Lethal activity of miconazole in Neisseria gonorrhoeae grown in pure and mixed cultures

Abstract Low concentrations (e.g. 2 × 10⁻⁶ M) of an imidazole derivative anti-fungal agent, miconazole, were lethal for the Gram-negative, facultative aerobic pathogen Neisseria gonorrhoeae grown either alone or in mixed culture with the yeast Candida albicans . Electron microscopic observation of Neisseria cells exposed to miconazole showed the presence of blebs in the outer wall and areas of separation between the wall and the cytoplasmic membrane. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell lysates did not reveal differences in major outer membrane proteins between the treated and the untreated cells of any one strain. Imidazole derivatives are frequently used in the treatment of candidiasis. Our in vitro results show that low concentrations of one of them, miconazole, can be bactericidal for N. gonorrhoeae , a bacterium that can colonise sites of the human body where Candida is often found.     

Regulation of human polymorphonuclear neutrophil (PMN) activity against Candida albicans by large granular lymphocytes via release of a PMN-activating factor

Using a 24-hr radiolabel microassay developed in our laboratory that measures [3H]glucose uptake in residual Candida, we have identified the effector cells responsible for in vitro inhibition of Candida albicans growth as mainly polymorphonuclear neutrophils (PMN) and monocytes within the human peripheral blood cells. Highly purified T cells and large granular lymphocytes (LGL) that mediate natural killer activity which were obtained by Percoll density gradient centrifugation were found to have no innate activity against C. albicans. The LGL could not be activated by interferon-alpha, interferon-gamma or interleukin 2 to inhibit Candida growth although their K562 tumor cytotoxic activity was readily enhanced by these cytokines. Stimulation with heat-killed C. albicans also did not activate fungal growth inhibitory function in LGL and the supernatant of these activated LGL had no direct fungicidal activity. However, the activated LGL supernatant had the capability to enhance PMN function against C. albicans growth. Addition of recombinant human tumor necrosis factor, affinity-purified interferon-alpha, or interferon-gamma to PMN caused increased antifungal activity in PMN. However, antibodies to these cytokines had only a partial adverse effect on the ability of the activated LGL supernatant to stimulate PMN anti-Candida function. Therefore, the activated LGL supernatant appeared to contain a potent stimulator of PMN function which is as yet unidentified. These data indicate that LGL did not directly mediate anti-Candida activity but could indirectly influence C. albicans growth by activating PMN against the fungi through the release of a specific PMN-activating factor. Our findings therefore add another role to LGL which is the regulation of PMN function, the consequence of which is regulation of fungal immunity.     

The first performance report for the Bio-Rad Dx CT/NG/MG assay for simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in urogenital samples

We evaluated the clinical performance of the Bio-Rad Dx CT/NG/MG assay for the detection of Chlamydia trachomatis, Mycoplasma genitalium and Neisseria gonorrhoeae in urogenital samples in comparison with the Roche COBAS? TaqMan? CT assay for C. trachomatis and an in-house TaqMan PCR assay for M. genitalium. Swab specimens were cultured for N. gonorrhoeae. In this prospective study, urogenital samples were obtained from symptomatic and asymptomatic patients attending the sexually transmitted disease clinic of Bordeaux, France, from January to April 2010. A total of 658 clinical specimens (259 male and 180 female urines, 191 vaginal, 21 endocervical and 7 urethral swabs) from 453 patients were analyzed. The prevalence of C. trachomatis and M. genitalium infections was 8.1% (21/260) and 1.9% (5/260) in men and 10.4% (20/193) and 2.1% (4/193) in women, respectively. The Bio-Rad Dx CT/NG/MG clinical sensitivity was 100% for C. trachomatis and M. genitalium in men and women. In male urine, the clinical specificity was 99.6% for C. trachomatis and 100% for M. genitalium. In women, the specificity was 99.5% for swabs and 100% for urines for detecting C. trachomatis and M. genitalium. All seven N. gonorrhoeae PCR-positive samples were also positive by culture. Patients were co-infected in 5/57 cases (8.8%), with C. trachomatis and M. genitalium in three cases, and C. trachomatis and N. gonorrhoeae in two cases. In conclusion, the Bio-Rad Dx CT/NG/MG assay can be recommended for the simultaneous detection of C. trachomatis, M. genitalium and N. gonorrhoeae in urogenital specimens of symptomatic and asymptomatic individuals.     

Increase in the in vitro susceptibility of Staphylococcus aureus to antimicrobial agents in the presence of Candida albicans.

In experiments with mixed cultures of Staphylococcus aureus and Candida albicans both in the absence and in the presence of 5-fluorocytosine (5-FC), we have observed that (1) there is an inhibition of S. aureus growth in mixed cultures with C. albicans in media supplemented with 1 microgram/mL of 5-fc and that 5-FC has no effect on staphylococci in pure cultures; (2) this inhibition occurred with clinically isolated and laboratory strains and could be reversed by specific metabolites; (3) Staphylococcus aureus was inhibited by filtrates of C. albicans cultures treated with 5-FC and this seemed to be favored by some C. albicans filterable product which can affect the cell wall and the permeability of the staphylococcal cells since they become sensitive to 5-FC; (4) nine other commonly used antimicrobials showed an increased inhibitory activity against S. aureus in mixed cultures with C. albicans; and (5) there is a decrease in the number of precipitating antigens of S. aureus and of the activity of alpha toxin when this species was grown with both C. albicans and 5-FC. Our results indicate that the susceptibility of some species to antimicrobials could be significantly modified in the presence of other species. One cannot exclude that a similar phenomenon could happen in hosts under treatment with antibiotics against infection.     

Determinants of nitric oxide steady-state levels during anaerobic respiration by Neisseria gonorrhoeae

Nitric oxide (NO) is an important host defence molecule that varies its immune stimulatory effects depending on the concentrations at which it is produced, with low concentrations (< 1 microM) promoting an anti-inflammatory host response while higher concentrations (>1 microM) lead to inflammatory responses. Neisseria gonorrhoeae grows anaerobically by anaerobic respiration using nitrite reductase (Nir) to convert nitrite to NO and nitric oxide reductase (Nor) to convert NO to nitrous oxide. As N. gonorrhoeae can both produce and degrade NO, we have begun a study of NO metabolism in this bacterium to understand how gonococcal manipulation of NO concentration may influence the inflammatory response during infection. N. gonorrhoeae has an apparent Nir Km of 33 microM nitrite and an apparent Nor Km of 1.2 microM NO. The maximum specific activities for Nir and Nor were 135 nmoles nitrite reduced per minute per OD600 (pH 6.7) and 270 nmoles NO reduced per minute per OD600 (pH 7.5) respectively. N. gonorrhoeae established a steady-state concentration of NO after nitrite addition that was dependent on the nitrite concentration until saturation at 1 mM nitrite. The NO steady-state level decreased as pH increased, and the ratio of activities of Nir and Nor correlated to the NO steady-state level. When the NO donor DETA/NO was used to simulate host NO production, N. gonorrhoeae also established a NO steady-state level. The concentration of NO at steady state was found to be a function of the concentration of NO generated by DETA/NO, with N. gonorrhoeae reducing the NO from proinflammatory (>1 microM) to anti-inflammatory (approximately 100 nM) concentrations. The implications of the ability of N. gonorrhoeae to maintain an anti-inflammatory NO concentration is discussed in relation to asymptomatic infection in women.     

Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a 'top-down' mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies.     

Amyloid of the Candida albicans Ure2p prion domain is infectious and has an in-register parallel β-sheet structure

Ure2p of Candida albicans (Ure2(albicans) or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2(glabrata)) cannot, even though the Ure2(glabrata) N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2(cerevisiae)) than Ure2(albicans) is. We show that the N-terminal N/Q-rich prion domain of Ure2(albicans) forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p(1-90), we show that this infectious amyloid has an in-register parallel β-sheet structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2(glabrata) does not readily form amyloid, and that formed upon prolonged incubation is not infectious.     

Application of Agglutinins for the Rapid and Accurate Identification of Medically Important Candida Species

Agglutinins have been prepared against the medically important Candida species. Crude antisera to the various species demonstrated intense cross-reactions with heterologous yeastlike fungi as well as with many true yeasts. However, carefully monitored adsorptions of selected antisera allowed the production of six factor sera that proved useful in a slide agglutination test. These six sera permitted the rapid and specific identification of C. guilliermondii, C. krusei, C. parapsilosis, and C. pseudotropicalis. They also allowed the delineation of two groups: (i) C. albicans (type A)-C. tropicalis and (ii) C. albicans (type B)-C. stellatoidea. C. albicans type A could be readily distinguished from C. tropicalis by its ability to form germ tubes in serum. C. stellatoidea could be distinguished from C. albicans type B by its predominantly filamentous growth on a nutritionally deficient medium. The medically important Candida species could be identified within 24 hr by the combined use of serological and morphological procedures.     

Rapid detection of fluoroquinolone resistance by isothermal chimeric primer-initiated amplification of nucleic acids from clinical isolates of Neisseria gonorrhoeae

To ensure a complete response to fluoroquinolone therapy against Neisseria gonorrhoeae infections, rapid susceptibility determinations are required. We assessed a new approach, an isothermal chimeric primer-initiated amplification of nucleic acids (ICAN)/hybrid-chromatography method to detect rapidly fluoroquinolone resistance in N. gonorrhoeae. Comparison of the amplification results with fluoroquinolone minimum inhibitory concentrations (MICs), which were determined by an agar dilution method, showed that the new method accurately determined fluoroquinolone resistance in all ciprofloxacin- and/or gatifloxacin-resistant isolates, but agreed with results based on MICs in only 6 of 8 (75.0%) ciprofloxacin-susceptible and 7 of 12 (58.3%) gatifloxacin-susceptible isolates. Our results suggest that this method can rapidly and reliably detect point mutations in the gyrA gene as well as fluoroquinolone resistance in resistant isolates of N. gonorrhoeae.     

Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation

We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.     

Evaluation of the Gonosticon Dri Dot Test in Females with a Low Incidence of Gonorrhea

A group of 765 females attending a Planned Parenthood Clinic was screened for gonorrhea by inoculating Thayer-Martin plates and Transgrow bottles with specimens from the cervix. Blood was obtained at the same time and tested for anti-gonococcal antibody by using the Gonosticon Dri Dot test. In this low-incidence group, 18 positive cultures were detected by culture on Thayer-Martin plates, whereas Transgrow detected only 15 positive cultures. Of the 18 patients with gonorrhea, 11 exhibited reactive serum (agglutination of the latex particles). In the total population, 64% of the patients had nonreactive serum (no agglutination) and negative cultures; 25% had reactive serum and negative cultures. When this latter group was subdivided on the basis of race, blacks and Latin Americans were found to have a higher incidence of reactive serum with a corresponding negative culture than was found in whites. Patients who were originally culture positive and nonreactive in the Gonosticon Test were retested; three out of four patients retested within 6 to 11 days after the initial screening had converted to a positive Gonosticon test.     

Distribution and evolutionary significance of mitochondrial plasmids in Neurospora spp.

The lethal and mutagenic effects of various mutagens on Neisseria gonorrhoeae were investigated. Lethality studies demonstrated that N. gonorrhoeae was relatively sensitive to ethyl methanesulfonate, UV light, and methyl methanesulfonate. Although N. gonorrhoeae was readily mutated by ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine for the three genetic markers assayed, no increase in the mutation frequency was observed for any of the selective markers after UV irradiation or methyl methanesulfonate treatment. These results suggest that N. gonorrhoeae lacks an error-prone repair mechanism.     

Effect of a polyene antibiotic on growth and phosphate uptake by Candida albicans.

The polyene antibiotic, amphotericin, inhibited phosphate uptake in Candida albicans more strongly than it inhibited growth. Cultures grown from an inoculum of young (2 h) cells were more affected than those inoculated with old (24 h) cells. Thus, the polyene displays a double effect on C. albicans (and presumably on other eukaryotic cells): it interferes with membrane sterols and also inhibits synthesis of a factor (or factors) during growth. Whether this factor(s) interferes with the uptake of the polyene antibiotic or neutralizes its effect by reacting with it remains unsolved.     

Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae

Lactoferrin (LF) and transferrin (TF) are postulated to be important physiological sources of iron for Neisseria gonorrhoeae. A dot binding assay involving the use of gonococcal total membranes derived from cells grown in iron-limited conditions demonstrated the presence of separate receptors for LF and TF. The ligand and functional specificities of these receptors were examined in competition-binding and growth experiments. The results indicate that the LF and TF receptors are highly specific for the human protein, suggesting that this property may be partially responsible for conferring the human host specificity of N. gonorrhoeae.     

Early steps in the biosynthesis of mycobactins P and S

1. Lysine is readily incorporated into mycobactins P and S. Incorporation is into the hydroxamic acid moieties only and is equal in the mycobactic acid and cobactin portions of the molecule. 2. 2-Amino-6-hydroxyaminohexanoic acid is not taken up by cells of Mycobacterium phlei and is not detectable in extracts of cells actively synthesizing mycobactin. 3. The most abundant material derived from lysine that can be detected in such cell extracts is an N(6)-acyl-lysine. Cells grown in the presence of iron contain markedly less of this material than do those grown under conditions of iron deficiency. 4. When added to growing cultures of M. phlei the N(6)-acyl-lysine is readily incorporated into mycobactin. 5. The hydroxy acid of cobactin P is derivable from propionate.     

The fungicidal activity of novel nanoemulsion (X8W60PC) against clinically important yeast and filamentous fungi

Surfactant nanoemulsions are water in oil preparations that proved to have a broad spectrum biocidal activity against a variety of microorganisms including Gram-positive and Gram-negative bacteria, spores and enveloped viruses. These preparations are non-toxic to the skin, mucous membrane and gastrointestinal tissues at biocidal concentrations. In this study, 0.1% of the nanoemulsion designated X8W60PC has shown fungicidal activity against yeast including Candida albicans and C. tropicalis in 15 minutes. C. tropicalis was more sensitive than C. albicans, which required a longer time or a higher concentration of the nanoemulsion to achieve killing. Neutral to slightly alkaline pH was more effective in killing the yeast cells than acidic pH. Using the minimum inhibitory concentration assay, 0.08% of the nanoemulsion was inhibitory to C. albicans, and parapsilosis and filamentous fungi including Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, Aspergillus fumigatus and Fusarium oxysporum. None of the individual ingredients was as effective a fungicidal as the nanoemulsion at equivalent concentration. This shows that the nanoemulsion structure is an important factor in the anti-fungal activity. The X8W60PC has great potential as a topical anti-fungal agent and further investigation into the mechanism of fungicidal action is warranted.