Attempted Isolation of Nanobacterium sp. Microorganisms from Upper Urinary Tract Stones

A single team has reported isolation of nanobacteria in humanand bovine blood products, as well as, more recently, kidneystones. This has raised controversy. To confirm the data, wesearched for nanobacteria from 10 aseptically removed upperurinary tract (UUT) stones. We used scanning electronic microscopy(SEM) with four stones and culture of stones on either 3T6 fibroblastmonolayers or liquid RPMI medium. Detection of nanobacteriawas made with a commercially available monoclonal antibody,16S ribosomal DNA amplification with specific primers, and transmissionelectronic microscopy (TEM) of inoculated cells. SEM showednanoparticles in four of four UUT stones similar to those recentlydescribed. TEM of inoculated 3T6 cell monolayers has shown transientintracytoplasmic vacuolar formations containing 200- to 500-nmparticles in 3 of 10 cell cultures. Gimenez staining, Hoechststaining, and specific monoclonal immunofluorescence failedto reveal nanobacteria. Finally, we could not grow Nanobacteriumsp. microorganisms by the techniques described. Although withSEM, we observed nanoparticles morphologically similar to nanobacteria,we failed to isolate Nanobacterium sp. microorganisms in cultureand to prove the bacterial nature of these nanoparticles instones.

INTRODUCTION

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Introduction

Nanoparticles detected in various hot spring sediments (7, 8)and a Martian meteorite (17) have been postulated to be bacterialmicroorganisms on the basis of their morphological features.In 1997, a Finnish team reported isolation of a hitherto-undescribedbacterial species sharing morphological characteristics in commonwith nanoparticles (14), thereby providing some evidence insupport of earlier suspicions. These microorganisms were thesmallest described bacteria to date, with dimensions of 0.08to 0.5 µm. Furthermore, these organisms were found toproduce a biofilm containing hydroxyl apatite or carbonate,preventing their effective staining (12). They were also isolatedfrom commercial serum used in cell culture (11). Nanobacteriahave been detected thereafter in blood and blood products derivedfrom horses, as well as blood from human blood donors. Two strains,one of Nanobacterium sanguineum and the other of Nanobacteriumsp., were isolated from kidney stones and human and bovine sera,respectively (14). Phylogenetic analysis based on comparisonof 16S ribosomal DNA (rDNA) sequences has placed the nanobacteriaisolated from fetal calf serum into the ${alpha}$ ₂ subgroup of Proteobacteria(14), closely related to Thiobacillus, a water contaminant,and Agrobacterium and Rhizobium, which are plant-associatedbacteria.

The controversy that surrounds this intriguing, apparently transmissiblemicroorganism (1) particularly stems from the fact that isolationof nanobacteria has been reported by only one team and has notbeen confirmed by others (D. Y. Chang, T. W. Jarrett, L. R.Kavoussi, and J. B. Nelson, abstract from the 95th Annual Meetingof the American Urological Association, J. Urol. 161:249, 2000).Although a strain has been deposited in the German Collectionof Microorganisms (DSM no. 5819-5821), it is not yet availableto the scientific community. The aims of our study were to confirmthe presence of nanoparticles in upper urinary tract (UUT) stonesby morphological evidence with scanning electron microscopy(SEM) and to try to culture nanobacteria in cell cultures inoculatedwith UUT stones.

MATERIALS AND METHODS

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Materials and Methods

Stones. Ten UUT stones were aseptically removed from 10 French patientswho received 1 g of cefotaxime as antibioprophylaxis. Serumwas collected from these patients. All patients gave informedconsent. One fragment of each stone was preserved for cultureanalysis, and the other fragment was used to determine its chemicalstructure by Fourier-transformed infrared (IR) spectroscopyaccording to the standard method for clinical use (3).

Immunostaining and SEM. Stones were manually ground and powdered with a Potter device.For immunostaining, stone fragments were deposited on a slide,air dried, heat fixed at 70°C for 10 min, rehydrated andblocked by soaking them in 2% fat dry milk-phosphate bufferedsaline (PBS), and then processed as previously described (3,4, 12) with a commercial monoclonal mouse antibody (NanoBacOy, Kuopio, Finland). For SEM, fragments collected from fourstones were fixed with 2.5% glutaraldehyde (Sigma Chemicals,Saint-Louis, Mo.) for 20 min and then extensively washed withsterile PBS and rinsed in distilled water. Fixed material wasdehydrated in a graded series of ethanol and dried in an EMscopeCPD 750 critical point CO₂ apparatus (Emscope Lab., Ashford,United Kingdom). The samples were placed in a JFC-1100 (JEOL,Tokyo, Japan) sputter coater for coating with gold-palladium.The UUT samples were examined with a JEOL 35CF SEM operatedat 15 kV, and micrographs were recorded.

Cell coculture. Stones were cocultured with 3T6 cells (ATTC CCL 96) in shellvials in Dulbecco-Vogt's modification of Eagle 's medium (DMEM)(Gibco, Paisley, United Kingdom) supplemented with 10% heat-inactivatedfetal bovine serum (FBS). Four different batches were tested:Eurobio, batch 941338; Gibco, batches 40G4093I and 40G1891K;and Sigma, batch F2442. 3T6 cells were incubated for 24 h beforeinoculation of UUT stones. One part of each powdered stone wasdemineralized by incubation in 10 µl of 1 M HCl for 10min at room temperature and then neutralized by addition of10 µl of 1 M NaOH and 2 ml of DMEM-10% FBS. Finally, thesuspension was filtered through a 0.22-µm-pore-size filter(Millipore, Saint-Quentin, France). The supernatant from eachshell vial was discarded, and then 0.5 ml of the UUT stone suspensionwas inoculated onto the 3T6 monolayer, and the inoculated vialswere centrifuged at 700 x g for 1 h and incubated at 37°Cunder 5% CO₂. The presence of bacteria was monitored weeklyby Gimenez staining (9) and Hoechst 33258 staining (Hoechststain kit; Flow Laboratories, Ayrshire, United Kingdom) as previouslydescribed (14). Immunological detection of nanobacteria in cellcultures was attempted with a commercial monoclonal mouse antibody(NanoBac Oy, Kuopio, Finland) and by incorporating the patient'sserum into an indirect immunofluorescence assay as previouslydescribed (18). Two powdered and demineralized stones were inoculatedin parallel in 5 ml of RPMI 1640 with L-glutamine (Gibco) supplementedwith 10% FBS in T-25 culture flasks and incubated at 37°Cin a 5% CO₂ environment for 4 weeks. Flasks were inspected macroscopicallyand microscopically weekly for biofilm formation and calcification.The presence of bacteria was monitored by Gimenez and Hoechststaining and by PCR-based detection of the universal 16S rRNAgene as previously described (6, 20) every week for 4 weeks.Transmission electron microscopy (TEM) of infected cells wasperformed at the 4th week. 3T6 cells cultured in DMEM with 10%FBS without inoculation of powdered stones under the same cultureconditions were used as negative controls.

RESULTS

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Results

SEM. The four UUT stones examined by SEM showed similar characteristics.Spherical coccoid particles were observed, which were groupedin coarse clusters and bound together to a mineral structure(Fig. 1). These spherical units were similar in size and morphology.The size of these particles varied between 200 and 300 nm, andthey appeared to have developed in stone cavities. Fourier IRspectroscopy analysis indicated the stones consisted of cystineor oxalate.

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FIG. 1. Details from a fractured UUT stone from SEM analysis showing nanobacterium-like particles. The particles appeared as coccoid structures grouped together in coarse clusters. Their diameter was between 200 and 300 nm. Bar, 1 µm. Magnification, x15,000.

Cell coculture. Gimenez staining of inoculated 3T6 cells and on inoculated RPMI1640 medium failed to reveal nanobacteria after 1 week of culturein any of the 10 stone samples (Table 1). However, after 3 weeksof culture, two to three Gimenez-positive vacuolar inclusionswere observed in the cytoplasm of 10 to 20% of 3T6 cells in9 of 10 inoculated cultures. After 5 weeks, 10 of 10 stone culturespossessed these Gimenez-positive inclusions. Hoechst stainingwas negative for all cultures and remained negative even after6 weeks of culture (Table 1). Immunological detection with acommercially available antinanobacterium monoclonal antibodyperformed with two cultures failed to detect Nanobacterium sp.antigen after either 3 or 5 weeks of culture. Also, immunodetectionwith the patient's serum failed to detect microorganisms in10 of 10 inoculated 3T6 cell cultures. TEM showed intracytoplasmicvacuolar formation containing 200- to 500-nm particles in 3of 10 UUT stone cultures (Table 1). The morphology of nanoparticleswas coccoid, and they lay within vesicles in vacuolized 3T6cells (Fig. 2). No calcification or biofilm formation was observedin the two RPMI T-25 flasks inoculated with stones, and Gimenezand Hoechst staining failed to reveal any microorganisms. PCRincorporating the universal 16S rRNA gene primers failed toproduce an amplicon in 10 of 10 inoculated cell cultures andin 2 of 2 inoculated RPMI flasks.

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TABLE 1. Results of cell culture of UUT stones by Gimenez and Hoechst staining, PCR, and TEM

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FIG. 2. TEM analysis of UUT stone-inoculated 3T6 monolayers. (A) Nanoparticles inside vesicles in 3T6 cells. (B) Capsular form of nanoparticles.

DISCUSSION

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Discussion

We have tried to demonstrate the presence of nanobacteria inUUT stones by using SEM and culture. Despite previously reportedsuccess for these approaches (3, 12, 13), in our hands, onlySEM yielded any evidence for the presence of nanoparticles.However, although we observed spherical nanoparticles groupedin clusters binding to the mineral surface and cavities of UUTstones, we saw no clear evidence to support a microbiologicalnature for these structures. Immunological detection with acommercially available anti-Nanobacterium sp. monoclonal antibodyfailed to detect nanobacteria as described by Kajander and colleagues(3, 4, 12). Although intracellular structures similar to nanoparticleswere transiently observed by TEM in 3T6 cells inoculated withmaterial derived from UUT stones (12, 14), specific immunologicaland molecular tools failed to confirm these cells containednanobacteria. Despite the fact that we strictly applied themethods described by Kajander and coworkers, we failed to obtaina nanobacterial culture from FBS and kidney stones. We thereforewonder if there is a culture parameter not mentioned in publicationsthat could explain discrepancy between our results and thosepreviously reported. Alternatively, the formal possibility existsthat our specimen series simply did not contain any nanobacteria.However, in an independent study (Chang et al., J. Urol. 161:249,2000), 16 different renal calculi and 16 human serum samplesfrom patients with nephrolithiasis were been cultured. Detectionof nanobacteria was attempted with an enzyme-linked immunosorbentassay (ELISA) and NanoDect-EIA kit (ACELL, Nano Oy, Kuopio,Finland), as well as with 16S rDNA amplification with primersderived from the published Nanobacterium sp. sequences. No evidencefor the presence of the specific 16S rDNA sequence or nanobacterialantigen in human kidney stones or serum specimen was found.Although transferable biomineralization has been observed afterculture of human serum, human saliva, and dental plaque in DMEM(5), molecular examination of decalcified biofilms failed todetect the nucleic acid or protein that would be expected fromgrowth of a living entity. Alternatively, initiation of biomineralizationby macromolecules and transfer by self-propagating microcrystallineapatite was demonstrated. Cell cultures without addition ofantibiotics have been performed in our laboratory for the last17 years. During this period of time, despite using numerouscell lines and different batches of reagents, we have nevernoticed formation of biofilms on plasticware surfaces. Furthermore,detection of cocultivated microorganisms on cell cultures isroutinely performed by PCR with universal 16S rDNA primers (6,20), and direct immunofluorescence is routinely performed withspecific antibodies, and these approaches have never led tothe identification of Nanobacterium sp. microorganisms. However,transient Gimenez-positive inclusions are frequently observedin our experience. The structures observed have been routinelyconsidered as artifacts, as are the structures we observed whenusing TEM on UUT stone material-inoculated 3T6 cells.

In a recent study of patients with polycystic kidney disease(10), the authors found 23 of 31 kidney cystic fluids and 10of 10 liver cyst fluids positive for Bartonella sp. antibodyby immunofluorescence; reactivity between decalcified nanobacteriaand hyperimmune serum to Bartonella henselae was observed. Wehave now performed 2,043 cell cultures of blood and biopsy samplesobtained from serologically positive B. henselae and Bartonellaquintana patients, using B. henselae and B. quintana polyclonalantibodies to detect positive cell cultures directly on coverslips(16). Not once have cross-reacting nanobacteria been encountered.Likewise, 100% of 22 cow serum samples demonstrated to reactwith the emerging Bartonella weisii species also reacted withN. sanguineum antigen (2). In three of these cow serum samples,B. weissii was indeed isolated. Based upon the respective 16SrDNA sequences, Bartonella and Nanobacterium spp. are membersof the ${alpha}$ ₂ subgroup of Proteobacteria and purportedly share cross-reactingepitopes. Also, Nanobacterium sp. 16S rDNA sequences share 97.8to 99% similarity with those of Phyllobacterium myrsinacearum,another microorganism identified as a source of contaminating16S rDNA in PCR studies (5, 19). These data may lead to thehypothesis of laboratory contamination as a possible explanationfor discrepancies regarding demonstration of the presence ofNanobacterium sp. Despite our application of strict laboratoryrules, we experienced cultured cell contamination by Methylobacterium,which is also a member of the ${alpha}$ ₂ subgroup of Proteobacteria,from three different clinical samples (unpublished data). Culturecontamination of clinical samples by the waterborne microorganismAfipia felis (15) has probably been responsible for the hypothesisthat A. felis is the etiologic agent of cat scratch diseasein humans. These bacterial species share the hospital watersupply in the same ecological niche, and thus the hypothesisof contamination by waterborne bacteria cannot be ruled out.

Finally, in trying to confirm the data previously reported fornanobacteria, we have observed spherical particles adherentto UUT stones by using SEM. The morphology and size were compatiblewith nanobacteria, but we failed to culture these particles.The infectious nature of these particles has to be elucidated,and transmissibility from infected cell culture has to be proven.More investigations have to be done to determine whether theparticles observed in UUT stones are bacteria. The availabilityof the nanobacterial strain for scientific investigators wouldbe an important step in making progress in our research on nanobacteria.

ACKNOWLEDGMENTS

We acknowledge the technical assistance of Sandrine Zampa.

FOOTNOTES

* Corresponding author. Mailing address: Unité des Rickettsies, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Phone: 33 (0)4 91 83 55 17. Fax: 33 (0)4 91 83 03 90. E-mail: Didier.Raoult{at}medecine.univ-mrs.fr.

REFERENCES

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