- Volume 52, Issue 3, 2003

Elementary bodies (EBs) of Chlamydia trachomatis serovar E are more toxic to sperm than those from serovar LGV. In this study, lipopolysaccharide (LPS) was prepared from the EBs of both serovars and incubated with human spermatozoa at concentrations that matched the LPS concentration of EBs. The effects of EBs and LPS on sperm motility, viability and acrosomal status were then determined. Sperm motility was measured by computer-assisted sperm analysis and the hypo-osmotic swelling test was used to determine the proportion of dead cells. Acrosomal status was examined using a standard mAb assay. Over a 6 h incubation, LPS from both serovars resulted in a marked reduction in sperm motility (and a concomitant increase in the proportion of dead spermatozoa) in a manner similar to that seen in response to EBs of serovar E. In addition, when sperm were incubated with a range of doses of EBs and LPS, probit analysis revealed that the greater spermicidal effects of EBs from serovar E (when compared with serovar LGV) were not observed when sperm were incubated with LPS from the two serovars. This suggests that the more potent effect of EBs of serovar E cannot be explained entirely by differences in the composition of LPS. Interestingly, Escherichia coli LPS was required in doses 500 times more concentrated than chlamydial LPS in order to kill a similar proportion of sperm, suggesting that bacterial LPSs may differ in their spermicidal properties. However, that chlamydial LPS was spermicidal was demonstrated by the use of polymyxin B (a polycationic antibiotic known to neutralize LPS effects), confirming that the effects observed were primarily a result of LPS activity.

The phosphorylation process is an important mechanism of cell signalling and regulation. It has been implicated recently in defence strategies against a variety of pathogens that alter host signalling pathways in order to facilitate their invasion and survival within host cells. In this study, the involvement of protein kinases (PKs) has been investigated in attachment and invasion by the pathogenic fungus Fonsecaea pedrosoi within epithelial cells and macrophages. The use of the PK inhibitors staurosporine, genistein and calphostin C prior to infection provided significant information about the role played by PKs in the F. pedrosoi–host cell interaction. All three PK inhibitors could reduce cell invasion by F. pedrosoi significantly. Pre-treatment of macrophages, epithelial cells or conidia with PK inhibitors decreased fungus invasion, and this effect could be overcome by okadaic acid, a phosphatase inhibitor. Immunofluorescence assays showed that tyrosine residues were phosphorylated in the first step of the interaction, while serine residues were phosphorylated in the subsequent step of entry of the parasite into the host cell. These results suggest that both host-cell and conidium PK activities are important in the interaction process, playing a significant role in cell invasion.

Helicobacter pylori is an aetiological agent of gastric disease. Although the role of urease in gastric colonization of H. pylori has been shown, it remains unclear as to where urease is located in this bacterial cell. The purpose of this study was to define the urease-associated apparatus in the H. pylori cytoplasm. H. pylori was incubated at both a neutral and an acidic pH in the presence or absence of urea and examined by double indirect immunoelectron microscopy. The density of gold particles for UreA was greatest in the inner portion of the wild-type H. pylori cytoplasm at neutral pH but was greatest in the outer portion at acidic pH. This difference was independent of the presence of urea and was not observed in the ureI-deletion mutant. Also, the eccentric shift of urease in acidic pH was not observed in UreI. After a 2 day incubation period at acidic pH, it was observed that the urease gold particles in H. pylori assembled and were associated with UreI gold particles. Urease immunoreactivity shifted from the inner to the outer portion of H. pylori as a result of an extracellular decrease in pH. This shift was urea-independent and UreI-dependent, suggesting an additional role of UreI in urease-dependent acid resistance. This is the first report of the intracellular transport of molecules in bacteria in response to changes in the extracellular environment.

The importance of hens eggs as a source of specific antibodies (IgY) is well recognized. The protective effect of IgY obtained from hens immunized with Helicobacter pylori whole-cell lysate has been reported for the control of H. pylori infection. However, IgY produced by whole-cell lysates presents the possibility of cross-reactivity with other bacteria, including the normal human flora, and this could decrease the efficiency of IgY. In the present study, the immunodominant proteins of H. pylori with reactivity to H. pylori-specific IgY (IgY-Hp) were identified. IgY obtained from hens immunized with various fractions of H. pylori proteins was isolated and purified, titres of IgY-Hp against H. pylori were determined and cross-reactivity between IgY-Hp and normal human bacteria was examined by Western blot analysis. Finally, immunodominant H. pylori proteins were identified by LC/MS analysis. IgY obtained 2 months after immunization with H. pylori whole-cell lysate showed the highest antibody titre. Five immunodominant proteins were identified that were strongly reactive to IgY-Hp: urease β-subunit (62 kDa), heat-shock protein 60 (60 kDa), urease α-subunit (26 kDa), probable peroxiredoxin (22 kDa) and probable thiol peroxidase (18 kDa). Immunization of hens with the immunodominant proteins identified would produce a more specific IgY against H. pylori.

In this study, the immune-modulatory and vaccine effects of using an interleukin (IL)-18 expression plasmid as a genetic adjuvant to enhance DNA vaccine-induced immune responses were investigated in a mouse herpes simplex virus 1 (HSV-1) challenge model. BALB/c mice were immunized by three intramuscular inoculations of HSV-1 glycoprotein D (gD) DNA vaccine alone or in combination with a plasmid expressing mature IL-18 peptide. Both the serum IgG2a/IgG1 ratio and T helper 1-type (Th1) cytokines [IL-2 and interferon (IFN)-γ] were increased significantly by the co-injection of the IL-18 plasmid compared with the injection of gD DNA alone. However, the production of IL-10 was inhibited by IL-18 plasmid co-injection. Furthermore, IL-18 plasmid co-injection efficiently enhanced antigen-specific lymphocyte proliferation and the delayed-type hypersensitivity response. When mice were challenged with HSV-1 at the cornea, co-injection of IL-18 plasmid with gD DNA vaccine showed significantly better protection, manifested as lower corneal lesion scores and faster recovery. These experiments indicate that co-injection of an IL-18 plasmid with gD DNA vaccine efficiently induces Th1-dominant immune responses and improves the protective effect against HSV-1 infection.

Due to the limitations of classical methods for the detection of systemic fungal infections and the high mortality rates associated with these infections, it has become essential to develop a quick, sensitive and specific detection assay. By using the Idaho Technologies Light-Cycler system, a qualitative real-time PCR system has been developed for the detection of the leading causes of systemic infection within the genus Candida. The sensitivity of the assay was comparable to previously described PCR methods (1–5 c.f.u. ml−1) and, by the use of a single Candida probe, it was able to detect, but not differentiate between, seven species of Candida (Candida albicans, Candida dubliniensis, Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis and Candida tropicalis). Single-round amplification on the Light-Cycler allowed rapid turn-around of clinical samples (within one working day) and it was shown to be more sensitive than classical procedures, exposing 39 possible systemic infections that were not detected by blood culture.

Sixty-three rifampicin-resistant (Rifr) isolates of Mycobacterium tuberculosis from Kaohsiung, Taiwan, were analysed for mutations in the core region (69 bp, codons 511–533) of the rpoB gene. Some 84.1 % (53/63) of the resistant isolates showed mutations in this region, especially in codons 531 (41.5 %), 526 (18.9 %), 516 (15.1 %) and 533 (7.5 %). Five novel alleles of a total of 16 different types of mutations were identified in Rifr isolates. Ten Rifr isolates (15.9 %) exhibited no mutations in the core region of rpoB. Also, they did not show mutations in another 365 bp fragment (codons 99–220) of rpoB. The agar proportion method was used to determine the relationship between the degree of rifampicin resistance and alterations in the core region of rpoB. The results revealed that the mean MIC was 92.38 μg ml−1 for the 53 isolates with a mutation in the core region, whereas the mean MIC of the other 10 isolates without mutations was only 24.8 μg ml−1. This indicates that the isolates with mutations in the core region had higher levels of resistance than those without mutations in this region. IS6110 restriction fragment length polymorphism (RFLP) was used for typing of 55 Rifr M. tuberculosis isolates. Isolates contained two to 19 copies of IS6110, with sizes ranging from 600 to 16 000 bp. The majority (85 %) contained six to 16 copies. No strains lacking IS6110 were found. A total of 54 of 55 RFLP types were defined at the 90 % similarity level. The observation of varied IS6110-associated banding patterns indicates that an outbreak of drug-resistant tuberculosis did not occur in this area.

The inhibitory effect of human immunodeficiency virus (HIV) proteinase inhibitors amprenavir and saquinavir and antifungal agents terbinafine, ketoconazole, amphotericin B and ciclopiroxolamine on aspartyl proteinases (Saps) secreted by Candida albicans was tested in an in vitro spectophotometric assay. As expected, both HIV proteinase inhibitors showed a significant inhibitory effect on Sap activity, which was comparable to that of the classical aspartyl proteinase inhibitor pepstatin A (P < 0.001). Antifungal drugs such as ketoconazole, terbinafine and amphotericin B had no, or only minor, inhibitory effects on proteolytic activity. In contrast, a significant reduction in Sap activity could be demonstrated during treatment with the antifungal agent ciclopiroxolamine (P < 0.001). These results point to a multiple effect of this antimycotic agent and might explain the reduced adherence of C. albicans to human epithelial cells at subinhibitory doses.

Penicillin has been the antimicrobial of choice for the treatment of Streptococcus pyogenes infections for almost six decades. Although penicillin-resistant isolates have not been described to date, clinical failures have been reported after treatment with β-lactams. In this study, we analysed the antimicrobial susceptibility and genetic diversity of S. pyogenes isolates obtained from healthy carriers or patients in different cities in the south and south east of Brazil. The MICs were determined for penicillin and seven other antimicrobials. Penicillin tolerance was also investigated. Genetic diversity was analysed by PFGE after SmaI fragmentation of the genomic DNA. All 211 isolates tested were susceptible to penicillin (MIC 0.0025–0.02 mg l−1). Four isolates were moderately penicillin-tolerant (MBC/MIC = 16 mg l−1). Most of the other drugs tested were very active against the strains examined, except for tetracycline, to which 50 % of strains were resistant. We also found extensive genetic diversity, in that 60 different patterns were recognized in the 96 strains studied. Indeed, we found no correlation between tetracycline resistance and clonality. Despite this diversity, some PFGE patterns persisted for up to 18 years and specific clone types were spread over different geographical locations

Clostridium difficile-associated disease continues to be a major problem in hospitals and long-term care facilities throughout the developed world. Administration of certain antibiotics such as amoxycillin, oral cephalosporins and clindamycin is associated with the greatest risk of developing C. difficile disease. The two antibiotics used for treatment of C. difficile disease are vancomycin and metronidazole, to which there is currently very little resistance. Randomly selected isolates (186) from 90 patients being investigated during an 18-month epidemiological study into the disease were tested for their susceptibility to vancomycin, metronidazole, amoxycillin, clindamycin, cefoxitin and ceftriaxone by the NCCLS agar dilution method. There was a narrow range of MIC for the two treatment agents (vancomycin and metronidazole), from 0.5 to 4 μg ml−1, with no evidence of resistance. All strains were resistant to cefoxitin (MIC 64–256 μg ml−1), the antibiotic used in most selective media. All strains were of similar sensitivity to amoxycillin (MIC90= 4 μg ml−1). Most strains were resistant to ceftriaxone (MIC ≥ 64 μg ml−1) or of intermediate resistance (MIC ≥ 32 μg ml−1), with only two sensitive strains (MIC 16 μg ml−1). Clindamycin resistance was common, with 67 % of strains resistant (MIC ≥ 8 μg ml−1), 25 % with intermediate resistance (MIC ≥ 4 μg ml−1) and only 8 % sensitive (MIC ≤ 2 μg ml−1). Twelve isolates from six different patients had very high resistance to clindamycin (MIC ≥ 128 μg ml−1). Multiple isolates from the same patient, taken at different times, showed changes in susceptibility patterns over time. The only major change in susceptibility over the time-period was in clindamycin resistance; some strains appeared to become more resistant while others became less resistant. No differences were seen in the MIC50 and MIC90 of the different S-types of C. difficile identified, although some S-types were present in very small numbers. There was no correlation between the antibiotics prescribed and susceptibility.

Chlamydia pneumoniae is a frequent causative agent of acute respiratory disease. To assess whether C. pneumoniae plays a role in persistent cough, the prevalence of C. pneumoniae infection in adult patients with persistent cough was investigated. Nasopharyngeal swabs and serology samples from 366 adult patients with a persistent cough lasting in excess of 2 weeks and 106 control subjects were analysed for bacterial isolation and by PCR. C. pneumoniae was isolated from two patients and from none of the controls and was detected by PCR in 20 patients and one control. Serological evidence of acute C. pneumoniae infection was present in 24 patients but in none of the controls. Of these 20 patients who were positive by culture and/or PCR, three were still positive by PCR after 2 weeks of treatment with clarithromycin and symptoms either continued or relapsed. However, when patients were treated with clarithromycin for 5–6 weeks, their symptoms disappeared completely and the results of their cultures and/or PCR for C. pneumoniae became negative. These data suggest that C. pneumoniae infection may cause persistent cough in adults. Furthermore, these data also indicate that it may be necessary to eradicate the organism when C. pneumoniae is detected by culture and/or PCR in patients with persistent cough.

A case of disseminated cryptococcosis caused by Cryptococcus neoformans var. grubii is presented in a male diabetic who had AIDS. The diagnosis was based upon the isolation and identification of the aetiological agent from a lymph-node biopsy, cerebrospinal fluid and sputum. The isolate formed spherical, encapsulated yeast cells, produced cherry-brown colonies on niger-seed agar, grew on canavanine-glycine-bromothymol blue (CGB) medium, changing its colour from greenish yellow to blue, and hydrolysed urea weakly in the presence of 100 μM EDTA. The strain was unable to assimilate d-proline and, serologically, it was untypable. The identity of the isolate as C. neoformans var. grubii, serotype A, possessing a mating-type allele Aα, was confirmed by crossing with standard laboratory test strains and by performing PCR with the mating-type α allele-specific primer of the STE12 gene and with serotype (A and D)- and mating type (a and α)-specific primers of the STE20 gene. To the best of our knowledge, this is the first report of disseminated cryptococcosis in an AIDS patient caused by a canavanine-resistant strain of C. neoformans var. grubii, serotype A, possessing mating type allele Aα; the strain is probably a hybrid. The report suggests that, in the absence of a clear-cut serotyping result, a positive CGB reaction alone is not sufficient for intervarietal discrimination and additional confirmatory evidence is required.