- Volume 42, Issue 3, 1995

Chemotactic responses of blood neutrophils and monocytes to media conditioned by eight strains of Escherichia coli with different virulence characteristics were measured in modified Boyden assay chambers to determine if these characteristics were associated with differences in leucocyte mobility. Responding neutrophils and monocytes were prepared on conventional density gradients, and in three instances, the chemotaxis of eosinophils isolated on metrizamide gradients was also studied. Media conditioned by enteroinvasive and non-enteroinvasive E. coli strains were tested as chemo-attractants and compared to the formylated peptide standard attractant. Chemotactic activity of neutrophils was greater than that of monocytes and eosinophils, and migration by all populations was significantly greater to conditioned media than to the control medium. Chemotactic responses to media conditioned by non-enteroinvasive E. coli and strains lacking virulence factors was greater than to media conditioned by plasmid- and Sereny-positive enteroinvasive organisms. The results suggest that virulence factors of E. coli that determine invasiveness did not augment the chemotactic responses of the leucocyte populations tested in vitro, and give no support to the hypothesis that they induce mucosal inflammation by directly increasing chemotaxis in vivo.

Ninety-two and 33 methicillin-resistant Staphylococcus aureus (MRSA) strains were isolated in Japan and China respectively. They were categorised as ofloxacin-susceptible (MIC < 12.5 mg/L), moderately (MIC 12.5-25 mg/L) or highly (MIC ≧ 50 mg/L) ofloxacin-resistant. 4-Quinolone concentrations required to inhibit purified DNA gyrase from the moderately and highly quinolone-resistant MRSA were at least 20 times higher than those required to inhibit the equivalent enzyme from quinolone-susceptible strains. Reconstitution assays demonstrated that the 4-quinolone-resistant MRSA had a mutation in subunit A of DNA gyrase. A portion of the gyrA gene from amino acids codons 40–115 was sequenced. Four moderately resistant and seven highly resistant MRSA contained a Ser → Leu substitution at amino acid 84; one moderately and one highly resistant MRSA and one moderately resistant methicillin-susceptible S. aureus (MSSA) strain contained a Glu → Lys substitution at amino acid 88. Eight MRSA, including one quinolone-susceptible strain and one MSSA contained a silent mutation at amino acid 86. Uptake of ofloxacin in moderately resistant strains was almost the same in the presence or absence of carbonyl cyanide mchlorophenylhydrazone (CCCP), whereas in highly resistant strains, uptake increased when CCCP was added. Restriction fragment length analysis of the norA gene with the restriction endonuclease SfcI showed a mutation of nucleotide position 1085 in all MRSA strains tested except for one highly quinolone-resistant strain. Thus the mechanisms of 4-quinolone-resistance in these MRSA isolates involved alterations in both DNA gyrase and antimicrobial uptake and efflux.

Bronchial secretions of patients with cystic fibrosis (CF) inevitably become colonised with Pseudomonas aeruginosa. This organism often exhibits multiple phenotypes with different antibiotic susceptibility profiles. Isolating each colonial morphotype and determining its antibiotic susceptibility profile is labour-intensive and time-consuming. Two disk diffusion methods for mixed morphotype susceptibility testing were examined; 134 morphotypes of P. aeruginosa from 50 respiratory specimens from CF patients were tested. Mixed cultures were prepared by (a) combining morphologically different colonies of P. aeruginosa directly from the sputum specimen primary culture plates from individual patients or (b) mixing colonies of individual morphotypes after isolation and subculture. Agreement with the results for testing morphotypes individually were 85.8% and 91.6%, respectively, for the two methods. These agreement rates rose to 95.6% and 99.4%, respectively, when minor errors (intermediate misclassified as susceptible or resistant or vice versa) were excluded. Mixed morphotype testing of P. aeruginosa directly from sputum specimen culture plates in chronically colonised CF patients is more efficient, reduces turn-around time and provides clinically meaningful information about antibiotic susceptibility.

A chronic bacteraemia due to Staphylococcus epidermidis occurred in a patient undergoing allogeneic bone marrow transplantation. Forty-two S. epidermidis isolates were obtained from blood cultures over a period of 5 months. Isolates were separated into three groups by SmaI macrorestriction characterisation with pulsed-field gel electrophoresis (PFE-1, one isolate; PFE-2, 32 isolates; PFE-3, nine isolates). Differences were detected in antimicrobial susceptibility patterns among isolates belonging to group PFE-2. The two strains, PFE-2 and PFE-3, were both responsible for the chronic bacteraemia and were isolated during different admissions to the hospital. A central venous catheter was the portal of entry for PFE-2. DNA macro-restriction analysis with pulsed-field gel electrophoresis was helpful in the epidemiological investigation of this S. epidermidis chronic bacteraemia.

The influence of six immunosuppressive agents on the occurrence of endogenous bacteraemia in mice was evaluated. The mortality rates in conventional ddY mice given cyclophosphamide (CY), fluorouracil (5-FU), methotrexate (MTX), cisplatin (CDDP) or FK-506 intraperitoneally, or dexamethasone (DXM) subcutaneously were 70, 100, 100, 100, 0 and 0%, respectively. Pseudomonas aeruginosa was isolated from 70% of mice treated with CY but from only 10% of mice treated with 5-FU and 30% treated with MTX. Enterobacteria were isolated from 90% of mice treated with 5-FU. Specific-pathogen-free (SPF) mice fed P. aeruginosa were also treated with these agents. All mice in the CY, 5-FU, MTX and CDDP groups died whereas mice treated with DXM and FK-506 showed 20% and 0% mortality, respectively. Pure cultures of P. aeruginosa were obtained from all of the mice treated with CY. Polymicrobial bacteraemia with P. aeruginosa and enterobacteria occurred in 5, 25, 5 and 5% of mice treated with 5-FU, MTX, CDDP and DXM, respectively. Enterobacterial bacteraemia was observed in 70% of mice treated with CDDP and in 5% of the DXM group. Different types of bacteraemia were induced by different immunosuppressive agents. The mechanism of immunosuppression may affect the frequency of bacteraemia and the causative organism.

Th1-and Th2-derived cytokine production in response to synthetic peptides of the fimbrial subunit protein (fimbrilin) from Porphyromonas gingivalis strain 381 was assessed in spleen mononuclear cells (MNC) of BALB/c mice (H-2d haplotype) immunised with the fimbrial protein antigen and adjuvant GM-53 in Freund’s incomplete adjuvant (FIA). Sixty-seven sequential overlapping 10-mer peptides covering the complete 337 amino-acids (AA) protein of P. gingivalis fimbrilin were synthesised. Stimulation of spleen MNC in vitro with these 10-mer peptides resulted in the production of murine interleukin-2 (IL-2), γ-interferon (IFN-γ), IL-4, IL-5 and IL-6. Peptides 13 (AA 61-70), 24 (AA 116-125), 31 (AA 151-160) and 64 (AA 316-325) markedly induced IL-2 production. In particular, peptide 24 (DPLKIKRVHA), which contained I-Ad, I-Ed and I-Ak binding motifs, was the most potent stimulator of IL-2, IFN-γ, IL-4, IL-5 and IL-6 production. Spleen MNC from C3H/HeN mice (H-2k) followed by BALB/c mice (H-2d) immunised with peptide 24 were high responders to peptide 24 in terms of both IFN-γ and IL-4 production, whereas A/J mice (H-2a) and C57BL/6 mice (H-2b) were very low responders, P. gingivalis fimbriae evoked higher delayed-type hypersensitivity (DTH) reactions in B10. D2 (H-2d) and B10. BR (H-2k) mice followed by C57BL/10 (B10, H-2b) and B10. A (H-2a) and in guinea-pigs immunised with the fimbriae and GM-53 in FIA. Thus, the Th1 and Th2 helper T cell cytokine-producing responses and the DTH reactions to P. gingivalis fimbriae in mice are restricted by H-2 haplotype and the amino-acid sequence (AA 116-125) within the fimbrial protein molecule acts as a common stimulator of cytokine production in both Th1 and Th2 cells.

A non-opsonic mechanism of binding and phagocytosis by human neutrophils of Trichophyton mentagrophytes arthroconidia is described. This was in direct contrast to the complement dependency of Candida albicans phagocytosis. Both serum complement and specific antibody to T. mentagrophytes promoted maximal phagocytosis (61% and 40% of neutrophils, respectively, contained arthroconidia). Increasing the ratio of arthroconidia to neutrophils did not increase non-opsonic phagocytosis (18-26%). Phagocytosis of arthroconidia exposed to trypsin in the absence of opsonin was not affected (18%). However, proteinase and chitinase reduced the level of non-opsonic and opsonic phagocytosis to negligible levels (6.3% and 4.5%, respectively). When mannose was added to neutrophils, mannose receptors on the phagocyte membrane were partially blocked when arthroconidia were opsonised, but this did not reduce the level of non-opsonic phagocytosis. The non-opsonic mechanism proposed here may have direct relevance in skin sites poor in opsonins.

A nested polymerase chain reaction (PCR) assay was developed to detect both rat-and human-derived Pneumocystis carinii DNA. The nested PCR product was 125 bp long and was representative of part of the gene coding for the large subunit of mitochondrial ribosomal RNA. Twenty serial blood samples and 24 tissues from six immunosuppressed Sprague-Dawley rats were examined by nested PCR. All lung samples were positive by PCR and Toluidine blue O staining. Buffy coat samples and all the other tissues were PCR-negative during up to 6 weeks of immunosuppression. Thirty-five clinical bronchoalveolar lavage, induced sputum or tracheal aspirate samples from human patients were tested. Twelve of 35 were positive by both PCR and indirect fluorescence assay (IFA) and 19 of 35 were both PCR-and IFA-negative. Four of 35 were IFA-negative but PCR-positive and there were good responses in these patients to specific therapy, indicating that PCR may be more useful than IFA in clinical samples. P. carinii DNA was not detected in three blood samples. The nested PCR is a sensitive and specific DNA amplification method suitable for the routine diagnosis of P. carinii in human respiratory samples.