Modification of Cycloserine Cefoxitin Fructose Agar to Suppress Growth of Yeasts from Stool Specimens

Cycloserine cefoxitin fructose agar is a widely used selective isolation medium for Clostridium difficile from stool specimens. Yeasts often colonize in the intestine of C. difficile disease patients and, if colonized heavily, pure culture of C. difficile can be delayed. The aim of this study was to modify cycloserine cefoxitin fructose agar to suppress the growth of yeasts. Antimicrobial activities of three commonly available antifungal agents were tested against recent clinical isolates of Candida species. Amphotericin B was most active in inhibiting all isolates by ≤0.5 mg/L concentration. Cycloserine cefoxitin fructose agar was modified by adding 2 mg/L of amphotericin B. Serial ten-fold dilution of stool specimens from 126 suspected C. difficile -associated diarrhea patients were cultured both on cycloserine cefoxitin fructose agar plates and modified agar plates. Yeasts grew from 60 specimens on cycloserine cefoxitin fructose agar, but none grew on the modified medium. Growth of C. difficile was detected from 37 and 39 of 126 specimens on cycloserine cefoxitin fructose agar and modified medium, respectively. The number of C. difficile colonies was similar on both media. In conclusion, 2 mg/L of amphotericin B supplementation to cycloserine cefoxitin fructose agar can facilitate the isolation of C. difficile from stool specimens which are densely colonized with yeasts.     

Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7 degrees C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca²⁺ concentrations in the mutant strain Aspergillus awamori 66A containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity of sporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 μM) stimulated spore germination: the number of germinating spores was 2–3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 μM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca²⁺ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 μM) were shown to cause an instantaneous increase in Ca²⁺ concentrations compared to the control and lower amphotericin concentration (2.5 μM). Ca²⁺ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.     

Cultural and Biochemical Characteristics of Mycobacterium Paratuberculosis Isolated from Goats in Norway

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant. The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria. Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

No evidence of oxidant events in amphotericin B cytotoxicity versus L. infantum promastigotes.

Amphotericin B is used for the treatment of systemic mycoses and visceral leishmaniasis. The objective of our study was to evaluate the impact of catalase, ascorbic acid and ketoconazole on the amphotericin B toxicity towards Leishmania promastigotes membrane by two flow cytometric tests, the membrane potential assay using a cationic dye, [DiOC5(3)], and the membrane permeability test using propidium iodide. The collapse of membrane potential appeared at amphotericin B concentrations weaker than those assessed by the membrane permeability test. The binding of amphotericin B to membrane sterol was not modified by catalase or ascorbic acid whereas amphotericin B-induced growth inhibition could be modulated by these products. The permeabilizing effect of amphotericin B on parasite membrane was strongly reduced in the presence of ketoconazole. These results confirmed the pore hypothesis of amphotericin B action and suggested that flow cytometric methods constituted a valuable alternative to conventional methods for assessing the effect of drugs on cellular membrane and evaluating parasite susceptibility to polyene antibiotics.     

Occurrence of Mycobacterium avium subsp. paratuberculosis in untreated water in Northern Ireland

Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.     

Persistence of Mycobacterium paratuberculosis during manufacture and ripening of cheddar cheese

Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 10(4) to 10(5) CFU/ml) and low (10(1) to 10(2) CFU/ml) inocula of three different Mycobacterium paratuberculosis strains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival of M. paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed in M. paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease in M. paratuberculosis CFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log(10)) of M. paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period. The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination, M. paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS. M. paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initial M. paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum.     

Effect of amphotericin B on cholesterol-containing liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. A refinement of the model for the formation of pores by amphotericin B in membranes

(1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes.     

Effect of the Current Antimicrobial Therapeutic Strategy on Fungal Colonization in Patients with Hematologic Malignancies

A “quasi-experimental” trial was carried out to investigate the effect of three antimicrobial regimens on oral and fecal yeast colonization in patients with hematologic malignancies. Fifty-four patients received ciprofloxacin and oral amphotericin B (group 1); 45 received ceftazidime, amikacin, vancomycin, and oral amphotericin B (group 2); and 30 received ceftazidime, amikacin, vancomycin, and intravenous amphotericin B (group 3). The oral yeast isolation rate showed a decrease in group 1 (from 59.3% to 40.7%) and group 3 (from 56.7% to 46.7%), and a marked increase in group 2 (from 51.1% to 84.4%). All the groups showed a reduction in their fecal yeast isolation rate. An overgrowth of Candida parapsilosis, C. krusei, and C. tropicalis was observed in all the groups, but it was much higher in group 2. Our findings provide evidence that ceftazidime, amikacin, and vancomycin, given with oral amphotericin B, induce an overgrowth/persistence of Candida species in the mouth and gut, which might be attributable to inclusion of vancomycin. Treatment with intravenous amphotericin B has at least the capacity of counterbalancing yeast proliferation induced by that antibacterial regimen. Received: 27 November 1995 / Accepted: 22 January 1996     

Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity

The increase in the use of antifungal agents for prophylaxis and therapy has led to the development of antifungal drug resistance. Drug combinations may prevent or delay resistance development. The aim of the present study was to investigate whether naturally and designed cationic antifungal peptides act synergistically with commonly used antimycotics. No enhanced activity was found upon addition of dhvar4, a designed analogue of the human salivary peptide histatin 5, or PGLa to fluconazole or 5–flucytosine, respectively. In contrast, strong synergism of amphotericin B with the peptides was found against several Aspergillus, Candida, and Cryptococcus strains, and against an amphotericin B-resistant C. albicans laboratory mutant in the standardised broth microdilution assays according to the NCCLS standard method M27–T. Amphotericin B showed synergism with dhvar5, another designed analogue of histatin 5, and with magainin 2 against all seven tested strains. Combinations of amphotericin B with histatin 5, dhvar4, and PGLa showed synergism against four of the seven strains. The growth inhibitory activity of amphotericin B was enhanced by sub-MIC concentrations of peptide, but its haemolytic activity remained unaffected, suggesting that its cytotoxicity to host cells was not increased and that peptides may be suitable candidates for combination therapy.     

New triplex real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan(mgb) and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10(2) CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.     

Effect of amphotericin B added to one side of a membrane

Amphotericin B which was added to the one side of a membrane in aqueous solutions with low pH 3.0 sharply increases the membrane conductance by the mechanism of channel forming. Long living ionic channels (20 sec) with large conductance dispersion (2-20 Ps) were found at the concentration 2 X 10(-8) M. Amphotericin B concentration which is necessary for obtaining single channels does not depend on cholesterol concentration in the membrane-forming solution in the range from 2 to 10 mg/ml. The effect of amphotericin B on the membrane depends on the phospholipid concentration in the membrane. The one-side selective permeability is the same as one under the symmetric action of the antibiotic. It is assumed that amphotericin B induced the membrane conductance by the mechanism of half-pores formation.     

Effect of amphotericin B on the metabolism of Aspergillus fumigatus

Action of amphotericin B on the growth and metabolism of Aspergillus fumigatus has been investigated. The fungus proved to be very sensitive to amphotericin B, showing complete inhibition of growth at 0.5 units/ml. Amphotericin B suppressed the exogenous and endogenous respiration and glycolysis of A. fumigatus as well as the assimilation of various glycolysis and TCA cycle intermediates. Addition of cations and cholesterol failed to reverse the action of amphotericin B. The treated mycelium released a variety of cellular constituents and it is inferred that the antibiotic effects the permeability of A. fumigatus cells. In experiments with ³²P labelled mycelium phosphorus compounds leached out in concentrations which were dependent on the antibiotic dose, period of contact, incubation temperature and metabolic state of the fungus.     

Voriconazole, Combined with Amphotericin B, in the Treatment for Pulmonary Cryptococcosis Caused by C. neoformans (Serotype A) in Mice with Severe Combined Immunodeficiency (SCID)

Cryptococcosis is a subacute or chronic systemic mycosis with a cosmopolitan nature, caused by yeast of the genus Cryptococcus neoformans. The model of systemic cryptococcosis in mice with severe combined immunodeficiency (SCID) is useful for immunological and therapeutic study of the disease in immunodeficient hosts. Amphotericin B, fluconazole and flucytosine are the drugs most commonly used to treat cryptococcosis. Voriconazole is a triazole with high bioavailability, large distribution volume, and excellent penetration of the central nervous system. The objective of this study was to evaluate treatment with amphotericin B (AMB), voriconazole (VRC), and AMB, used in combination with VRC, of experimental pulmonary cryptococcosis in a murine model (SCID). The animals were inoculated intravenously (iv) with a solution containing 3.0 × 10(5) viable cells of C. neoformans ATCC 90112, (serotype A). Treatments were performed with amphotericin B (1.5 mg/kg/day), voriconazole (40.0 mg/kg/day) and AMB (1.5 mg/kg/day) combined with VRC (40.0 mg/kg/day); began 1 day after the initial infection; were daily; and lasted 15 days. Evaluations were performed using analysis of the survival curve and isolation of yeast in the lung tissue. There was a significant increase in survival in groups treated with AMB combined with VRC, compared with the untreated group and groups receiving other treatments (P < 0.05). In the group treated only with VRC and AMB combined with VRC, there was a significant reduction (P < 0.05) in the isolation of C. neoformans in lung tissue. Amphotericin B combined with voriconazole may be an effective alternative to increasing survival and may reduce yeast in the lung tissue of mice with pulmonary cryptococcosis and SCID.     

Amphotericin B-induced apoptosis and cytotoxicity is prevented by the Na+, K+, 2Cl(-)-cotransport blocker bumetanide

Amphotericin B is the most commonly used antifungal drug although it exhibits poor effectiveness and considerable toxicity during treatment. It acts as a ionophore inducing cellular potassium efflux. The efflux of potassium, which is necessary for cell shrinkage during apoptosis, is counteracted by increased inward pumping of potassium ions. Modulation of potassium pump activity could therefore interact with programmed cell death depending on the nature of the disruption of cellular potassium homeostasis and subsequently affect the cytotoxicity of various drugs. We explored the role of apoptosis in amphotericin B-induced cytotoxicity in a mesothelioma cell line (P31) and investigated the role of K+ influx inhibitors of Na+, K+, ATPase and Na+, K+, 2Cl(-)-cotransport in these processes. Clone formation was used to determine the cytotoxicity of amphotericin B, ouabain (Na+, K+, ATPase blocker), and bumetanide (Na+, K+, 2Cl(-)-cotransport blocker), alone or in combination. Apoptosis was estimated by quantifying free nucleosomes. Amphotericin B (3.2 micromol/L, 3 mg/L) per se reduced the percentage of surviving clones to 64% and increased the number of nucleosomes by 31% compared to untreated control. When ouabain (100 micromol/L) was added to amphotericin B a less than additive effect on clone formation was seen but no reduction of nucleosomes was noted. Bumetanide (100 micromol/L) per se was not cytotoxic but increased cellular nucleosome expression. Bumetanide eradicated amphotericin B-induced reduction of formed clones and generated nucleosomes. In conclusion, the induction of apoptosis seems to be of significant importance in amphotericin B-induced cytotoxicity. Amphotericin B-induced cytotoxicity and apoptosis was eradicated by the Na+, K+, 2Cl(-)-cotransport inhibitor bumetanide. The changes of cellular K+ fluxes induced by bumetanide combined with amphotericin B needs further elucidation. Bumetanide could possibly be used in antifungal therapy to increase amphotericin B effectiveness doses without increasing its adverse effects.     

Testing for Mycobacterium avium subsp. paratuberculosis infection in asymptomatic free-ranging tule elk from an infected herd

Forty-five adult tule elk (Cervus elaphus nannodes) in good physical condition were translocated from a population located at Point Reyes National Seashore, Marin County (California, USA), to a holding pen 6 mo prior to release in an unfenced region of the park. Because infection with Mycobacterium avium subsp. paratuberculosis (Mptb) had been reported in the source population, the translocated elk underwent extensive ante-mortem testing using three Johne's disease assays: enzyme linked immunosorbent assay (ELISA); agar gel immunodiffusion assay (AGID), and fecal culture. Isolation of Mptb was made from fecal samples in six of 45 elk (13%). All AGID results were negative while ELISA results for 18 elk (40%) were considered elevated. Elevated ELISA results or Mptb isolation from fecal samples were obtained for 22 of 45 elk (49%); these elk were euthanized and necropsied. Mycobacterium avium subsp. paratuberculosis was isolated from tissue in 10 of 22 euthanized elk (45%); of these 10 cases of confirmed infection, eight had elevated ELISA results (80%) and four were fecal culture positive (40%). One of 10 cases had histopathologic lesions consistent with Mptb infection. Mycobacterium avium subsp. paratuberculosis was also isolated from tissue from one of eight fetuses sampled. The number of tule elk found to be infected was unexpected, both because of the continued overall health of the source herd and the normal clinical status of all study animals.     

Antifungal pharmacodynamic characteristics of amphotericin B against Trichosporon asahii, using time-kill methodology

We determined the MIC of amphotericin B against 45 Trichosporon asahii isolates from various clinical and environmental sources, and used in vitro time-kill methods to characterize the relationship between amphotericin B concentrations and MIC for four representative T. asahii isolates. Amphotericin B had concentration-dependent antifungal activity. MICs ranged from 0.5 to 16 microg/ml, and most T. asahii isolates (76%, 34/45) were inhibited at safely achievable amphotericin B serum concentrations (< or = 2 microg/ml). However, 40% (18/45) of isolates were not killed at these concentrations (MFCs from 1.0 to 32 microg/ml). At concentrations > or = 2 x MIC, amphotericin B exhibited fungicidal activity (< 99.9% reduction in CFU) over a 12-hr time-period; the maximal effect was achieved at > or =4 x MIC. Susceptibility testing confirmed the resistance of T. asahii to amphotericin B, and in vitro pharmacodynamic results also suggest that amphotericin B is not suitable therapy for T. asahii infection.     

Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium

Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20 degrees C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log(10) concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.     

Polymerase Chain Reaction for the Detection of Chlamydia psittaci in the Feces of Budgerigars

The polymerase chain reaction (PCR) targeting the ompA gene of Chlamydia psittaci was evaluated for its ability to detect chlamydiae in fecal specimens of budgerigars as compared with isolation procedures using cell culture and embryonated egg inoculations. Several procedures for PCR template DNA preparation were compared so as to determine their detection levels for chlamydiae propagated in cell culture in the presence of fecal materials. Tween-20 and proteinase K treatments followed by centrifugation of the template DNA were found to be an appropriate procedure for DNA preparation for primary PCR. Subsequent nested PCR was shown to detect 4.8 IFU/ml or 84 particles/ml of chlamydiae. Chlamydiae in 50 fecal specimens from apparently healthy budgerigars were examined by nested PCR and several other methods. Nested PCR detected chlamydiae at a higher rate (12/50, 24%) than the isolation procedure in embryonated eggs (6/50, 12%). Primary PCR combined with the isolation procedure in cell culture gave a detection rate (5/50, 10%) similar to that of isolation from embryonated eggs. Detection rates by primary PCR (1/50, 2%) and in cell culture (0%) were inferior to the other procedures.     

The antifungal drug amphotericin B promotes inflammatory cytokine release by a Toll-like receptor- and CD14-dependent mechanism

Amphotericin B is the most effective drug for treating many life-threatening fungal infections. Amphotericin B administration is limited by infusion-related toxicity, including fever and chills, an effect postulated to result from proinflammatory cytokine production by innate immune cells. Because amphotericin B is a microbial product, we hypothesized that it stimulates immune cells via Toll-like receptors (TLRs) and CD14. We show here that amphotericin B induces signal transduction and inflammatory cytokine release from cells expressing TLR2 and CD14. Primary murine macrophages and human cell lines expressing TLR2, CD14, and the adapter protein MyD88 responded to amphotericin B with NF-kappaB-dependent reporter activity and cytokine release, whereas cells deficient in any of these failed to respond. Cells mutated in TLR4 were less responsive to amphotericin B stimulation than cells expressing normal TLR4. These data demonstrate that TLR2 and CD14 are required for amphotericin B-dependent inflammatory stimulation of innate immune cells and that TLR4 may also provide stimulation of these cells. Our results provide a putative molecular basis for inflammatory responses elicited by amphotericin B and suggest strategies to eliminate the acute toxicity of this drug.