Inhibition of Nanobacteria by Antimicrobial Drugs as Measured by a Modified Microdilution Method

Abstract

Compounds from 16 classes of antimicrobial drugs were tested for their abilities to inhibit the in vitro multiplication of nanobacteria (NB), a newly discovered infectious agent found in human kidney stones and kidney cyst fluids from patients with polycystic kidney disease (PKD). Because NB form surface calcifications at physiologic levels of calcium and phosphate, they have been hypothesized to mediate the formation of tissue calcifications. We describe a modified microdilution inhibitory test that accommodates the unique growth conditions and long multiplication times of NB. This modified microdilution method included inoculation of 96-well plates and determination of inhibition by periodic measurement of the absorbance for 14 days in cell culture medium under cell culture conditions. Bactericidal or bacteriostatic drug effects were distinguished by subsequent subculture in drug-free media and monitoring for increasing absorbance. NB isolated from fetal bovine serum (FBS) were inhibited by tetracycline HCl, nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxazole, and ampicillin at levels achievable in serum and urine; all drugs except ampicillin were cidal. Tetracycline also inhibited multiplication of isolates of NB from human kidney stones and kidney cyst fluids from patients with PKD. The other antibiotics tested against FBS-derived NB either had no effect or exhibited an inhibitory concentration above clinically achievable levels; the aminoglycosides and vancomycin were bacteriostatic. Antibiotic-induced morphological changes to NB were observed by electron microscopy. Bisphosphonates, aminocaproic acid, potassium citrate-citric acid solutions, and 5-fluorouracil also inhibited the multiplication of NB in a cidal manner. Insights into the nature of NB, the action(s) of these drugs, and the role of NB in calcifying diseases may be gained by exploiting this in vitro inhibition test system.

The role of calcifying nanoparticles in biology and medicine

“…1 Resistant to a wide spectrum of antimicrobial therapeutics: 30 aminoglycosides (in pharmacological concentrations), chloramphenicol, lincosamides, cephalosporins, macrolides, fluoroquinolones, glycopeptides (in pharmacological concentrations), polymyxins, antituberculous agents, aminocyclitol, spectinomycin. Sensitive to tetracyclines, 30 ampicillin, 30 trimethoprim, 30 trimethoprim-sulfamethoxazole, 30 nitrofurantoin, 30 5-fluorouracil, 30 cytosine arabinoside, 30 antimycin A, 22 sodium azide, 22 potassium cyanide, 22 bisphosphonates (etidronate, clodronate), 30 6-aminocaproic acid; 30 in-vitro growth can be inhibited by EDTA, EGTA, and citrate 22 Culture properties Doubling time is 3 days; in media without serum, 6 days. 1,16 Passage can be performed in DMEM or RPMI-1640 independently of the presence of serum.…”

HIV and nanobacteria

“…Nanobacterial infection could present a massive additional challenge to the extremely stressed immune system of HIV-infected patients. Additional stress could come, in particular, from inflammatory processes triggered by [8] and/or exposure to biostimulatory light intensities, applied at virtually non-destructive doses via NASA-LEDs (light emitting diodes) or low-level lasers [9,10], may help to reduce the nanobacteria-related disease burden in HIVinfected patients.…”

Clinical implications of calcifying nanoparticles in dental diseases: a critical review

“…In another report, contamination by other bacteria, eg, Phyllobacterium myrsinacearum, was presumed possible. 13 Ciftçiog lu et al 40 described morphological changes of CNPs caused by antimicrobial drugs under electron microscopy. In addition, they demonstrated the inhibition of CNP replications by aminocaproic acid, potassium citratecitric acid solutions, and 5-fluorouracil.…”