Bismuth-mediated disruption of the glycocalyx- cell wall of Helicobacter pylori:ultrastructural evidence for a mechanism of action for bismuth salts

Charles W. Strattona,*, Ronald R. Warnerb, Philip E. Coudronc and North A. Lillyb,{dagger}

a Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232-5310; b Miami Valley Laboratories, Procter & Gamble Co., Cincinnati, OH 45242; c Pathology and Laboratory Medicine Service, Veteran' s Administration Medical Center, Richmond, VA 23249-0001, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mechanism of bismuth' s bactericidal activity against Helicobacter pyloriwas investigated using transmission electron microscopy (TEM) and analytical electron microscopy (AEM); time- kill kinetic methods evaluated the effect of excess divalent cations. TEM analysis of untreated H. pylori revealed a normal morphology. In contrast, H. pylori exposed to bismuth salts had swollen, distorted cells with membrane—cell wall blebbing and a cytoplasm containing electron-dense, sometimes crystalline aggregates. By AEM, swollen cells contained bismuth at the cell periphery, whereas bacillary forms contained cytoplasmic bismuth localizations. Time—kill studies showed that the bactericidal activity of bismuth could be prevented by pretreatment with divalent cations. The effects of bismuth salts on the glycocalyces—cell walls of H. pylori with reversal of bactericidal activity by divalent cations are identical to those produced by other polycationic agents on various Gram-negative bacilli. We conclude that disruption of the glycocalyces—cell walls of H. pylori is one mechanism of action for bismuth salts.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The gastroduodenal effects of bismuth salts were first described in the 15th century, with 1785 being the date of first recorded use of these salts as medicinal agents. 1,2 Today, bismuth salts continue to be widely used in the treatment of duodenal ulcer disease caused by Helicobacter pylori.

Despite a long history of use for bismuth as a medicinal agent for gastrointestinal disorders and duodenal ulcers, the mechanism of action of bismuth salts is not completely understood. In efforts to understand the antimicrobial action of bismuth, we reported previously that the presence of fetal calf serum (FCS) in broth blocks the lethal effect of bismuth salts (at low concentration) on cultured H. pylori, in contrast to starch-containing broth, which did not inhibit bismuth action. 3 This blocking of the action of bismuth by FCS appeared to be related to fetuin, an anionic sialoprotein constituent of FCS and a known chelator of multivalent cations. This phenomenon suggested that the lethal effect of bismuth salts may be related to their cationic nature. This was confirmed, in part, when the addition of fetuin to starch-containing broth blocked the antibacterial effect of bismuth salts. 3

In gastric infections H. pylori is found encased in a flocculent gel layer (glycocalyx) 4 in which individual H. pylori microorganisms can be seen as densely stained curved, oval, or circular bodies with the surface of all organisms covered with a thick layer of glycocalyx material. Glycocalyx is a recognized target of polycations, with disruption resulting in cellular damage or death. 5

To evaluate the possibility that the antibacterial action of bismuth salts is via a cation-mediated interaction with the glycocalyx- cell wall of H. pylori, we used transmission electron microscopy (TEM) and analytical electron microscopy (AEM) to perform ultrastructural and bismuth localization studies on H. pylori in culture. Because polycation-mediated disruption of cell walls can be decreased or reversed by pretreatment with excess divalent cations, we also used time- kill kinetic methods to evaluate the effects of excess divalent cations on the bactericidal activity of bismuth salts against H. pylori.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Micro-organisms

Four isolates of H. pylori were used: these included two ATCC isolates (43504 and 11638) as well as two clinical isolates from gastric biopsies of patients with ulcer disease.

Media

The broth medium selected for the time- kill kinetic studies was cation-adjusted Mueller- Hinton broth (CA-MHB, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) which contains physiological concentrations of Ca 2+ and Mg2+ ions. This was supplemented with 0.5% starch which, unlike FCS, has been shown to support the growth of H. pyloriwithout suppressing the activity of bismuth salts. 3 CA-MHB has been used extensively in our laboratory for time- kill kinetic studies with H. pylori. 6,7 For TEM studies where antimicrobial suppression was not an issue because of the high concentration (1000 mg/L) of bismuth salts, a standard brucella broth (Becton Dickinson) supplemented with 5% FCS was used.

Inoculum preparation

Inocula used for time- kill kinetic studies were prepared as follows: organisms were grown overnight at 37°C in 10% CO 2 using CA-MHB containing 10% FCS. Samples from these overnight broth cultures were then inoculated onto campylobacter base agar (Becton Dickinson) supplemented with whole sheep blood, horse serum, cholesterol, and cations. 3 These plates were incubated overnight at 37°C under microaerophilic conditions. Cell suspension inocula were prepared from this overnight growth on agar plates and represented microorganisms in late logarithmic, early stationary growth phase as shown by an initial inoculum size of 5 x 10 6 cfu/mL increasing to 5 x 10 7 cfu/mL after 24 h. Cells in the inocula suspension showed typical curved and spiral morphology when viewed by phase microscopy and many were motile.

Inocula used for TEM were grown overnight at 37°C in 10% CO 2 using brucella broth containing 5% FCS. Samples from these overnight broth cultures were then inoculated into fresh broth and incubated overnight at 37°C in 10% CO 2. Final cell suspensions were prepared from this second overnight growth in broth using a 1:10 dilution in fresh broth (control) or fresh broth containing colloidal bismuth subcitrate (CBS) at 1000 mg/L.

Antimicrobial agents

A standard powder for CBS (Brocades Pharma bv, Delft, The Netherlands) was kindly supplied by the manufacturer.

Transmission electron microscopy

Conventional TEM sample preparation techniques similar to those used by Armstrong et al. 8 were employed for fixation and embedment. Culture samples were fixed in 2% glutaraldehyde for 2 h at room temperature. The samples were spun at 1000 rpm for 5 min, the pellet was resuspended in 10% sterile bovine albumin, respun at 1000 rpm for 5 min and the resulting pellet overlaid with fixative. Fixation was performed overnight at room temperature. The gelled pellets were divided into two groups; one was post-fixed in 1% buffered OsO 4 before being dehydrated and embedded in Spurr' s resin. The other was directly dehydrated and embedded in LR White resin. For morphological studies, thin (90 nm) sections were cut on water; Spurr' s-embedded sections were stained with uranium acetate and lead citrate, whereas LR White-embedded sections were left unstained to better identify electron-dense bismuth complexes. Sections were examined with a Zeiss EM-902 (Carl Zeiss, Inc., Thornwood, NY, USA) operated in the zero loss mode at 80 kV.

Analytical electron microscopy

Before cryo-fixation, aliquots from cultures at each time point were centrifuged at 1000 rpm for 5 min, resuspended in 0.3 mL droplets of 10% albumin, repelleted, and 10 µl aliquots snap-frozen in a stirred bath of 80% propane- 20% isopentane cooled by liquid nitrogen. Thick (0.3 m) sections were cut dry on a cryoultramicrotome at -115°C, placed on carbon-coated Formvar-covered nickel slot grids and freeze-dried overnight in a Denton 502 vacuum evaporator (Denton Vacuum, Moorestown, NJ, USA). Thick (0.3–1.0 µm) sections of unstained LR White-embedded bacteria were cut dry, mounted on Formvar-covered nickel slot grids, and coated with a thin coating of carbon in a Denton 502 vacuum evaporator (Denton Vacuum). Analysis was done at 100 kV in the scanning transmission electron microscopy (STEM) mode using an Hitachi H500 TEM- STEM (Hitachi Scientific Instruments, Inc., Mountain View, CA, USA) and Tracor Northern TN-5500 (Noran Instruments, Middletown, WI, USA) energy dispersive spectrometer (EDS) system, or a Philips CM12 (Philips Electronic Instruments, Mahwah, NJ, USA) and Link eXL (Oxford Instruments, Concord, MA, USA) EDS system.

Time- kill kinetic studies

Time- kill kinetic studies were performed in CA-MHB containing 0.5% starch and CBS salts. 6 The inoculation of media-containing sealed bottles was done with a syringe and yielded approximately 5 x 10 6 cfu/mL. Following inoculation, the bottles were flushed with a microaerobic mixture of gases (nitrogen, oxygen and carbon dioxide) and shaken continuously at 150 rpm at 37°C. Samples of the H. pylori were removed at 24 h, diluted serially, and plated for colony counts (cfu/mL). Carryover of bismuth salts was addressed by adding EDTA to the samples before dilution. After 4–6 days of incubation under microaerophilic conditions at 37°C, the plates were read for colony counts. The change in log 10 cfu/mL after 24 h incubation with bismuth salts relative to the log 10 cfu/mL at time zero was determined. Thus, a zero value means no change, -2 represents a 2 log 10 decrease and 99% killing of the initial inoculum, etc. Maximal killing was defined as <30 cfu recovered from a 1.0 mL sample aliquot and represents a decrease of at least 5.2 in log 10 cfu/mL at 24 h.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Viability studies

Viability studies on the in-vitro cultures used in the microscopy investigations demonstrated that after 4 h of bismuth exposure, the H. pylori cfu increased at a rate similar to that seen for the untreated culture. At 24 h, however, there was a complete loss of viability for the bismuth-treated culture whereas the untreated culture continued to grow (data not shown).

Transmission electron microscopy

TEM examination of untreated cultures of H. pylori after 4 or 24 h of incubation revealed coccoid, spiral, and slightly curved bacillary forms in which the cellular membranes adhered closely to the smooth cell walls, as illustrated in Figure 1a. The bacterial cytoplasm is relatively homogeneous. The glycocalyx is not visualized in Figure 1a- d because neither a stabilizing method nor ruthenium staining was used. As shown in Figure 1b, H. pylori exposed to bismuth salts at 1000 mg/L for 4 h have a morphology that is very similar to the control cultures although the cells were larger, suggesting they were becoming swollen; these observations are consistent with the viability data. However, H. pylori cells exposed to bismuth for 24 h were markedly swollen and distorted, with blebbing in the cellular membrane- cell wall, as shown in Figure 1c. These degenerative cells are surrounded by micelles of thrown-off cell wall blebs; the narrow stalks of membrane continuity between bacterium and bleb that can be seen in a few cells should be noted (arrows, Figure 1c). In some cells, the cytoplasm appears to separate from the cell wall. In most cells, the cytoplasm is not homogeneous and contains electron-dense cytoplasmic aggregates (arrows in Figure 1d). Thin sections not stained by osmium, uranyl acetate and lead citrate revealed a variety of electron-dense structures within the cytoplasm of these bacteria (Figure 2a). Fine, somewhat diffuse but still localized particulates were present in most bacteria. Some cytoplasmic structures were nearly circular and extremely electron dense (top insert of Figure 2a). Lighter particulates in the cytoplasm, some with linear, very geometric shapes (lower insert in Figure 2a) were also frequently observed. Occasionally, electron-dense particles were also observed around the membrane of H. pylori(arrow, Figure 2b) as previously described by Armstrong et al. 8 As shown below, electron-dense structures may be a consequence of the presence of iron or bismuth.






View larger version (733K):
[in this window]
[in a new window]
 
Figure 1. (a) Transmission electron micrograph of untreated H. pylori culture grown for 24 h. The cytoplasm is relatively uniform, and there is close adherence of the cytoplasm to the cytoplasmic membrane except in the normal polar lucent zone where there is an attached flagellum (arrow). Stained with uranyl acetate- lead citrate. (b) Transmission electron micrograph of H. pylori culture exposed to CBS 1000 mg/L for 4 h. The morphology is very similar to the control culture although the cell appears larger and is perhaps swollen. Stained with uranyl acetate–lead citrate. (c) Transmission electron micrograph of H. pylori culture exposed to CBS 1000 mg/L for 24 h. Many cells are swollen (not evident in this image) with cell wall distortion and blebbing. The development of cell wall extrusions (arrows), which often become micelles (arrowheads) after separation from the cell wall, should be noted. In most cells the cytoplasm is less homogeneous, and in some cells begins to separate from the cytoplasmic membrane. Stained with uranyl acetate–lead citrate. (d) Transmission electron micrograph of H. pylori culture exposed to CBS 1000 mg/L for 24 h. Many bacteria contain electron-dense cytoplasmic aggregates that exhibit variable staining and ultrastructure (arrows). Stained with uranyl acetate- lead citrate.

 



View larger version (350K):
[in this window]
[in a new window]
 
Figure 2. (a) Transmission electron micrograph of H. pylori culture exposed to CBS 1000 mg/L for 24 h. The preparation is unstained, and although the bacteria are barely discernible, the presence of localized, electron-dense particulates within the cytoplasm of bacteria is very evident. The inserts show two different morphologies of the cytoplasmic electron-dense structures. (b) Transmission electron micrograph of H. pylori culture exposed to CBS 1000 mg/L for 24 h. Around the interior of the membrane of one (swollen) bacterium are 6 nm electron-dense particles (arrows). Unstained preparation.

 
Analytical electron microscopy

AEM examinations of cryosections and of plastic-embedded sections provided similar results, although the absence of an embedding medium in cryosections gave better X-ray counting statistics and sample visualization. STEM (morphological) images and associated X-ray maps for phosphorus, iron and bismuth of H. pylori bacteria in albumin or plastic matrices were examined using control untreated cultures of H. pyloriand cultures exposed to bismuth. Bismuth was not detected in untreated bacteria, either in elemental maps or in spectra from analysed points within the bacteria (data not shown). Cytoplasmic iron and phosphorus distributions were noted to overlap usually in both the cryosections and the plastic-embedded bacteria; sulphur was usually distributed homogeneously throughout the bacteria, although occasional localizations were observed (data not shown).

Bismuth was not detected in H. pylori exposed to bismuth salts at 1000 mg/L for 4 h (data not shown). However, after 24 h of exposure bismuth was readily detected by the X-ray spectrum from both cryosections and plastic-embedded bacteria (data not shown). Bismuth concentration was found to be variable within a bacterium with two distinct forms of bismuth appearing in H. pylorifollowing 24 h of exposure. When H. pylori had a bacilliform morphology, bismuth was detectable throughout the cytoplasm but was primarily present in discrete localizations at high concentration (data not shown). In swollen, distorted cells bismuth was primarily associated with the cell periphery (data not shown). The bismuth localizations within bacilliform H. pyloridid not coincide with the iron (or phosphorus) localizations.

Time–kill kinetic study results

The results of the time–kill kinetic studies are summarized in Table I. After 24 h of exposure, CBS was bactericidal at 8 or 16 mg/L. However, the addition of Ca 2+ ions at six times the physiological concentration of calcium present in CA-MHB broth totally prevented any bactericidal effect.


View this table:
[in this window]
[in a new window]
 
Table. Effect of calcium cations on the bactericidal effects of 24 h of exposure to bismuth salts in two isolates of H. pylori
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The reported viability studies are similar to those of Armstrong et al. 8 The TEM sample preparation was based on that of Armstrong et al., 8 and we agree with them that no morphological changes in H. pyloriultrastructure were observed after 4 h of bismuth exposure. However, in contrast to the study by Armstrong et al., 8 marked changes in H. pyloriultrastructure were observed after 24 h of bismuth exposure, including formation of dense cytoplasmic aggregates, cell wall blebbing, and cell swelling. This morphological progression correlated well with the viability data. This major discrepancy in morphology between our results and those of Armstrong et al. 8 is difficult to explain. Small differences in these two studies exist with regard to the bacterial cultures, broth composition, CO 2 concentration, and number of subcultures, but these differences hardly seem sufficient to explain such disparate results. Perhaps the vagaries of sampling, such as a selection of viable cells during centrifugation in the studies by Armstrong et al., 8 may have led to their observations.

An additional difference between our and Armstrong' s results concerns their lone morphological change induced by bismuth; namely, the presence in unstained sections of a distinctive pattern of approximately 6 nm electron-dense particulate deposits at the periphery of bismuth-exposed bacterial cells, referred to as ‘bismuth complex’ . 8 In their preparations the presence of these particulate deposits increased with time of bismuth exposure, and at 24 h of exposure virtually all bacteria exhibited this membrane deposit. 8 In our preparations the presence of electron-dense particulates at bacterial membranes, although seen, was far less common.

The presence of electron-dense material within the cytoplasm of the 24 h bismuth-exposed cultures supports the AEM results showing the presence of high levels of bismuth within the H. pyloricytoplasm. Although bismuth clearly enters these cells, we do not fully understand the two different distributions that we see. When H. pylori cells had a bacilliform morphology, bismuth was present within the cytoplasm, primarily at discrete localizations. When H. pylori cells were swollen, bismuth was associated with the bacterium cell periphery. These distributions may relate to the progression of cell death, moving from a bacilliform to a swollen morphology. Alternatively, bismuth may have multiple mechanisms of antibacterial activity.

A possible mechanism of bactericidal activity for bismuth salts against H. pyloriis suggested by the environment in which this pathogen exists. A study of human gastric mucosa in patients with chronic gastritis 4 has demonstrated that the preferred location in the gastric antrum for H. pylori is close to the surface of epithelial cells lining the gastric pits, where large numbers of microorganisms clump together and occasionally form colonies within the finely granular mucus of the gastric pit. Within this mucus, the H. pyloriare themselves encased in a glycocalyx that forms a flocculent gel layer. Glycocalyx is a polyanionic matrix-supported gel composed of 99% water and can be thought of as an anionic polymeric diffusion barrier as well as an ion exchange resin of almost infinite surface area. 9 The glycocalyx is probably a protective repository for urease produced by H. pylori and may protect this organism from osmotic, pH, or enzymatic dangers found in the inhospitable milieu of the stomach. H. pylori, like other microorganisms living within glycocalyx-encased microcolonies, 9 replicates slowly and therefore is difficult to eradicate with most antimicrobial agents.

The glycocalyx surrounding an individual microbial cell is anchored by lipooligosaccharides which are attached to the inner cytoplasmic membrane or to the peptidoglycan layer. 9 These lipooligosaccharide stalks protrude from the cell wall and provide a matrix by ionically linking their branching polysaccharide side-chains with divalent cations. Because these polysaccharide side-chains in the matrix are hydrophilic, water is absorbed into the matrix, which transforms this outer layer into a gel.

The microbial glycocalyx–cell wall is a recognized target 10 for the physiochemical action of chelating agents as well as certain polycationic antimicrobial agents. 11 This mechanism of action for glycocalyx-disrupting agents has been well described 12 ,13 ,14 ,15 ,16 ,17 ,18 ,19 ,20 ,21 and is briefly summarized. Bacterial cells normally have an internal hydrostatic pressure that is higher than that of the external milieu. 22 The presence of this gel matrix as an outer layer helps maintain the shape of many bacteria including Gram-negative bacilli such as H. pylori. 9,23,24 The integrity of this hydrated matrix depends upon Ca 2+ and Mg 2+ions, which ionically link the polysaccharide side-chains. When a portion of this glycocalyx is disrupted, the high internal pressure of the cell is focused directly on the weak area. As a result, the cell membrane- wall may bulge and a portion of the cell membrane may protrude through this weak area. 21 This results in finger-like protrusions containing cytoplasmic contents. The stress on the cell membrane- wall as it protrudes through this ‘ hole’ is thought to activate autolytic enzymes present in the cell wall that normally dissolve the wall septum during replication. The premature dissolution of cell wall peptidoglycan at the point of protrusion effectively severs this protruding bleb and leaves a transient hole which the microbial cell may repair. If enough holes are formed, leakage of cytoplasmic contents results in cellular death.

The disruption of bacterial glycocalyx can be accomplished by a number of physiochemical mechanisms which displace divalent cations from the glycocalyx. The loss of divalent cations can be accomplished by the use of chelating agents such as EDTA 25,26,27 or fluoroquinolones, 28 or by the use of polycationic agents which compete for divalent cation binding sites. 25 Microbial glycocalyces–cell walls are known to be disrupted by polycationic complexes such as polymyxin, 12,16,19,21 aminoglycosides, 14,15 amphiphilic peptides, 13,17,18,20,29 and heavy metals. 5 Disruption of the glycocalyx- cell wall increases cell permeability and improves penetration of hydrophilic antimicrobial agents. 30 Severe or prolonged disruption of the cell wall may result in cell death. 23,25 The disruptive effect of polycations caused by displacement of divalent cations may be decreased or prevented by increasing the concentration of divalent cations before the polycationic assault. 5,31

Bismuth salts in aqueous solutions form hydrated polycations with inorganic ligands or with certain organic ligands such as citrate, malate, and tartrate. 2 For example, CBS, commonly used in combination therapy for infections caused by H. pylori, is a complex bismuth salt of citric acid; the trivalent bismuth and the trivalent citric acid have many possibilities for salt formation resulting in molecules of different size with the average molecules so large that the aqueous solution becomes colloidal. 32 Unlike the inorganic bismuth salts, which are largely insoluble in aqueous solutions between pH 1.0 and pH 7.0, bismuth salts with organic ligands are readily soluble in aqueous solutions at neutral pH. In acid solutions (pH< 5.0), organic bismuth salts precipitate, forming various insoluble salts, the smallest being bismuth oxychloride (BiOCl). In addition, more complex structures containing Bi +groups are formed. 1,2 The Bi +groups in complex bismuth salts are known to ionically bind to anionic surfactants such as glycerolipids and glycoproteins, the predominant component of glycocalyx. 1,2 This binding of Bi + groups may displace divalent cations such as Mg 2+ and Ca 2+. Such a physiochemical property of polycationic bismuth salts might explain its antimicrobial activity against H. pylori.

In our ultrastructural studies, H. pylori was exposed over a 24 h period to bismuth salts at 1000 mg/L, which approximates clinical concentrations achieved with usual bismuth-based eradication therapies. The ultrastructural changes of the glycocalyx- cell wall noted in our study are identical to those ultrastructural changes of glycocalyx–cell wall caused by disrupting agents as previously described in the literature. 12 ,13 ,14 ,15 ,16 ,17 ,18 ,19 ,20 ,21 The phenomenon in which bismuth salts enter the cytosol of H. pylori may mimic the self-promoted uptake pathway proposed for cationic compounds by Hancock and co-workers. 33,34 Those investigators proposed that the displacement of magnesium by cations or chelators might temporarily expose and/or disrupt the lipid domains of the outer membrane and allow increased passage of hydrophobic agents. Displacement of magnesium is known to disrupt outer cell membranes 25 and may do so by first disrupting the glycocalyx. 5,9,24,26

A number of other theories for the mechanism of action for bismuth salts have been proposed, the first being that bismuth salts form a protective coating over the ulcers, which protects the ulcer from the peptic activity of gastric juices. 35,36 Bismuth salts also bind to mucus glycopeptides and may impair H+ back-diffusion across the gastric mucosa. 1 Other studies suggest that bismuth salts inhibit the adherence of H. pylori to human gastric mucosal cells. 37 The first two ‘mechanisms’ may exist and perhaps assist in ulcer healing, but would not be expected to provide bactericidal activity against H. pylori. Decreased adherence to human gastric mucosal cells would be consistent with alterations in glycocalyces–cell walls. Another mechanism has been suggested by Beil et al., 38 who have demonstrated that bismuth salts form stable complexes with dithiols and thus inhibit dithiol enzymes such as Na +/K+ -ATPase. The Na +/K+ -ATPase is a surface enzyme located on the microbial cell wall; its inhibition is likely to lead to osmotic effects that could disrupt the glycocalyx–cell wall. Finally, Baer et al. 39 have demonstrated that bismuth may have an antibacterial effect as a consequence of inhibiting the respiratory chain of H. pylori. Entry of positively charged bismuth salts into the microbial cytosol would be expected to interfere with electron coupling and may well be a secondary mechanism of action in rapidly replicating microbes that are able to repair their cell walls. This scenario, for instance, could explain the different bismuth distributions that we see associated with the two different bacterial morphologies after 24 h of exposure. Bacteria that withstand a bismuth-mediated glycocalyx–cell wall disruption leading to swelling and death would retain their bacillary form but nevertheless would have taken up sufficient bismuth via an altered permeability to cause secondary metabolic or anabolic interventions leading to death.

In summary, the morphological effects of physiochemical disruption of glycocalyx–cell wall have previously been described and are identical to the morphological effects of bismuth salts on the glycocalyx–cell wall of H. pylori observed in this study. The prevention of the bactericidal effect of bismuth salts by pre-exposure to excess divalent cations is consistent with the effect of excess divalent cations on the disruption of glycocalyces–cell walls by polycationic agents. At least in bacteria with a swollen morphology, bismuth is associated with the membrane periphery. It would appear that disruption of the glycocalyx–cell wall of H. pylori is an important mechanism of action for bismuth salts.

This physiochemical mechanism of action for bismuth salts has important implications in terms of microbial resistance. Because disruption of glycocalyx–cell wall is the result of a physiochemical reaction, it is very difficult for microorganisms to become resistant to this action. Although the development of microbial resistance to polycationic agents has been described in vitro for mutated microorganisms with diminished production of glycocalyx, such resistance is rare. 40 Moreover, such mutations are not likely to become important clinical problems because microorganisms with diminished glycocalyx are generally poor pathogens having low virulence. 40 This may explain why no H. pyloriisolates with resistance to bismuth salts have been reported to date.

Our data are consistent with bismuth salts having a mechanism of action involving the disruption of the glycocalyx–cell wall of H. pylori.Bismuth salts thus appear to be an excellent cornerstone for any combination antimicrobial therapy directed against this important pathogen.


    Notes
 
* Correspondence address. Clinical Microbiology Laboratory, Room 4525-TVC, The Vanderbilt Clinic, 21st and Edgehill, Nashville, TN 37232-5310, USA. Tel: +1-615-343-9144; Fax: +1-615-343-8420. Back

{dagger} Present address. School of Library and Information Science, Kent State University, PO Box 5190, Kent, OH 44242, USA Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Lambert, J. R. (1991). Pharmacology of bismuth-containing compounds. Reviews of Infectious Diseases 13, Suppl. 8, S691–5.[ISI][Medline]

2 . Paradies, H. H. (1990). Bismuth— from the element to the tablet: the general chemistry of bismuth with relevance to pharmacy and medicine. In Helicobacter pylori, Gastritis, and Peptic Ulcer , (Malfertheiner, P. & Ditsehuneit, H., Eds), pp. 409–426. Springer-Verlag, New York.

3 . Coudron, P. E. & Stratton, C. W. (1995). Factors affecting growth and susceptibility testing of Helicobacter pylori in liquid media. Journal of Clinical Microbiology 33,1028 –30[Abstract]

4 . Thomsen, L. L., Gavin, J. B. & Tasman-Jones, C. (1990). Relation of Helicobacter pylori to the human gastric mucosa in chronic gastritis of the antrum. Gut 31,1230 –6[Abstract]

5 . Stratton, C. W. (1996). Mechanisms of action for antimicrobial agents: general principles and mechanisms for selected classes of antibiotics. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed.), pp. 579–603 Williams & Wilkins, Baltimore, MD.

6 . Coudron, P. E. & Stratton, C. W. (1995). Use of time- kill methodology to assess antimicrobial combinations against metronidazole-susceptible and metronidazole-resistant strains of Helicobacter pylori. Antimicrobial Agents and Chemotherapy39 , 2641–4[Abstract]

7 . Coudron, P. E. & Stratton, C. W. (1995). Utilization of time- kill kinetic methodologies for assessing the bactericidal activities of ampicillin and bismuth, alone and in combination, against Helicobacter pylori in stationary and logarithmic growth phases. Antimicrobial Agents and Chemotherapy 39, 66–9[Abstract]

8 . Armstrong, J. A., Wee, S. H., Goodwin, C. S. & Wilson, D. H. (1987). Response of Campylobacter pyloridis to antibiotics, bismuth and an acid-reducing agent in vitro — an ultrastructural study. Journal of Medical Microbiology 24, 343–50[Abstract]

9 . Costerton, J. W., Lewandowski, Z., DeBeer, D., Caldwell, D., Korber, D. & James, G. (1994). Biofilms, the customized microniche. Journal of Bacteriology 176, 2137–42[ISI][Medline]

10 . Magnusson, K.-E. (1989). Physiochemical properties of bacterial surfaces. Biochemistry Society Transactions 17, 454–8.

11 . Vaara, M. & Vaara, T. (1983). Polycations as outer membrane-disorganizing agents. Antimicrobial Agents and Chemotherapy 24, 114–22[ISI][Medline]

12 . Dixon, R. A. & Chopra, I. (1986). Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide. Antimicrobial Agents and Chemotherapy29 , 781–8[ISI][Medline]

13 . Haynie, S. L., Crum, G. A. & Doele, B. A. (1995). Antimicrobial activities of amphiphilic peptides covalently bonded to a water- insoluble resin. Antimicrobial Agents and Chemotherapy 39, 301–7[Abstract]

14 . Iida, K. & Koike, M. (1974). Cell wall alterations of Gram- negative bacteria by aminoglycoside antibiotics. Antimicrobial Agents and Chemotherapy 5, 95–7[ISI][Medline]

15 . Kadurugamuwa, J. L., Clarke, A. J. & Beveridge, T. J. (1993). Surface action of gentamicin on Pseudomonas aeruginosa.Journal of Bacteriology 175, 5798–805[Abstract]

16 . Kaye, J. J. & Chapman, G. B. (1963). Cytological aspects of antimicrobial antibiosis. III. Cytologically distinguishable stages in antibiotic action of colistin sulfate on Escherichia coli. Journal of Bacteriology 86 ,536 –43[ISI][Medline]

17 . Lehrer, R. I., Barton, A., Daher, K. A., Harwig, S. S., Ganz, T. & Selsted, M. E. (1989). Interaction of human defensins with Escherichia coli : mechanism of bactericidal activity. Journal of Clinical Investigation 84, 553–61[ISI][Medline]

18 . Lehrer, R. I., Lichtenstein, A. K. & Ganz, T. (1993). Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annual Review of Immunology 11, 105–28[ISI][Medline]

19 . Rosenthal, K. S. & Storm, D. R. (1977). Disruption of the Escherichia coli outer membrane permeability barrier by immobilized polymyxin B. Journal of Antibiotics 30,1087 –92[ISI][Medline]

20 . Sawyer, J. G., Martin, N. L. & Hancock, R. E. (1988). Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas aeruginosa.Infection and Immunity56 , 693–8

21 . Schindler, P. R. G. & Teuber, M. (1975). Action of polymyxin B on bacterial membranes: morphological changes in the cytoplasm and in the outer membrane of Salmonella typhimurium and Escherichia coli B. Antimicrobial Agents and Chemotherapy 8, 95–104.

22 . Lugtenberg, B. & Van Alphen, L. (1983). Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochimica et Biophysica Acta 737, 51–115[ISI][Medline]

23 . Hancock, R. E. W. (1991). Bacterial outer membranes: evolving concepts. American Society of Microbiology News 57, 175–82

24 . Leive, L. (1974). The barrier function of the Gram-negative envelope. Annals of the New York Academy of Sciences 235, 109–29[ISI][Medline]

25 . Hancock, R. E. W. (1984). Alterations in outer membrane permeability. Annual Review of Microbiology 38, 237–64[ISI][Medline]

26 . Leive, L. (1968). Studies on the permeability change produced in coliform bacteria by ethylenediaminetetraacetate. Journal of Biological Chemistry 243, 2373–80[Abstract/Free Full Text]

27 . Rogers, S. W., Gilleland, H. E. & Eagon, R. G. (1969). Characterization of a protein- lipopolysaccharide complex released from cell walls of Pseudomonas aeruginosa by ethylene-diaminetetraacetic acid. Canadian Journal of Microbiology 15, 743–8[ISI][Medline]

28 . Yassien, M., Khardori, n., Ahmedy, A. & Toama, M. (1995). Modulation of biofilms of Pseudomonas aeruginosa by quinolones. Antimicrobial Agents and Chemotherapy39 , 2262–8[Abstract]

29 . Martin, N. L. & Beveridge, T. J. (1986). Gentamicin interaction with Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 29, 1079–87[ISI][Medline]

30 . Vaara, M. & Vaara, T. (1983). Polycations sensitize enteric bacteria to antibiotics.Antimicrobial Agents and Chemotherapy 24, 107–13[ISI][Medline]

31 . Newton, B. A. (1953). Reversal of the antimicrobial activity of polymyxin by divalent cations. Nature 172, 160–1[ISI][Medline]

32 . Wieriks, J., Hespe, W., Jaitly, K. D., Koekkoek, P. H. & Lavy, U. (1982). Pharmacological properties of colloidal bismuth subcitrate (CBS, DE-NOL). Scandinavian Journal of Gastroenterology 17, Suppl. 80, 11–16[Medline]

33 . Hancock, R. E. W. & Bellido, F. (1992). Antibiotic uptake: unusual results for unusual molecules. Journal of Antimicrobial Chemotherapy 29, 235–9[ISI][Medline]

34 . Hancock, R. E. W., Raffle, V. J. & Nicas, T. I. (1981). Involvement of the outer membrane in gentamicin and streptomycin uptake and killing in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 19 , 777–85[ISI][Medline]

35 . Gregory, M. A., Moshal, M. G. & Spitaels, J. M. (1981). The effect of tri-potassium di-citrato bismuthate on the duodenal mucosa during ulceration. An ultrastructural study. South African Medical Journal 62, 52–5[ISI]

36 . Rokkas, T. & Sladen, G. E. (1988). Bismuth: effects on gastritis and peptic ulcer. Scandinavian Journal of Gastroenterology 23, Suppl. 142, 82–6.

37 . Wagner, S., Beil, W., Mai, U. E. H., Bokemeyer, C., Meyer, H. J. & Manns, M. P. (1994). Interaction between Helicobacter pylori and human gastric epithelial cells in culture: effect of antiulcer drugs. Pharmacology 49, 226–37[ISI][Medline]

38 . Beil, W., Bierbaum, S. & Sewing, K. F. (1993). Studies on the mechanism of action of colloidal bismuth subcitrate. I. Interaction with sulfhydryls. Pharmacology47 , 135–40[ISI][Medline]

39 . Baer, W., Koopmann, H. & Wagner, S. (1993). Effects of substances inhibiting or uncoupling respiratory-chain phosphorylation of Helicobacter pylori. International Journal of Medical Microbiology, Virology, Parasitology, and Infectious Diseases 280, 253–8

40 . Nicas, T. I. & Hancock, R. E. W. (1980). Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin. Journal of Bacteriology 143 , 872–8[ISI][Medline]

Received 19 May 1998; returned 2 July 1998; revised 27 August 1998; accepted 24 December 1998