Med Microbiol Immunol (1992) 181:145-152

9 Springer-Verlag 1992

Deposition of bismuth by Yersinia enterocolitica Owen W. Nadeau 1, Dieter W. Gump 2, Gregory M. Hendricks 3, and Diane H. Meyer l Departments of 1Biochemistry, ZMedicine,Infectious Diseases Unit, and 3Anatomy, The Universityof Vermont College of Medicine, Burlington, VT 05405, USA ReceivedMarch 3, 1992

Abstract. Yersiniaenterocolitica 8081c cultures in exponential growth were incubated for 1 h in 0.1% microcrystalline bismuth subsalicylate (BSS) suspensions. Scanning electron microscopy (SEM) revealed microcrystals directly bound to BSS-treated bacteria. Energy dispersive spectroscopy (EDS) X-ray microanalysis of the attached microcrystals confirmed that the crystals were the microcrystalline BSS. X-ray spectra positive for bismuth were also obtained by SEM-EDS X-ray microanalysis of whole bacteria, suggesting metal incorporation into the bacteria in regions absent of bound microcrystals. Transmission electron microscopy of thin sections of embedded preparations of BSS-treated exponential-growth-phase bacteria showed electron-dense deposits in the periphery of the bacteria. Y. enterocolitica cultures that were in stationary phase at the time of incubation with microcrystalline BSS showed no evidence of the electron-dense deposits and EDS spectra were negative for bismuth. Bacteria incubated in the absence of microcrystalline BSS also lacked electron-dense deposits. Scanning transmission electron microscopy used in conjunction with EDS X-ray microanalysis to view and analyze semi-thick sections (250-300 nm) of embedded preparations of BSStreated bacteria in exponential growth confirmed that the electron-dense deposits at the periphery of the bacteria are the sites of bismuth depositions.

Introduction There is considerable evidence for the antimicrobial effects of bismuth subsalicylate (BSS), an insoluble salt of trivalent oxybismuth and salicylate, yet the mechanism of action remains unclear [2, 8, 13]. Sox et al. [13] have demonstrated that BSS binds bacteria with subsequent killing; the effect is accompanied by a reduction in intracellular ATP levels. Further evidence that bismuth affects antimicrobial activity is found in both in vivo and in vitro studies. Three reports Correspondence to: D. W. Gump

146 indicate t h a t there is p r o b a b l e d e p o s i t i o n of b i s m u t h within the cell walls of

Helicobacterpylori ( p r e v i o u s l y k n o w n as Campylobacterpylori a n d Campylobacter pyloridis) r e c o v e r e d f r o m biopsies o f patients t r e a t e d with c o l l o i d a l b i s m u t h subcitrate [ 1, 9, 11 ]. O u r o b s e r v a t i o n o f electron-dense particles in the p e r i p h e r y o f Yersiniaenterocolitica following i n c u b a t i o n in BSS suspensions also suggests b i s m u t h d e p o s i t i o n in this strain o f b a c t e r i a [4]. I n v a s i o n o f epithelial cells by Y. enterocolitica is r e d u c e d following p r e t r e a t m e n t o f the b a c t e r i a with BSS at s u b i n h i b i t o r y c o n c e n t r a t i o n s [4]. E l e c t r o n - d e n s e m a t e r i a l is o b s e r v e d in Y. enterocolitica which have succeeded in i n v a d i n g epithelial cells [4]. C u r r e n t evidence, thus, indicates t h a t BSS a n d o t h e r b i s m u t h c o m p o u n d s exert a n t i m i c r o b i a l activity t h r o u g h a variety o f m e c h a n i s m s which m a y involve b o t h the m e t a l a n d its ligand. One i m p o r t a n t aspect o f the a n t i m i c r o b i a l activity o f these c o m p o u n d s a p p e a r s to be directly r e l a t e d to the central m e t a l ion. In this s t u d y we have focused on d e t e r m i n i n g if the electron-dense d e p o s i t s o b s e r v e d in the a r e a o f the cell wall o f B S S - t r e a t e d Y. enterocolitica are b i s m u t h .

Materials and methods BSS in microcrystalline form was manufactured by Procter and Gamble Co., Cincinnati, Ohio. Y. enterocolitica 808 lc was provided by Dr. Pamela Small, Middlebury College, Middlebury, Vt. The bacteria were maintained in frozen glycerol stocks and grown in tryptic soy broth or on tryptic soy agar plates (Difco Laboratories, Detroit, Mich.).

Bismuth treatment of bacteria Bacteria from overnight stationary cultures were diluted in fresh broth and incubated at 25~ with shaking until reaching exponential growth phase. The bacteria were enumerated by measuring the optical density of the culture at 575 nm. Aliquots of the cultures were diluted to densities of 2 • 10V/ml with Dulbecco's modified Eagle's medium supplemented with 5 % fetal bovine serum (DMEM-FBS) (both Sigma Chem. Co., St. Louis, Mo.). Aliquots of bacterial DMEM-FBS suspensions were added to sterile tubes containing microcrystalline BSS in quantities to yield 0.1% suspensions and rotated on a Labquake shaker for 1 h. The suspensions were then centrifuged at 1,000 g for 5 min to pellet the microcrystalline BSS. The supernatant was decanted, centrifuged at 3,000g for 15 min, and the bacterial pellet was resuspended in 0.5 ml phosphate-buffered saline (PBS). Overnight cultures were also made 0.1% in microcrystalline BSS and treated as described above to compare bismuth uptake in stationary versus exponential phase cultures of Y. enterocolitica. Disks containing 0.2 mg of microcrystalline BSS were placed on agar plates inoculated with Y. enterocolitica. The plates were incubated for 48 h at 25~ and examined for bismuth mirrors [9].

Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) X-ray mieroanalysis Suspensions of Y. enterocolitica in PBS were fixed by adding 2.5% glutaraldehyde in 0.1 M Mollonig's phosphate buffer (MPB; pH 7.2) to the culture tubes one drop at a time until the initial volume of medium was doubled. The bacterial suspensions were allowed to stabilize in this solution for 10min. The bacteria were then pelleted at 3,000g for 10min. The pellet was resuspended in fresh 2.5% glutaraldehyde in 0,1 M MPB and allowed to fix for 30 rain at room temperature. Following primary fixation, the bacterial suspensions were pelleted by centrifugation at 3,000 g and resuspended in fresh buffer for 10 min. The process was repeated three times to

147 wash the bacterial suspensions. Following the final wash, the pellet was resuspended in 10% ethanol and dehydrated through a graded series of ethanol to 100 % (two changes). Several drops of the bacterial suspensions were then placed on Poretics (Livermore, Calif.) 0.08-1am PVDF (polyvinyldifluoride)discrete pore filters and allowed to air dry overnight in covered petri dishes. The dried filters containing the bacterial suspensions were then secured to a pure carbon sample bar (SPI#1687) with conductive colloidal graphite and sputter coated with 5nm Au/Pd to stabilize the filters in the electron beam. The filters were then examined at 8,000 x for the presence of bacteria using a JEOL 100CX II TEMSCAN equipped with a high-resolution ASID unit and a Tracor-Northern (Noran Instruments, Middleton, Wis.) X-ray analyzer. All scans were made at 20 kV accelerating voltage, 30 IJA filament current, 0.135 pA probe current, and a confined raster of 0.5 lam2. Microanalyses were counted for 200s over an energy range of 0-20keV. The detector resolution was approximately 155 eV.

Transmission electron microscopy (TEM) Suspensions of Y. enterocolitia in PBS were fixed and washed as described above for SEM. Following the last wash, the pellet was resuspended in 1.0% osmium tetroxide in MPB for 30 rain at 25 oC to postfix the bacteria. The bacterial suspensions were then washed three times in MPB as described above, dehydrated through a graded series of ethanol (10%-100%; two changes), transferred through two changes of propylene oxide (10rain each), and put into propylene oxide:Embed 812/Arildite 502 resin (50/50 V/V) for 12h at 25~ Following infiltration in the resin, the bacteria were pelleted by centrifugation and resuspended in pure Embed 812/Arildite 502 epoxy resin (two changes, 30 min each). The bacteria were pelleted again, transferred to Beem capsules containing the final embedding resin mixture, and pelleted at the terminus of the capsules. The samples were polymerized overnight at 70~ and ultrathin sections were cut. No further contrasting was done on the sections prior to examination on a JEOL 100CX II TEMSCAN at 80 kV.

Scanning transmission electron microscopy (STEM) and EDS X-ray microanalysis Bacterial suspensions were fixed, postfixed, dehydrated, and embedded as described above for TEM. Semi-thick sections (250-300 nm thick) were cut and placed on copper, 200-mesh support grids without further contrasting. They were carbon coated to stabilize the sections in the beam and to prevent excessive charging of the semi-thick sections. These sections were then examined on a JEOL 100CX II TEMSCAN at 100kV accelerating voltage at 60,000X. EDS X-ray microanalysis was counted and collected for 1,000 s over an energy range of 0-20 keV, with 100 pA emission current, 0.135 pA probe current, and a confined STEM raster of 50 nm 2.

Results

Y. enterocofitica cultures g r o w n o n a g a r plates t h a t h a d disks o f m i c r o c r y s t a l l i n e BSS p l a c e d on t h e m p r o d u c e d reflective m i r r o r s , indicative o f b i s m u t h r e d u c t i o n [9]. T h e reflective m i r r o r s were o b s e r v e d in a 15-ram r a d i u s a r o u n d the BSS disks on the a g a r plates. B i s m u t h m i r r o r s were n o t p r e s e n t on plates w i t h o u t bacteria. S E M o f whole m o u n t e d b a c t e r i a t r e a t e d with m i c r o c r y s t a l l i n e BSS a n d s u p p o r t e d on discrete p o r e filters revealed crystals b o u n d to the surface o f the b a c t e r i a (Fig. 1 A , a r r o w B). These crystals were identified as the m i c r o c r y s t a l l i n e BSS b y E D S X - r a y m i c r o a n a l y s i s (Fig. 1 B). H o w e v e r , X - r a y m i c r o a n a l y s i s o f regions o f the b a c t e r i u m t h a t were n o t a s s o c i a t e d with the m i c r o c r y s t a l l i n e BSS (Fig. 1 A, a r r o w C), were also positive for b i s m u t h at a very low level ( p e a k

148

A

Fig. 1. A Scanning electron micrograph (SEM) of a dividing Yersinia enterocolitica treated with microcrystalline bismuth subsalicylate (BSS) and supported on a discrete pore filter. Numerous microcrystals can be seen attached to the bacterium (upper right, arrowB). B SEM-energy dispersive spectroscopy (EDS) X-ray spectrum of the attached microcrystals in A (indicated by arrow B). A series of spectral peaks characteristics for bismuth (BI) are indicated. The vertical full scale (VFS) of this spectrum is 256 X-ray counts and the spectrum was collected over 200 s. The BI-Mct I peak area is 1205 X-ray counts above background. C SEM-EDS X-ray spectrum of the bacterium at arrow C(lower left) in A. Again the characteristic BI-MccI peak is present while small it is detectable (BI-Mc~l peak area is only 226 X-ray counts above background). The VFS is the same as in B

a r e a = 226 X - r a y counts a b o v e b a c k g r o u n d ) , suggesting b i s m u t h i n c o r p o r a t i o n even in regions u n a s s o c i a t e d with b o u n d microcrystals. T E M o f thin sections o f e m b e d d e d , m i c r o c r y s t a l l i n e B S S - t r e a t e d b a c t e r i a revealed the presence o f very fine, electron-dense d e p o s i t s in the region o f the cell wall o f the b a c t e r i a as well as n u m e r o u s large, electron-dense, i r r e g u l a r l y s h a p e d , rod-like structures (Fig. 2A). E D S X - r a y m i c r o a n a l y s i s o f the e l e c t r o n - d e n s e d e p o s i t s a s s o c i a t e d with the b a c t e r i a l cell wall p r o v e d to be i m p o s s i b l e due to their e x t r e m e l y small size (10-30 n m in d i a m e t e r ) a n d the fragility o f the thin sections. H o w e v e r , E D S X - r a y m i c r o a n a l y s i s o f the large, r o d - l i k e structures s h o w e d the

149

Fig. 2. A Transmission electron micrograph of a thin section of an embedded Y. enterocolitica culture treated with microcrystalline BSS. BSS treatment was carried out during exponential growth. Note the presence of electron-dense deposits in the region of the bacterial cell wall (arrows) of this unstained section. Also, numerous large BSS microcrystals are present throughout this preparation. B Scanning transmission electron micrograph of a semi-thick, unstained section of the same bacterial preparation of Y. enterocolitica as seen in A. The electrondense deposits are confined to the periphery of the bacterium (arrow). C STEM-EDS X-ray spectrum of a single electron-dense deposit seen in B. A spectral peak characteristic for bismuth (BI-Mct 1)is indicated. The vertical full scale (VFS) of this spectrum is 1024 X-ray counts, collected over 1000 s. The bismuth (BI-Mct t)peak area, while small, is discernable (peak area is 454 X-ray counts above background). X-ray spectral peaks characteristic for Cu, Au, and Si are background generated by the support grid characteristic bismuth spectrum (data not shown), demonstrating that these large structures are deposits o f microcrystalline BSS embedded with the bacteria. To obtain spectra f r o m regions o f the bacterial wall containing the very fine, electron-dense deposits, semi-thick sections ( 2 5 0 - 3 0 0 n m thick) were cut and viewed utilizing S T E M (Fig. 2B). The electron-dense deposits were analyzed simultaneously using E D S X - r a y microanalysis. A bismuth-positive spectrum was obtained f r o m the region o f the electron density (Fig. 2C). While it appears very

150 small on the full spectrum (vertical full scale 1024), the BI-Mct 1 peak area is 454 X-ray counts above background. Areas devoid of electron-dense deposits showed no evidence of bismuth accumulation (spectrum not shown). The semi-thick sections were viewed unstained to avoid any possible interactions between the heavy metal stains and the very fine electron-dense deposits. However, this does result in a loss of resolution with respect to the fine structure of the bacterial cell wall and thus, even at 80,000 X the exact location of the electron-dense deposit associated with the bacterial cell wall could not be determined.

Discussion In this study we have shown that there is deposition of bismuth in Y. enterocolitica harvested from cultures in exponential growth treated with microcrystalline BSS. This finding appears to be directly related to the reduction of BSS and the formation of metal mirrors adjacent to viable colonies on agar plates. Although other studies have documented the presence of electron-dense particles in bacteria treated with bismuth salts [1, 9, 11], previous investigations had failed to identify the electron-dense particles as bismuth. It is clear from the results of the STEMEDS X-ray microanalysis of the semi-thick sections, that the amount of bismuth in the electron-dense deposits is very small, possibly at the limit of detectability for our instrumentation (10 16g of an element) [3]. Further, the ability to detect bismuth in the semi-thick sections is most likely enhanced by the superposition of several bismuth deposits aligned in the periphery of the bacterial cell [5]. STEM of semi-thick sections suggests that the bismuth deposits are confined to the region of the cell wall and possibly the outer layer of the cell membrane. Metal deposition in active bacteria may occur by a number of processes [7]. Our results most probably rule out mechanisms involving intracellular accumulation. This is supported by the fact that electron-dense deposits were not observed in the protoplasm. EDS X-ray microanalysis of areas within the protoplasm or even in areas on the periphery of the bacteria devoid of electron-dense deposits showed no evidence of bismuth accumulation. Furthermore, penetration of the plasma membrane and the accumulation of toxic metals in the protoplasm is rare and generally associated with induction of chromosomal expression of transport sites within the plasma membrane following chronic exposure [12]. This type of metal induction is unlikely to have occurred during the 1 h of BSS exposure in our experiments [10]. The lack of metal deposition in stationary-phase cultures of Y. enterocolitica treated with microcrystalline BSS is not consistent with a simple metal absorption process, but does suggests a cell-mediated process. The fact that only small amounts of this highly insoluble bismuth salt are detected may suggest that the BSS was solubilized prior to being taken up by the bacterium. The reduction of metals by bacteria is well documented [6, 7]. Woolfolk and Whiteley d~monstrated that Micrococcuslactilyticus reduces di-and tri-valent bismuth complexes to elemental bismuth in hydrogenase-coupled reactions [14]. Marshall et al. [9] reported the formation of bismuth mirrors formed adjacent to disks of bismuth salts in contact with bacterial plaques on agar plates suggesting that bismuth salts are being reduced to the base metal by metabolic products formed by the bacteria. We have observed the formation of metal mirrors by Y. enterocolitica grown on agar plates in the presence of microcrystalline BSS

151 demonstrating that the microcrystalline BSS is being reduced by Y. enterocolitica. This process may be further enhanced by the fact that the microcrystalline BSS is apparently bound directly to the metabolically active bacteria, as seen in Fig. 1 A, facilitating the reduction and uptake of the base metal. The metabolic relevance of bismuth uptake is unknown, although it has been suggested that the observed reduction of metals may be related to the ability of certain bacteria to reduce nitrogen compounds [6]. Macaskie and Dean [7] proposed that the reduction of metal compounds and their subsequent uptake may serve as a defense mechanism for some strains of bacteria. In conclusion, this study confirms the deposition of bismuth in Y. enterocolitica exposed to microcrystalline BSS during exponential growth phase. Further, the apparent binding of the microcrystalline BSS directly to the bacteria appears to facilitate the subsequent reduction of the insoluble bismuth salt and uptake of the metallic bismuth by the bacteria. It is possible that the uptake of bismuth by the bacteria results in alteration of metabolic processes of the bacterium sufficient to interfere with its ability to invade eucaryotic cells (see previous publication). Future studies will include the use of high voltage electron microscopy to determine the precise location of bismuth deposition in the bacteria. Acknowledgements. Grand support was provided by the Procter and Gamble Co., Cincinnatti, OH 45241-2421.

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152 12. Silver S, Misra TK (1988) Plasmid-mediated heavy metal resistances. Annu Rev Microbiol 42:717-743 13. Sox TE, Olson CA (1989) Binding and killing of bacteria by bismuth subsalicylate. Antimicrob Agents Chemother 33:2075-2082 14. Woolfolk CA, Whiteley HR (1962) Reduction of inorganic compounds with molecular hydrogen by Micrococcus lactilyticus. I. Stoichiometry with compounds of arsenic, selenium, tellurium, transition and other elements. J Bacteriol 84:647-658

Deposition of bismuth by Yersinia enterocolitica.

Yersinia enterocolitica 8081c cultures in exponential growth were incubated for 1 h in 0.1% microcrystalline bismuth subsalicylate (BSS) suspensions. ...
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