The interaction of Pneumocystis carinii purified from rat lungs with rat peritoneal macrophages and human circulating polymorphonuclear leucocytes was studied by amplified chemiluminescence and examination of stained cytospin preparations. A polyclonal rat antiserum to P. carinii was opsonic with both types of phagocyte. Complement had no opsonic properties alone but produced a synergic effect in combination with antibody.

Adherence to and entry of the parasite into the host is one of the essential elements of microbial pathogenicity. We investigated the adherence to and entry into primate kidney epithelial (Vero) cells of Borrelia burgdorferi by radiolabelling techniques, immunofluorescence and electronmicroscopy. The attachment to and subsequent entry of both untreated and heat (50°)-treated B. burgdorferi into Vero cells occurred at cell-surface sites associated with aggregated coated pits. In contrast, there was minimal attachment of spirochaetes heated at 60°. Radiometric studies showed that, with untreated cells, there was incorporation of both 14C-glucose-1-phosphate and 14C-thymidine, whereas with the 50°-treated spirochaetes only glucose-1-phosphate was incorporated, and with the 60°-treated spirochaetes neither radionuclide was incorporated. Spirochaetes heated at 50° or 60° did not grow at 35° in culture medium. These results suggest that the presence of certain metabolic activities of the spirochaete but not viability (ability to grow) are necessary for the attachment process. After entry of untreated B. burgdorferi, most of the spirochaetes were either free in the cytoplasm or tightly bound to the host membrane. In contrast, 50°-treated spirochaetes remained bound to host membrane in large phagosome-like vesicles.

The sequence of events involved in haemagglutination and lysis of erythrocytes by washed cells, vesicles and the culture supernate of Porphyromonas gingivalis strain W83 was monitored by 51Cr release and transmission electronmicroscopy. All preparations, except capsular material and lipopolysaccharide, caused haemagglutination and, by a slow process of attachment and specific attack on the surface structures of the red blood cells, produced minute pores and eventual leakage of cellular contents. N-acetylglucosamine, N-acetylgalactosamine and several other sugars such as glucose and sucrose had no effect on haemagglutination. Antiserum raised against a cloned haemagglutinin of P. gingivalis strain 381 inhibited the activity of strain W83 cells, vesicles and supernate. The antiserum-neutralised supernate lost 70–80% of its hydrolytic activity towards α-N-benzoyl-L-arginine-4-nitroanilide but the residual activity behaved in a manner similar to the native supernate in that it was completely inhibited by the addition of 2,2′-dipyridyl disulphide and was fully restored upon addition of a low-Mr mercaptan. Binding of the antiserum to the haemagglutinin epitope of P. gingivalis still permitted titration of the active centre cysteinyl thiol group of the proteinase. Purified gingivain caused lysis of erythrocytes and was not neutralised by antiserum to the haemagglutinin. These results suggest that, although the haemagglutinin and gingivain are probably separate molecules, they are closely associated on the outer membrane of P. gingivalis and may be functionally related.

Protection against experimental Serratia marcescens infection in mice was enhanced by prior injection of formalin-killed or viable bacteria of the same strain. From the first to the fourth week after vaccination, specific immunity was involved in the host defence against systemic serratia infection. The transfer of antiserum specific for S. marcescens increased bacterial clearance from the liver, but did not increase the survival of the mice. Bacterial clearance from the liver was also increased by the transfer of spleen cells from immunised mice, but, again, survival was not increased. However, the transfer of both antiserum and spleen cells from vaccinated mice increased both bacterial clearance from the liver and survival (p <0.01). These results suggest an additive effect of humoral immunity and T-cell-mediated immunity in protection against systemic serratia infection.

Klebsiella strains possessing capsule type K7 are found predominantly in respiratory secretions. To investigate the importance of this K antigen in virulence, 13 K7 strains were compared with K2 capsulate isolates which are generally regarded as highly virulent. The toxicity of the strains was determined in a mouse peritonitis model. Generally, K7 isolates were significantly less toxic for mice than K2 strains. In the absence of serum, neither capsule type showed much stimulation of leucocytes, measured as the chemiluminescence (CL) response of human polymorphonuclear leucocytes (PMNL). However, in the presence of normal human serum, CL values with K7 strains increased considerably, whereas the CL response to K2 isolates was unaffected. Correspondingly, intracellular killing by PMNL was observed with K7 strains only, whereas K2 isolates proved to be relatively resistant to phagocytic destruction. No correlation was found between capsule type K7 and serum resistance. These data suggest that, in contrast to K2, capsule type K7 may not be a critical factor in the virulence of K7-capsulate Klebsiella strains nor does it seem to act as an antiphagocytic barrier.

A probe based on 16S ribosomal RNA (rRNA) sequences was developed to detect Mycobacterium paratuberculosis, the causative agent of Johne’s disease in cattle. Three universal primers were used to sequence the amplified fragments of the 16S rRNA gene of various species of mycobacteria. When the nucleotide sequences were analysed, a deletion was detected in the sequence of the fast-growing species. An oligonucleotide probe (P) directed to this region was synthesised and hybridised directly with total RNA of various mycobacterial strains in a dot-spot assay. The probe detected M. paratuberculosis, some other slow-growing mycobacteria of the M. avium-intracellulare (MAI) complex, and one atypical strain, M. gordonae. To increase the sensitivity of the probe, a 413-bp fragment of the 16S rRNA gene of M. paratuberculosis between P and a second oligonucleotide primer was amplified and hybridised with a M. paratuberculosis/M. avium-specific probe. When faecal samples of cattle were tested, all culture-positive samples were positive in the PCR assay.

Escherichia coli adherent to HEp-2 and HeLa cells were isolated from the faeces of 43 (19.7%) of 218 hospitalised infants aged below 6 months with acute diarrhoea. No conventional virulence factors, including enterotoxin production—heat-labile (LT) or heat-stable (ST), the verotoxin (VT) or shiga-like toxin (SLT)—or the invasive phenotype (determined by the Sereny test) could be detected among these isolates. Out of the 43 isolates, 16 (37.2%) were of the known enteropathogenic O:K serogroups—enteropathogenic E. coli (EPEC). The remaining 27 (62.8%) isolates showed different types of adherence to HEp-2 and HeLa cells which was diffuse (40.7%), localised (37.0%), or both (22.3%); they were identified as entero-adherent E. coli (EAEC). The EAEC isolates adhered to HEp-2 and HeLa cells in the presence of mannose, lactose, fucose, galactose, and fetuin, indicating that adhesion was not specific for these sugars or glycoprotein. Haemagglutination and the salt aggregation test (SAT) did not correlate with patterns of adherence. The results of this study indicate that LA-EAEC is an important aetiological agent of acute diarrhoea in infants aged below 6 months in Calcutta.

Of 147 isolates of three species of Aeromonas, 54 were from clinical and 93 from environmental sources. When tested for enterotoxin production, most of the isolates (56%) caused accumulation of fluid in rabbit ileal loops (RILs). Although large proportions of clinical and environmental isolates of A. caviae (55% and 65%, respectively) elicited such a response in RILs, isolates of A. hydrophila and A. sobria produced significantly more fluid (p<0.05). Furthermore, the environmental strains of A. hydrophila and A. sobria produced more fluid than the clinical isolates (p<0.05). The strains of Aeromonas spp. that caused little or no fluid accumulation in the initial experiments became enterotoxin producers after 1–3 passages through RILs, regardless of their source, and showed gradual enhancement of fluid outpouring after each passage. The present study suggests that all strains of these species of Aeromonas are potentially enterotoxigenic, whether from clinical or environmental sources.

Citrobacter diversus brain abscess occurred in two infants in Aberdeen, 5 months apart. These are the first reported cases of this condition in the UK since 1976. Restriction endonuclease analysis with SacI enzyme showed blood and CSF isolates from both patients to be identical and different from 10 other clinical isolates of C. diversus and one C. amalonaticus strain. Furthermore, isolates of C. diversus from patients belonged to biotype “d” whereas control isolates were of biotypes “a” or “e”. Because both infants attended the same central and peripheral maternity units, this raised suspicions of long term contamination of the hospital environment by this organism. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of whole-cell proteins and immunoblotting with normal human serum were remarkably homogeneous for all 13 C. diversus strains and thus were not useful for typing. However, the only C. amalonaticus strain was clearly differentiated from C. diversus strains by SDS-PAGE. Management of the infants included multiple intravenous antibiotic therapy for 4–6 weeks and repeated computerised tomography (CT) scanning and drainage of the abscess cavity. Both children survived albeit with some minor degree of brain damage.

A total of 2401 isolates of Haemophilus parainfluenzae was isolated from respiratory secretions of 36 healthy adults and 128 patients with chronic bronchitis over a period of 1 year. The isolates were allocated to eight biotypes, by their production of indole, urease and ornithine decarboxylase. Biotypes I and II constituted most of the isolates of H. parainfluenzae from the oropharynx of controls (75%) and chronic bronchitics (c. 90%). Among the patients, there was no difference in the isolation rate between oropharyngeal swabs and sputum specimens. Biotypes III, IV, VI, VII and VIII were isolated less frequently, as was a new taxon defined here as bioptype V which does not produce indole, urease or ornithine decarboxylase. Biotype III was isolated significantly less frequently from cases of chronic bronchitis than from controls, whereas biotype II was isolated somewhat more frequently from the patients, especially during acute episodes.

Epinephrine and norepinephrine inhibit attachment of Leishmania donovani promastigotes to cultured hamster peritoneal macrophages. The inhibition was significant at catecholamine concentrations of 10−4 and 10−5M and occurred when they were added to the cell mixtures, or after pre-treatment of either macrophages or parasites. Inhibition of attachment after pre-treatment was less marked than when the catecholamines were added to parasite-cell mixtures. Similar results were obtained with dibutyryl cyclic AMP, cholera toxin, theophylline, and cadaverine which raise intracellular cyclic AMP (cAMP). Pre-treatment of parasites or macrophages with the bioamines elevated the intracellular cAMP concentration. It is suggested that the inhibitory effect on the host-parasite interaction is mediated through cAMP.

Staphylococcal abscesses contain two types of lipids which are bactericidal for Staphylococcus aureus. These include a group of long chain unsaturated free fatty acids and another as yet unidentified lipid with unique properties. When abscess homogenates are incubated with S. aureus culture filtrates, the amount of bactericidal activity is increased. This phenomenon is called activation. To determine the source of increased bactericidal activity during activation, individual types of lipid were isolated from abscess homogenates and examined for their ability to be activated. Activation was found to result from the release of long chain unsaturated fatty acids from glycerides, presumably by the action of staphylococcal lipase.

Certain strains of Staphylococcus aureus produce an enzyme capable of inactivating the bactericidal fatty acids produced in staphylococcal abscesses by esterification to various alcohols. The enzyme, called FAME (fatty acid modifying enzyme), has a pH optimum between 5.5 and 6.0 and a temperature optimum of about 40°. Enzyme activity is not affected by edetic acid or by the presence or absence of sodium and potassium ions. Although FAME can utilise methanol, ethanol, 1-propanol, 2-propanol, 1-butanol or cholesterol as substrates, cholesterol appears to be the preferred substrate. FAME esterifies without being an esterase operating in reverse. Strains capable of producing the enzyme can synthesise it in trypticase soy broth and in a chemically defined medium, but not necessarily in equal amounts. FAME production is correlated with the ability of a strain to grow and survive within the tissues.