- Volume 44, Issue 5, 1996

The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was evaluated for species identification among mycobacteria by analysis of the dnaJ gene. Nine clinical isolates of Mycobacterium tuberculosis with different fingerprint patterns all gave the same distinct SSCP banding pattern and could be distinguished from other mycobacteria, such as M. avium. In contrast, considerable strain-specific dnaJ gene variations were observed amongst 42 clinical isolates of M. avium and 13 other atypical mycobacterial strains. Only 62% of the M. avium isolates hybridised to an M. avium-specific probe and only 14% could be identified correctly as M. avium by both probe and restriction fragment length polymorphism analysis. This finding was supported by direct sequence analysis. Variations were also observed in M. gordonae and M. scrofulaceum isolates. Computerised analysis of M. avium samples broadly identified three clusters. Results suggest that although the SSCP procedure may be useful for distinguishing M. tuberculosis from other mycobacteria, this technique applied to the dnaJ gene may not be suitable for strain identification. The results stress the importance of testing a large collection of clinical isolates before new molecular procedures are introduced into routine laboratories.

A retrospective survey was conducted of the characteristics of acinetobacter infections in Hong Kong — seasonal and geographic distributions, frequency of isolation from various body sites, antimicrobial susceptibility and molecular epidemiology. Most (80%) isolates of Acinetobacter spp. belonged to DNA groups 2 (A. baumannii) or 13, as defined by growth at 44°C. An increased isolation rate in summer was related to higher ambient temperatures. The notion that acinetobacters are opportunist nosocomial pathogens was supported by the body site-and ward-specific distributions, which were similar to those of Pseudomonas aeruginosa and in marked contrast to those of coagulase-negative staphylococci and Escherichia coli. Typing of Acinetobacter isolates by arbitrary-primed polymerase chain reaction revealed extensive genotypic polymorphism, suggesting that numerous unrelated strains were circulating between patients. In view of the association with a high incidence of polymicrobial bacteraemia and multiresistance to antibiotics, a careful selection of appropriate antibiotics in combination is necessary for empirical therapy of infections caused by Acinetobacter spp.

Streptococcus intermedius, part of the ‘Streptococcus milleri group’, has the ability to produce glycosaminoglycan depolymerising enzymes (hyaluronidase and chrondroitin sulphate depolymerase) which is unique amongst the viridans streptococci and may contribute to their virulence in brain and liver abscesses. The growth of S. intermedius strain UNS 35 was studied in basal medium supplemented with chondroitin sulphate A (CS-A, sulphated at position 4 of the N-acetylgalactosamine moiety) or chondroitin sulphate C (CS-C, sulphated at position 6 of the N-acetylgalactosamine moiety) as the major carbohydrate source. CS-A but not CS-C supported the growth of S. intermedius. Extracellular degradation of CS-A resulted in the initial accumulation of 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-δenepyranosyluronic acid)-D-galactose (δUA GalNAc-0S), and low levels of 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-δenepyranosyl uronic acid)-4-O-sulpho-D-galactose (δUA GalNAc-4S) in the medium with GalNAc-0S being subsequently utilised during bacterial growth. Metabolic end-products included formate and ethanol but not lactate, indicating that growth was probably carbon-limited. The CS-A contained 30% CS-C, which was also depolymerised resulting in the formation of 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-δenepyranosyluronic acid)-6-O-sulpho-D-galactose (δUA GalNAc-6S) in the culture supernate, but this unsaturated disaccharide was apparently not utilised during growth. The results indicate that S. intermedius produced CS-AC depolymerase, which was inducible and extracellular, and sulphatase activity. Experiments with authentic δUA GalNAc-4S and δUA GalNAc-6S demonstrated that δUA GalNAc-4S rather than δUA GalNAc-6S was the preferred substrate for the sulphatase. Therefore, it is suggested that the CS-AC depolymerase of S. intermedius may play a role in the destruction of CS in host tissues, facilitating bacterial spread, and also in bacterial nutrition by the liberation of nutrients at the site of infection.

A glycosaminoglycan (GAG) depolymerase that acts on chondroitin sulphate A (CS-A), chondroitin sulphate C (CS-C) and hyaluronic acid (HA) was purified to apparent homogeneity from a culture of Streptococcus intermedius, strain UNS 35, grown in minimal medium supplemented with CS-A as the sole carbon source. The enzyme was purified by ammonium sulphate precipitation followed by serial chromatography on DEAE Trisacryl M, CM Trisacryl M and heparin-agarose. SDS-PAGE analysis of the purified enzyme yielded a single band with a mol. wt of c. 83 000. The purified GAG depolymerase was unusual in its substrate specificity. The enzyme was initially regarded as a CS depolymerase because of its induction by CS-A. However, the GAG depolymerase exhibited greatest activity against HA, whereas the degradation rates of CS-A and CS-C were c. 8% and 2%, respectively, of the rate with HA. On this basis the enzyme could be classified as a hyaluronidase rather than a CS depolymerase. The pH optimum was around neutrality and the enzyme was unusual in having a high pl of approximately 9.3.

The potential of exocellular carbohydrate antigens of Staphylococcus epidermidis as markers of infection in bone was investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Exocellular antigens were prepared by gel filtration chromatography of concentrated brain heart infusion culture supernates. The antigenic material appeared as diffuse bands between 24 and 32 kDa on the immunoblots and was not susceptible to digestion with trypsin, indicating that the response in the patients was to non-protein (polysaccharide or teichoic acid, or both) exocellular material. Significant differences were detected between the immunoblot antigen profiles for serum IgG from patients with S. epidermidis bone infection and those with an uninfected prosthetic joint. Thirteen of 16 patients with S. epidermidis prosthetic joint infection showed an elevated serum IgG level by ELISA compared with controls with uninfected joints. However, the antigen was not specific for S. epidermidis bone infection; high levels of IgG were also detected in patients with other serious staphylococcal and streptococcal infections. The ELISA test may be valuable in distinguishing between staphylococcal infection of joints and aseptic loosening by excluding cases of infection.