An in vitro study of the activity of telithromycin against mobile and cystic forms of Borrelia afzelii.

In this study the new ketolide telithromycin was tested in vitro against motile and cystic forms of Borrelia afzelii, one of the species of Borrelia burgdorferi sensu lato. Acridine orange staining, dark field microscopy, and transmission electron microscopy were the techniques used to study the influence of telithromycin on the bacteria. The activity was unexpectedly high, 0.0003 microg/ml < MBC < or = 0.0006 microg/ml for the mobile forms after 7 days of incubation at 34 degrees C. MIC was < 0.00015 microg/ml. It is likely that the agent works bacteriostatically and kills in a time-dependent and concentration-independent way, by binding tightly to the ribosomes. The agent was not able to prevent cyst formation, and the cysts were not affect ed at an in vivo achievable concentration. Electron microscopy also supports the hypothesis of telithromycin being an effective agent against the mobile bacteria.

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine.

In this work the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine (HCQ) was studied. The minimal bactericidal concentration (MBC) of HCQ against the mobile spirochetes was > 32 microg/ml at 37 degrees C, and > 128 microg/ml at 30 degrees C. Incubation with HCQ significantly reduced the conversion of mobile spirochetes to cystic forms. When incubated at 37 degrees C, the MBC for young biologically active cysts (1-day old) was > 8 microg/ml, but it was > 32 microg/ml for old cysts (1-week old). Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that the contents of the cysts were partly degraded when the concentration of HCQ was > or = MBC. At high concentrations of HCQ (256 microg/ml) about 95% of the cysts were ruptured. When the concentration of HCQ was > or = MBC, core structures did not develop inside the cysts, and the amount of RNA in these cysts decreased significantly. Spirochetal structures inside the cysts dissolved in the presence of high concentrations of HCQ. When the concentration of HCQ was > or = MBC, the core structures inside the cysts were eliminated. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of HCQ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi.

Association between multiple sclerosis and cystic structures in cerebrospinal fluid.

BACKGROUND: The aim of the study was to search for infectious agents in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS). PATIENTS AND METHODS: CSF from ten patients with the diagnosis relapsing remitting MS and from five controls without MS were examined by transmission electron microscopy (TEM), dark field microscopy (DF), interference contrast microscopy (ICM) and UV-microscopic examination of acridine orange staining (AO). All CSF samples from patients and controls were cultured. RESULTS: Cystic structures were observed in CSF of all ten patients by AO and TEM. DF revealed eight cyst-positive patients out of nine. One of five control persons had such structures in the CSF; this person had suffered from erythema migrans. Spirochete or rod-like structures emerged after culturing two of the MS patient CSF samples and these structures could be propagated. CONCLUSION: A significant association of CSF cysts and MS was identified in this small study among residents in a coastal area of southern Norway. The cysts could be of spirochetal origin. Our study may encourage other researchers to study larger patient groups.

Susceptibility of motile and cystic forms of Borrelia burgdorferi to ranitidine bismuth citrate.

Gastrointestinal symptoms accompanying Lyme disease have not been considered in the treatment of Lyme patients yet. Here we examine the effect of ranitidine bismuth citrate (RBC) on motile and cystic forms of Borrelia burgdorferi in vitro, to determine whether it could cure this bacterial infection in the gastrointestinal tract. When motile forms of B. burgdorferi were exposed to RBC for 1 week at 37 degrees C, the minimal bactericidal concentration (MBC) was > 64 mg/ml. At 30 degrees C, the MBC was > 256 mg/ml. When the incubation lasted for 2 weeks at 37 degrees C, the MBC dropped to > 2 mg/ml. Bismuth aggregates were present on the surface of B. burgdorferi when RBC > or = MBC, as shown by transmission electron microscopy (TEM). Cystic forms of B. burgdorferi, exposed to RBC for 2 weeks at 37 degrees C, were examined by cultivation in BSK-H medium (Sigma B3528). They were stained with acridine orange (pH 6.4, pH 7.4) and studied by TEM. The MBC for RBC for young cystic forms (1 day old) and old cysts (8 months old) was estimated to be > 0.125 mg/ml and > 2 mg/ml, respectively. Bismuth aggregates were attached to the cysts and, in some, the pin-shaped aggregates penetrated the cyst wall. The bismuth aggregates also bound strongly to blebs and granules of B. burgdorferi when RBC > or = MBC. When B. burgdorferi is responsible for gastrointestinal symptoms, bismuth compounds may be candidates for eradication of the bacterium from the gastrointestinal tract.

Peptic ulcer disease in south Ethiopia is strongly associated with Helicobacter pylori.

Helicobacter pylori infection was detected in 93% of 174 patients with a peptic ulcer compared with 63% of 116 patients with normal findings (chi 2 = 37.3; P < 0.001) in a cohort of 834 consecutive patients examined by gastroscopy in Yirga Alem Hospital in south Ethiopia. Fourteen patients were given 14 days' treatment with metronidazole 500 mg t.i.d., doxycycline 100 mg b.i.d. and bismuth subnitrate mixture 150 mg q.i.d. Of 10 patients who returned for follow-up, only 2 patients were free from H. pylori and cured. Nineteen strains of H. pylori from 19 consecutive patients in the same hospital were tested for resistance in vitro against metronidazole, doxycycline and ampicillin. All but 1 were highly resistant to metronidazole; 2 were fully and 14 intermediate resistant against doxycycline. All strains were fully sensitive in vitro to ampicillin. Thus, peptic ulcer was strongly associated with H. pylori in south Ethiopia, but eradication of the infection was hampered by antibiotic resistance.

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole.

The aim of this study was to examine the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole. Because B. burgdorferi is a microaerobic bacterium like Helicobacter pylori, metronidazole (MZ) was chosen in the susceptibility test. For both microaerobic and aerobic incubation the normal mobile spirochetes were resistant to this antibiotic with an MBC > or = 512 microg/ml. Conversion of mobile spirochetes to cystic forms was not observed when they were incubated with MZ. When they were incubated under microaerobic conditions, the biologically active cystic forms had an MBC > or = 4 microg/ml, but the MBC was > or = 32 microg/ml with aerobic incubation at 37 degrees C. Staining with acridine orange (AO), dark field microscopy (DFM), and transmission electron microscopy (TEM) revealed that the contents of the cysts were degraded when the concentration of MZ was > or = MBC. Some cysts were also ruptured. When incubated with a sufficient concentration of MZ, core structures did not develop inside the cysts, and AO revealed less RNA in the cysts. Our observations may help efforts to treat resistant infections caused by B. burgdorferi with a combination of MZ and other antibiotics in order to eradicate both cystic and mobile forms of B. burgdorferi.

In vitro conversion of Borrelia burgdorferi to cystic forms in spinal fluid, and transformation to mobile spirochetes by incubation in BSK-H medium.

The purpose of this study was to examine the structural alterations of Borrelia burgdorferi when exposed to spinal fluid. Normal, mobile spirochetes were inoculated into spinal fluid, and the spirochetes were converted to cysts (spheroplast L-forms) after 1-24 h. When these cystic forms were transferred to a rich BSK-H medium, the cysts were converted back to normal, mobile spirochetes after incubation for 9 to 17 days. The cultures were examined by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). When neuroborreliosis is suspected, it is necessary to realize that B. burgdorferi can be present in a cystic form, and these cysts have to be recognized by microscopy. This study may also explain why cultivation of spinal fluid often is negative with respect to B. burgdorferi.

A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes.

Mobile Borrelia burgdorferi were transferred to distilled water (10(6) per ml). The cultures were observed by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). 95% of the spirochetes were converted to cysts after 1 min, and after 4 h no normal mobile borreliae were observed. When transferred to growth medium (BSK-H), the cysts became smaller and more irregular, and were filled with organic substances. After 1 day, 1-5 thin structures sprouted from the cysts. They continued to grow in both length and thickness until they attained a normal spirochetal structure. Finally, these new-born spirochetes detached from the cysts, by which time their mobility had become normal. The present method for producing large amounts of cystic forms of B. burgdorferi is well suited for further studies of this unique microbe.

Transformation of cystic forms of Borrelia burgdorferi to normal, mobile spirochetes.

The purpose of this study was to evaluate the behaviour of Borrelia burgdorferi under controlled conditions. The occurrence of cystic forms of Borrelia burgdorferi in vitro was noted, and these cysts were able to be transformed to normal, mobile spirochetes. B. burgdorferi was cultivated in a commercial culture medium without serum. The spirochetes multiplied only slowly in this medium, and transformation to encysted forms was observed after 1 week. When these cysts were transferred to the same culture medium with rabbit serum, the encysted forms developed into regular, mobile spirochetes after 6 weeks, and their regeneration time was normal. Examination of these cysts in the transmission electron microscope revealed transverse fission inside the cysts. It is probable that similar phenomena may occur in vivo under conditions unfavourable for spirochetes. These observations may help to explain why diagnosis and treatment of B. burgdorferi infections in humans can be difficult.

A simple method for determining metronidazole resistance of Helicobacter pylori.

The reliability of methods for determination of Helicobacter pylori resistance to metronidazole has been found to depend upon the incubation time. Because the disk diffusion method is more vulnerable than other methods to prolonged incubation, this method has not been recommended for H. pylori. However, because media designed for rapid growth of H. pylori have been introduced, the time has come to look at the clinical usefulness of this inexpensive and simple method again. The correlation of readings obtained with the E test (AB Biodisk, Solna, Sweden) and Rosco's (Taastrup, Denmark) disk diffusion method for in vitro metronidazole resistance determination for H. pylori with a short incubation time (24 to 31 h) was studied. Plates which could not be read after 24 to 31 h were reincubated for another night. Fifty-seven consecutive clinical strains were tested. Because the rate of regrowth of H. pylori depends upon the age of the colonies inoculated, the reproducibility of resistance test results for young colonies versus old colonies was also studied. Resistance plates could be read after 24 to 31 h of incubation for 28 of 29 strains when the inoculum consisted of young colonies (3 to 4 days old). For these 29 strains, a high correlation (r = -0.937) was found between results obtained with the E test and those obtained with the disk diffusion test. A poorer correlation was found for old colonies (> or = 5 days old) (r = -0.742), which required a prolonged incubation for 8 of 23 strains. In conclusion, short incubation was successfully applied with young colonies. Results obtained with the simple and inexpensive disk diffusion method correlated well with those obtained with the E test.

Risks related to lack of standardization of tests to detect in vitro metronidazole resistance in Helicobacter pylori.

The correlation between metronidazole MIC readings obtained in vitro on two different media for two different inoculum dilutions was studied after two, three, and five days of incubation using 20 clinical isolates of Helicobacter pylori. The PDM epsilometer (E test) was used. After two days of incubation, log MIC values on chocolate PDM and Vestfold charcoal medium, a new charcoal medium, showed good correlation. Charcoal media can thus be used for metronidazole sensitivity tests. In terms of double-dilution gradients, prolongation of incubation time from two to three days and change of inoculum dilution from 0.5 to 4 McFarland had little impact when studied separately. However, the combined effect of the two variables was marked. The matching of a sparse inoculum incubated for two days against a heavy inoculum incubated for three days resulted in readings which, for 30% of the pairs, differed by two double-dilution gradients or more. On the other hand, the separate influence of both variables was found to affect the correlation coefficients significantly. Five days of incubation was associated with a poor correlation when the variables on MIC readings was not exposed in a scale of double-dilution gradients because the intervals were too large. For this reason, scales without thresholds are recommended.

Rapid growth of Helicobacter pylori.

The ability of six variants of a new charcoal medium and Skirrow's medium to grow Helicobacter pylori in 3 and 5 days was studied using 20 different strains of Helicobacter pylori. The main admixtures for the charcoal media were serum, whole blood, and egg yolk emulsion. For this purpose, serum was significantly better and egg yolk emulsion significantly worse than whole blood. The addition of Iso Vitalex resulted in significantly improved growth on the charcoal media. Skirrow's medium showed very poor performance after three days of incubation and needed a long incubation time.