- Volume 48, Issue 7, 1999

Twenty-eight phenotypically separate strains of clinically isolated vancomycin-resistant enterococci have been investigated with two API identification kit systems (20 Strep and Rapid ID 32 Strep) and two BBL Crystal kits (Gram Positive and Rapid Gram Positive). All strains were identified as Enterococcus faecium by a reference laboratory. The Rapid ID 32 kit positively identified 15 of 28 strains (54%), but only two (7%) were identified correctly: 11 were identified as ‘doubtful’ or ‘to genus level’ and two gave ‘unacceptable’ profiles. The API 20 Strep kit identified 27 strains (96%), but only 16 (57%) were identified correctly as E. faecium. The Rapid ID 32 kit erred by either positively misidentifying vancomycin-resistant E. faecium as E. casseliflavus or E. gallinarum, or indicated that this was the most likely identification, while the API 20 Strep kit more commonly produced a misidentification as E. casseliflavus. The Crystal Gram Positive and Rapid Gram Positive kits correctly identified 26 (93%) and 27 (96%) of the strains, respectively.

The purpose of this study was to evaluate the anti-chlamydial activities in vitro of liposome-encapsulated doxycycline (Dox) and tetracycline (Tet) in comparison with free Dox and Tet. Dox and Tet encapsulated in cationic (CAL), anionic (ANL) and neutral (NTL) liposomes by sonication, were quantified by high-performance liquid chromatography. Anti-chlamydial activities were determined by addition of serial dilutions of antibiotics (MIC 0.12-0.007 mg/L; MBC 4-0.25 mg/L) to HeLa 229 cell monolayers inoculated with Chlamydia trachomatis L2/434/Bu (103 ifu/well). After incubation for 72 h at 37°, chlamydial inclusions were stained by the May-Grünwald Giemsa method to establish MICs. MBCs were determined in chlamydial agent-free medium after second passages. Dox-encapsulation efficiencies were 28.6 SEM 6.4% in cationic (CAL-Dox), 49.1 SEM 6.7% in anionic (ANL-Dox) and 21.0 SEM 0.8% in neutral (NTL-Dox) liposomes. Tet-encapsulation efficiencies were 3.5 SEM 0.3% in anionic (ANL-Tet) and 2.2 SEM 0.6% in neutral (NTL-Tet) liposomes; no Tet was detected in cationic (CAL-Tet) liposomes. MIC values were 0.06 mg/L for Dox, 0.12 mg/L for Tet, 0.03 mg/L for CAL-Dox, NTL-Dox and NTL-Tet, and 0.01 mg/L for ANL-Dox and ANL-Tet. MBCs were 4 mg/L for Tet, 0.5 mg/L for CAL-Dox and NTL-Dox, and 1 mg/L for Dox, ANL-Dox, ANL-Tet, NTL-Tet and NTL-Tet. For MICs, the relative increase in anti-chlamydial activity observed with liposomal formulations compared to the corresponding free antibiotic ranged from 2- to 6-fold with Dox and from 4- to 10-fold with Tet. For MBCs, the relative increases in anti-chlamydial activity were 2- and 4-fold with liposome-encapsulated Dox and Tet, respectively. Dox was better encapsulated than Tet in all liposomes. Liposome-encapsulated drugs showed greater anti-chlamydial activities than their free forms; thus, these drug formulations have potential in the treatment of chlamydial infections.

A new commercial serotyping set based on heat-stable Penner's antigens was compared with pulsed-field gel electrophoresis (PFGE) with SmaI and SacII restriction endonucleases. Among 50 isolates of Campylobacter jejuni from Finnish patients, which represented predominant PFGE patterns selected from isolates from sporadic cases and isolates associated with small outbreaks, 11 different serotypes were demonstrated from 43 typable isolates. Several PFGE patterns could be found within one serotype; on the other hand, several serotypes could be demonstrated within one PFGE type. Most isolates originated from sporadic cases; however, some isolates were epidemiologically associated and showed identical serotypes and PFGE patterns. Although the new serotyping set would have been useful in the few epidemic cases studied, several isolates (14%) representing the major PFGE patterns remained untypable or gave weakly positive agglutination reactions only suggesting a plausible serotype (18%). This might restrict the use of the novel serotyping set, at least in Finland.

Many diverse clinical isolates of Proteus mirabilis (48 strains), P. penneri (25), P. vulgaris biogroup 2 (48) and P. vulgaris biogroup 3 (21) from man were examined for their ability to produce proteolytic enzymes and the nature and characteristics of the proteases were studied. All the P. penneri isolates, most (94-90%) of the P. mirabilis and P. vulgaris biogroup 2 isolates, but only 71% of the P. vulgaris biogroup 3 isolates, secreted proteolytic enzymes. These were detected most readily at pH 8 with gelatin as substrate. A strong correlation was found between the ability of a strain to form swarming growth and its ability to secrete proteases. Non-swarming isolates invariably appeared to be non-proteolytic. However, some isolates, particularly of P. vulgaris biogroup 3, were non-proteolytic even when they formed swarming growth. Analysis of the secreted enzymes of the different Proteus spp. on polyacrylamide-gelatin gels under various constraints of pH and other factors showed that they were all EDTA-sensitive metalloproteinases. Analysis of the kinetics of production of the proteases revealed the formation of an additional protease of undefined type and function that was cell-associated and formed before the others were secreted. The secreted protease was subsequently modified to two isoforms whose mass (53-46 kDa) varied with the Proteus spp. and the strain. There was no evidence that the secreted proteases of strains of Proteus spp. were of types other than metalloproteinases.

PCR and restriction fragment length polymorphism (RFLP) typing of flagellin genes (fliC) from 57 clinical isolates of Burkholderia cepacia indicated that only type II flagellins were present. Twenty-two isolates previously identified as the epidemic UK cystic fibrosis strain were indistinguishable by this method, as were 11 isolates from a pseudo-outbreak in Senegal. Other clinical isolates, including 19 from disparate sources in Malaysia, were separated into nine fliC RFLP groups, exhibiting a large degree of divergence. When isolates were indistinguishable by fliC genotyping, their similarity was confirmed by whole genome macro-restriction analysis with pulsed-field gel electrophoresis following XbaI digestion. The variation in fliC sequences of B. cepacia was far greater than that with B. pseudomallei, supporting the view that ‘B. cepacia’ as currently defined, may comprise several different genomic species.

A commercially available disk diffusion procedure was used in a large-scale study to evaluate the susceptibility of a wide range of Candida isolates to polyenes and azoles. With almost all isolates of C. glabrata resistant colonies were present within the inhibition zones for the azole compounds fluconazole, ketoconazole and miconazole, and less frequently for isoconazole, econazole and clotrimazole. Ten randomly selected isolates were cloned by limiting dilution and the susceptibility of the resulting strains to polyenes and azoles was determined. All strains presented a similar susceptibility pattern with sensitivity to polyenes and the presence of resistant colonies for all azole compounds except tioconazole. For each strain and each antifungal agent, one of these resistant colonies was subcultured and studied for antifungal susceptibility. All these colonies showed similar properties regardless of which antifungal agent allowed their selection, with increased sensitivity to polyenes and cross-resistance to the azole compounds except tioconazole. Similar results were obtained on Shadomy's modified medium and on synthetic medium. Likewise, determination of MICs by the Etest method confirmed the resistance to fluconazole. Comparative growth studies revealed a respiratory deficiency in the mutants caused by mitochondrial DNA (mtDNA) deletions. In addition, ‘petite’ mutants were obtained from a wild-type strain by exposure to ethidium bromide, and these respiratory mutants were shown to be resistant to azoles. These results demonstrate the relationship between mtDNA deficiency and resistance to azoles, and provide an interesting model to study the mechanisms of action of these antifungal agents.

Sera from patients with likely and possible Pneumocystis carinii pneumonia (PCP) on the basis of clinical information and laboratory investigations were tested by immunoblotting to assess the usefulness of trophozoites in the serodiagnosis of PCP. IgG antibodies to 50-60- kDa proteins were demonstrated with cyst antigen, but antibodies to additional proteins of 61, 70, 82, 95, 99 and 116 kDa were found with trophozoite antigen. These bands were not demonstrated with control sera. IgG antibody to the 116-kDa protein was found in 18 (46%) of 39 sera from patients with possible PCP compared with 5 (17%) of 30 sera from patients with likely PCP. There was no other significant difference between the two patient groups in detection of these proteins. Sera with higher indirect immunofluorescence assay (IFA) IgG titres were more likely to be immunoblot positive. Only 4 of 16 patients with likely PCP were IgG negative in the IFA; three of these were IgG immunoblot positive. In 4 of 10 patients with likely PCP and 6 of 15 patients with possible PCP, demonstration of IgM or IgA, or both, by IFA or immunoblotting provided evidence suggestive of current infection. This study confirms the usefulness of rat-derived antigen, especially trophozoite antigen, in PCP serology. The IgG IFA remains the most useful test, but IgM and IgA testing and immunoblotting can support the diagnosis.