- 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.

The immunogenicity of several Brucella melitensis cell-wall (CW) fractions was tested in BALB/C mice. These CW fractions were smooth lipopolysaccharide (S-LPS) fraction from smooth (S) B. melitensis strain 16M, sodium dodecyl sulphate-insoluble (SDS-I) CW fraction from B. melitensis strain 16M (S) undigested or digested with pepsin, and SDS-I CW fraction from rough (R) B. melitensis strain H38. The B. melitensis SDS-I CW fraction contained two major outer-membrane proteins (OMPs) of 25–27 kDa and 31–34 kDa, peptidoglycan (PG) and a small quantity (1.5%) of LPS. One month after immunisation, mice were challenged with virulent B. melitensis strain H38 (S) and Brucella spleen counts were recorded on days 28 and 49 after challenge. Before challenge, as measured by ELISA, the highest antibody responses to S-LPS were observed in mice immunised with SDS-I CW fraction from B. melitensis strain 16M (S), whether digested with pepsin or undigested. All immunised mice, except those immunised with the SDS-I CW fraction from the R strain, showed higher IgG1 than IgG2a antibody responses to S-LPS (IgG1:IgG2a ratio 3.64–7.71). Antibody responses to the 25–27-kDa OMP were very low, with the highest responses in the mice immunised with the SDS-I CW fraction from the R strain. These results indicated that, in BALB/c mice, these CW fractions probably induced Th2-dependent more than Th1-dependent antibody responses. The highest protection levels were produced by immunisation with the pepsin-digested SDS-I CW fraction from B. melitensis strain 16M (S) and by passive transfer of an anti-S-LPS monoclonal antibody (MAb 2E11; M epitope specificity). S-LPS fraction from B. melitensis strain 16M (S) or SDS-I CW fraction from B. melitensis strain H38 (R) did not induce significant protection. The absence of protection by S-LPS fraction could be explained by the low antibody titres induced by it. On day 49, protection conferred by pepsin-digested SDS-I CW fraction from B. melitensis strain 16M (S) was significantly higher (p < 0.05) than by the untreated SDS-I CW fraction from the corresponding strain. Furthermore, the level of protection in mice immunised with the SDS-I CW fraction from the R strain and which were also passively immunised, 1 day before challenge, with the S-LPS specific MAb was lower than in mice treated with the anti-S-LPS MAb alone. These results indicate that immunity induced by the S strain B. melitensis SDS-I CW fraction is probably mainly S-LPS dependent and that even one or more protein components of this fraction may play an antagonistic role to immunity conferred by S-LPS.

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.

Serotyping of 50 streptococcal strains of serological group B isolated from human clinical specimens in Chile revealed mainly the serotypes Ia, II and III, either alone or in combination with protein antigens c or R. No significant difference in serotype distribution was detected between group B streptococci isolated from cervical swabs from clinically healthy women and those isolated from various pathological processes. Determination of antibiotic susceptibility of the bacteria demonstrated resistance to tetracycline and minocycline in 29 isolates. All 29 tetracycline-resistant cultures hybridised with a gene probe for tet(M). Again, no differences were detected between the group B streptococcal isolates of various origins.

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.

Previous studies with three isolates from diarrhoeal stools suggested that Providencia alcalifaciens is an invasive enteric pathogen that also causes actin condensation in infected cells. These findings were extended in the present study with a further 14 diarrhoeal stool isolates of P. alcalifaciens and HEp-2 cell monolayers for invasion assays. Studies on invasion characteristics with two selected isolates suggested that P. alcalifaciens required prior growth at 37°C for better invasion. Invasion and actin condensation were inhibited by an agent that inhibits microfilament formation, but not by agents that inhibit receptormediated endocytosis, microtubule formation, endosome acidification or receptor recycling. In time-course assays with HEp-2 cell monolayers maintained in medium containing gentamicin, P. alcalifaciens showed a small degree of multiplication after invasion of the cells, but viable bacteria could not be recovered over a 24-h period although the integrity of the cell monolayer was preserved during this period.

To elucidate whether Chlamydia trachomatis and C. pneumoniae infections occur to a significant extent in monocytes-macrophages, the human monocytic cell line, U-937, was infected with C. trachomatis L2 or C. pneumoniae TW-183. Chlamydial DNA and genus-specific antigens of the lipopolysaccharides (LPS) in epitopes of the chlamydial cell wall were detected from C. trachomatis L2-inoculated monocytes over a period of 150 days after inoculation and from the C. pneumoniae TW-183-inoculated cells during a period of 14 days. C. trachomatis-infected U-937 cells expressed significantly lower levels of CD4+, CD45RA+, CD11b+ and CD33+ cells, determined by flow cytometry, than control uninoculated cells on the seventh day after inoculation and they expressed a slightly increased level of CD4+ cells and lower levels of CD45RA+ and CD11b+ cells on the 14th day after inoculation. C. pneumoniae-infected U-937 cells expressed significantly lower levels of CD4+, CD45RA+, CD11b+ and CD33+ cells than controls on the seventh day after inoculation and an increased level of CD4+ and a lower level of CD45RA+ cells on the 14th day after inoculation. Unlike infection with C. trachomatis L2 strain, chronic persistent infection with C. pneumoniae appears not to occur in monocytes-macrophages.

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.

Eighteen strains of Aeromonas hydrophila from patients with bacteraemia were investigated for possible virulence factors. Cytotoxin and haemolysin were produced by all strains, whereas cholera toxin-like factor was produced by 33% of strains only. Enterotoxin production was not detected. Haemagglutination of guinea-pig, fowl and rabbit erythrocytes was demonstrated by 83%, 67% and 61% of strains, respectively. Fucose- and mannose-sensitive haemagglutinins were predominant. None of the strains agglutinated sheep erythrocytes. Extrachromosomal DNA was detected in 17 strains, 16 of which had a plasmid (3.6-5.1 MDa), the majority being between 4.6 and 5.1 MDa.

There are several methods for the detection of haemolytic activity in campylobacters. However, we found the haemolytic effect of campylobacters on conventional blood agar plates to be variable, inconsistent and difficult to interpret. Blood agarose plates showed campylobacter haemolytic activity more clearly. The incubation conditions (temperature and gaseous) appear to be important for the expression of this activity. Ninety four percent of the Campylobacter isolates examined were found to be haemolytic by the microplate assay with minimal haemolytic units that ranged from 1 to 64. Haemolytic activity was detected only from live bacterial cultures and not from any of the 50 bacterial culture supernates, which suggests that campylobacters may possess a cell-associated haemolysin. The identification of such haemolytic activity in a large number of campylobacters (94%) suggests its potential role as a virulence factor in campylobacter gastroenteritis.

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.