Journal of Antimicrobial Chemotherapy (2008) 61, 353 – 361doi:10.1093/jac/dkm468Advance Access publication 10 December 2007
Broad-spectrum in vitro antibacterial activities of clay minerals
against antibiotic-susceptible and antibiotic-resistant bacterial
Shelley E. Haydel1,2*, Christine M. Remenih1 and Lynda B. Williams3
1Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ,
USA; 2School of Life Sciences, Arizona State University, Tempe, AZ, USA; 3School of Earth and Space
Exploration, Arizona State University, Tempe, AZ, USA
Received 10 September 2007; returned 15 October 2007; revised 5 November 2007; accepted 12 November 2007
Objectives: The capacity to properly address the worldwide incidence of infectious diseases lies in theability to detect, prevent and effectively treat these infections. Therefore, identifying and analysinginhibitory agents are worthwhile endeavours in an era when few new classes of effective antimicrobialshave been developed. The use of geological nanomaterials to heal skin infections has been evidentsince the earliest recorded history, and specific clay minerals may prove valuable in the treatment ofbacterial diseases, including infections for which there are no effective antibiotics, such as Buruliulcer and multidrug-resistant infections.
Methods: We have subjected two iron-rich clay minerals, which have previously been used to treatBuruli ulcer patients, to broth culture testing of antibiotic-susceptible and antibiotic-resistant patho-genic bacteria to assess the feasibility of using clay minerals as therapeutic agents.
Results: One specific mineral, CsAg02, demonstrated bactericidal activity against pathogenic Escherichiacoli, extended-spectrum b-lactamase (ESBL) E. coli, Salmonella enterica serovar Typhimurium, Pseudo-monas aeruginosa and Mycobacterium marinum, and a combined bacteriostatic/bactericidal effectagainst Staphylococcus aureus, penicillin-resistant S. aureus, methicillin-resistant S. aureus (MRSA) andMycobacterium smegmatis, whereas another mineral with similar structure and bulk crystal chemistry,CsAr02, had no effect on or enhanced bacterial growth. The <0.2 mm fraction of CsAg02 and CsAg02heated to 200 or 5508C retained bactericidal activity, whereas cation-exchanged CsAg02 and CsAg02heated to 9008C no longer killed E. coli.
Conclusions: Our results indicate that specific mineral products have intrinsic, heat-stable antibacterialproperties, which could provide an inexpensive treatment against numerous human bacterialinfections.
Keywords: infections, nanominerals, therapeutics, natural, bactericidal, bacteriostatic
aiding digestive processes and cleansing and protecting the skin.4,5Due to the small particle size (,2.0 mm), these natural geological
Medicinal and therapeutic use of mineral products has impacted
products have a vast surface area (hundreds of m2/g of clay) with
human health for thousands of years, and pure clay minerals, such
high concentrations of ions and compounds located on the surfaces.
as smectite and illite, are nanomaterials of geological origin. The
Despite the clear, beneficial effects on human health related to
intentional consumption of earth materials, such as clays, by
ridding the body of foreign substances, few studies have investi-
humans and animals is known as geophagy, a complex behaviour,
gated the antibacterial properties of clay minerals.6 – 10 Moreover,
largely attributed to religious beliefs, cultural practices, psychologi-
to our knowledge, there have been no published scientific reports
cal disorders, cosmetics, dietary/nutritional needs and medicinal
that examine the antibacterial activities of clay minerals on a
benefits.1 –3 Early research focused on the extraordinary adsorptive
broad-spectrum panel of bacterial pathogens, including antibiotic-
properties of clay minerals and the health benefits recognized in
resistant strains, that infect and cause disease in humans.
*Corresponding author. Tel: þ1-480-727-7234; Fax: þ1-480-727-0599; E-mail: [email protected]
# The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]
Haydel et al.
Documented use of the two clay minerals described in this
Salmonella enterica serovar Typhimurium ATCC 14028, P. aer-
study as a therapeutic treatment of Buruli ulcer11 suggests that
uginosa ATCC 27853, S. aureus ATCC 29213, Mycobacterium
these natural nanomaterials have significant effects on infectious
smegmatis ATCC 19420 and M. marinum ATCC 927. With the
bacteria and wound healing. In 2001, a French humanitarian
exception of the mycobacterial strains, the CLSI (formerly
working in the Ivory Coast of Africa began treating children with
NCCLS) recommends these bacteria as quality control strains
Buruli ulcer with the two clay minerals described herein. Within
for laboratory testing of antimicrobials.15 Upon receipt, all bac-
days of initiating treatment with clay poultices, the therapeutic
terial cultures were grown in the appropriate liquid medium
properties of the clay minerals were demonstrated with the
(described below) and stored at – 708C prior to use. ESBL E.
initiation of rapid, non-surgical debridement of the destroyed
tissue. Extended treatment with the clay minerals resulted in con-
Laboratories (Tempe, AZ, USA) and confirmed by MicroScan
tinued debridement of the ulcer, tissue regeneration and wound
healing. After several months of daily clay applications, the
Sacramento, CA, USA) testing to be resistant to 11 antibiotics
Buruli ulcer wounds healed with soft, supple scarring and the
(Table 1). Penicillin-resistant S. aureus (PRSA) was obtained
return of normal motor function.9,11 These therapeutic observations
from the ASU School of Life Sciences microbiological culture
are highly relevant since antibiotic treatment is only effective for
collection, subjected to MIC disc diffusion susceptibility
pre-ulcerative lesions and has generally been unsuccessful with the
testing by Sonora Quest Laboratories and confirmed to be
ulcerative form of Buruli ulcer disease.12 Currently, the only
resistant to penicillin (data not shown). MRSA was obtained
accepted treatment of an advanced Mycobacterium ulcerans infec-
from Sonora Quest Laboratories and confirmed by MicroScan
tion, the causative agent of Buruli ulcer disease, is surgical exci-
Positive MIC Panel Type 20A (Dade Behring) testing to be
sion of the ulcerative lesion along with extended healthy tissue to
resistant to 10 antibiotics (Table 2).
prevent persistent subcutaneous infection.13 This costly anddangerous treatment often leads to significant loss of tissue and
Table 1. Antimicrobial susceptibility patterns of the ESBL E. coli
possible permanent disability. To begin understanding how these
ATCC 51446 strain used in the CsAg02 and CsAr02 antimicrobial
two mineral products are effective at healing patients infected with
assays; the susceptible concentration of the ESBL E. coli strain and
M. ulcerans, we have initiated antimicrobial susceptibility testing
the qualitative susceptibility interpretation for each antibiotic are
of several human bacterial pathogens.
Here, we report an assessment of the broad-spectrum antibac-
terial properties of two different clay minerals: CsAg02, an iron-rich smectite and illite clay mineral enriched with magnesium
and potassium, and CsAr02, an iron-rich smectite and illite clay
mineral enriched with calcium.9 To assess the usefulness of thesenanominerals as antibacterial agents, we tested a range of human
bacterial pathogens, including Pseudomonas aeruginosa, extended-
spectrum b-lactamase (ESBL) Escherichia coli, methicillin-resistant
Staphylococcus aureus (MRSA) and Mycobacterium marinum.
M. marinum is genetically closely related to M. ulcerans and is
a cutaneous pathogen that can cause nodular and ulcerated skin
lesions, and can invade deeper structures including synovia,
bursae and bone.14 M. marinum infection generally occurs when
traumatized skin of an extremity is exposed to contaminated
aquariums, salt water or marine animals.14
Our in vitro microbiological investigations indicate that the
two clay minerals, although similar in major phases and bulk
chemistry,9 have striking and opposite effects on bacterial popu-
lations, ranging from enhanced microbial growth to complete
growth inhibition. This research documents the therapeutic
effect of the clay minerals previously used to treat Buruli ulcer
patients,9,11 and represents initial investigations aimed at identi-
fying the mechanism by which particular clay nanomaterials
exhibit antibacterial behaviour. Our goal is to identify new
inhibitory agents in an era when bacterial antibiotic resistance
continues to challenge human health and the availability of new
antimicrobial compounds is limited.
Materials and methods
intermediate, 32 – 64
The following bacterial strains, obtained from the American Type
Culture Collection (ATCC), were used as target antibiotic-susceptible organisms in these studies: E. coli ATCC 25922,
aThe endpoint of susceptibility interpretation is indicated for each antibiotic.
Antibacterial activities of clay minerals
Table 2. Antimicrobial susceptibility patterns of the MRSA strain
808C) and grinding, before use. X-ray diffraction analyses
used in the CsAg02 and CsAr02 antimicrobial assays; the
(limits of detection ,5%) of the French clay samples indicate
susceptible concentration of the MRSA strain and the qualitative
that they are composed predominantly of smectite and illite min-
susceptibility interpretation for each antibiotic are shown
erals. The bulk CsAg02 clay is composed of 24% Fe-illite and50% Fe-smectite, whereas the bulk CsAr02 clay is composed of27% Fe-illite, 30% Fe-smectite, 3% kaolinite and 3% chlorite.9
The surface areas of CsAg02 and CsAr02 are 115.4 and
90.5 m2/g, respectively,9 which provides a potentially large reac-tive area for ion exchange. Major element analyses of the clay
sized fraction (,2.0 mm) of CsAg02 and CsAr02 showed that
both are iron-rich ( 6% wt),9 while other clays obtained from
the supplier of CsAg02 have lower iron content (data not
shown). Levels of trace elements, including silver, arsenic and
lead, in the clay minerals were all below MICs and will be
reported elsewhere. Before use in any susceptibility testing, all
mineral samples were sterilized by autoclaving at 1218C for 1 h.
Particle size fractionation of clay minerals
Since clay samples are not pure clay minerals and can contain
numerous detrital minerals, such as quartz, feldspar and carbon-
ates, clay-sized particles of ,2.0 mm in diameter were collected
and separated by particle size. After a series of washes and ultra-
sonication of a clay suspension in distilled-deionized water to
remove chloride salts, the clay sample suspension (at room
temperature) was centrifuged in 50 mL conical tubes in a swing-
ing arm rotor with an axial distance of 15 cm from the centre
axis to the bottom of the tube. According to Stokes' law, a cen-
trifuge speed of 750 rpm for 3.3 min will settle particles
.2.0 mm as long as the water temperature, viscosity and settling
distance are fixed.17 The suspended material, which contains
,2.0 mm particles, was centrifuged at 2000 rpm for 1.8 min to
settle the 1.0 – 2.0 mm particles. For purification, this process
was performed in triplicate. After collecting the 1.0 – 2.0 mm
particles, the remaining suspension containing ,1.0 mm par-
ticles was centrifuged for 29 min at 2500 rpm to settle the 0.2 –
The endpoint of susceptibility interpretation is indicated for each antibiotic.
1.0 mm particles. The remaining ,0.2 mm particles, in theresulting suspension, were dried at 608C or less. All fractionated
Bacterial growth media and growth conditions
and collected CsAg02 clay particles were dried, crushed with a
E. coli, S. enterica serovar Typhimurium and P. aeruginosa
mortar and pestle, and sterilized by autoclaving at 1218C for 1 h.
were cultured using Luria – Bertani (LB) broth or agar, nutrient
broth or agar and trypticase soy broth or agar, respectively, at378C. S. aureus strains were cultured using trypticase soy broth
To remove the natural exchangeable cations, CsAg02 (1 g) was
or agar at 378C. Bacterial cultures of M. smegmatis and M.
saturated with 1 M KCl (25 mL) and shaken for 24 h. The clay
marinum were grown in supplemented Middlebrook 7H9 broth
was then washed and dialysed to remove Cl2, leaving Kþ ions
[Middlebrook 7H9 broth supplemented with 10% OADC (oleic
in the exchangeable sites of the clay. Potassium-exchanged
acid, albumin, dextrose and catalase), 0.2% glycerol and 0.05%
CsAg02 was dried, crushed with a mortar and pestle, and steri-
Tween 80] at 37 and 308C, respectively.16 Viable cell counting
lized by autoclaving at 1218C for 1 h.
of mycobacteria was performed on supplemented Middlebrook7H10 agar (Middlebrook 7H10 supplemented with 10% OADC)
Thermal treatment of clay minerals
after 48 h of growth at 378C for M. smegmatis and after 5 days
To gradually destroy the water and hydroxyl bonds in the
of growth at 308C for M. marinum. For the assays described
mineral structure, separate aliquots of CsAg02 were heated to
below, all bacteria were cultured with gentle rotary mixing to
200 and 5508C for 24 h. Heating to 2008C dehydrates the inter-
layer of smectite,18 whereas heating to 5508C dehydroxylates
most iron-rich clays.19 Finally, heating to 9008C for 24 hdestroys the clay structure completely, leaving only oxide com-
ponents of the clay. For this procedure, 1 – 2 g of clay was
The CsAg02 and CsAr02 minerals used in this study were both
placed in a porcelain crucible, fitted with a porcelain lid and
supplied by Brunet de Courssou11 from the batches used in the
heated to 200, 550 or 9008C in a muffle furnace. All thermal
Buruli ulcer clinics located in western Africa. The clays were
treatments were performed in air to generate a highly oxidizing
subjected to factory processing, which included drying (below
environment. Although microorganisms would be destroyed by
Haydel et al.
thermal treatments, the heated CsAg02 minerals were sub-sequently sterilized by autoclaving at 1218C for 1 h before per-forming antimicrobial assays.
Bacterial strains were grown overnight and diluted with freshmedium to achieve an approximate density of 1 107 cfu/400 mL. To confirm the initial bacterial counts, serially dilutedbacterial cultures were plated on the appropriate agar plates andenumerated. After dilution, sterilized clay minerals (200 mg)were introduced into 400 mL of media containing the initialpathogen inoculum to achieve a consistency similar to thehydrated clay poultices used to treat Buruli ulcer patients. Thebacteria – mineral mixtures were incubated with constant rotaryagitation for the appropriate times and temperatures, asdescribed above. Positive controls for growth of bacteria in theabsence of clay minerals were included in each series of inde-pendent experiments. To ensure that the clay samples were steri-lized after autoclaving and maintained sterilization duringstorage, negative control growth experiments with clay mineralsin LB broth were performed several times throughout the courseof the study. Incubation of the bacteria – clay mixtures was per-formed as follows: E. coli, S. enterica serovar Typhimurium, P.
aeruginosa, S. aureus and MRSA were incubated for 24 h at378C, M. smegmatis was incubated for 48 h at 378C and M.
marinum was incubated for 5 days at 308C. After incubation, themixtures were subjected to successive 10-fold serial dilutions in
Figure 1. Bactericidal activity of CsAg02 against susceptible and resistant
the appropriate medium, mixed with a vortex shaker to ensure
Gram-negative pathogens: (a) E. coli ATCC 25922; (b) ESBL E. coli ATCC
dispersion and quantitatively cultured in duplicate onto agar
51446; (c) S. enterica serovar Typhimurium ATCC 14028; and (d) P.
plates to determine the number of viable bacteria. Additionally,
aeruginosa ATCC 27853. The reported values represent the average and SD
100 mL of the bacteria – clay suspension was directly plated onto
of at least three independent experiments. Statistical significance ( paired
agar plates to assess the bacterial viability in undiluted samples.
t-test) of Cs-inoculated 24 h bacterial growth compared with standard 24 h
At least three independent antimicrobial assays with specific
bacterial growth: ***P ¼ 0.0006; **P ¼ 0.007.
clay minerals and specific bacterial strains were performed.
Viable cell counts are expressed as log10 cfu per 400 mL.
and P. aeruginosa (Figure 1d). Most notably, CsAg02 demon-strated a bactericidal effect against ESBL E. coli (Figure 1b),
Statistical analysis was performed using Prism 4 (GraphPad
which was resistant to 11 of the 27 tested antibiotics (Table 1).
Software, San Diego, CA, USA) and was calculated using a two-
In contrast, in the presence of the CsAr02 clay minerals, growth
tailed paired Student's t-test. A P value of ,0.05 was con-
of the susceptible and resistant E. coli strains was significantly
sidered statistically significant.
enhanced (Figure 1a and b), whereas growth of S. entericaserovar Typhimurium and P. aeruginosa was not significantlydifferent compared with bacterial growth in media alone
(Figure 1c and d). No microbial growth was evident in mineralsthat were subjected to sterilization before use in the antimicro-
To assess the effect of the CsAg02 and CsAr02 minerals on the
bial susceptibility assays (data not shown).
growth of clinically relevant Gram-negative bacteria, suscepti-
Susceptibility testing with S. aureus ATCC 29213 was per-
bility testing of E. coli ATCC 25922, ESBL E. coli ATCC
formed as described above to assess the potential broad-spectrum
51446, S. enterica serovar Typhimurium ATCC 14028 and P.
inhibitory activity of CsAg02. In addition, to assess the inhibitory
aeruginosa ATCC 27853 was performed in liquid cultures. To
activity of CsAg02 against antibiotic-resistant S. aureus, a single
achieve poultice consistencies similar to those used to treat
antibiotic-resistant strain, PRSA, and a multidrug-resistant strain,
Buruli ulcer patients, initial bacterial cultures (107 bacteria/
MRSA, were also subjected to susceptibility testing. The MRSA
400 mL) were mixed with 200 mg of clay minerals and incu-
strain exhibited resistance to 10 of the 23 tested antibiotics, includ-
bated at 378C on a rotating drum for 24 h. After incubation, the
ing methicillin (oxacillin) (Table 2). Incubation with CsAg02
bacteria – clay mixtures were diluted and plated onto agar to
minerals resulted in partial growth inhibition of all three S. aureus
determine the number of viable bacteria (Figure 1). Based on
strains while growth was unaffected in the presence of CsAr02
these susceptibility experiments performed in triplicate, incu-
minerals (Figure 2). In contrast to the complete bactericidal effect
bation with the CsAg02 clay minerals resulted in complete
demonstrated with Gram-negative bacteria, incubation with CsAg02
killing of several antibiotic-sensitive Gram-negative bacteria:
appears to have more of a bacteriostatic effect on the S. aureus
E. coli (Figure 1a), S. enterica serovar Typhimurium (Figure 1c)
strains. Compared with the initial S. aureus cfu, viability of the
Antibacterial activities of clay minerals
Figure 3. Effects of CsAg02 and CsAr02 on mycobacterial growth. Forantimicrobial susceptibility testing, M. smegmatis ATCC 19420 (a) and M.
marinum ATCC 927 (b) were grown in the presence of CsAg02 or CsAr02for 48 h at 378C and 5 days at 308C, respectively. The reported valuesrepresent the average and SD of at least three independent experiments.
Statistical significance ( paired t-test) of Cs-inoculated bacterial growthcompared with standard bacterial growth: *P , 0.05.
ulcerans, and significantly reduce the growth of the non-pathogenic M. smegmatis, whereas CsAr02 clay partially inhib-ited M. marinum growth, but had no effect on M. smegmatisgrowth. The bactericidal effect of CsAg02 and bacteriostaticeffect of CsAr02 on M. marinum growth (Figure 3b) stronglysuggest that these minerals will have a detrimental effect on the
Figure 2. Effects of CsAg02 and CsAr02 on S. aureus ATCC 29213 (a),
growth of genetically similar M. ulcerans, as indicated by the
PRSA (b) and MRSA (c) growth. Average values and SDs from three
topical application of these two minerals to effect a cure for
independent experiments are shown. Statistical significance ( paired t-test) of
Buruli ulcer patients.11 These results are especially promising
Cs-inoculated 24 h bacterial growth compared with standard 24 h bacterial
considering that there is no therapeutic cure for the ulcerative
growth: **P , 0.004; *P , 0.05.
form of Buruli ulcer disease.
To eliminate the potential that a non-clay mineral in the bulk
susceptible S. aureus strain and the PRSA and MRSA strains
clay sample is responsible for killing bacteria and to determine if
after 24 h was decreased 10-fold. The comparable effect on
the crystal structure of the minerals is important in the antibacterial
antibiotic-susceptible S. aureus and MRSA indicates that the
effectiveness, we separated the clay minerals by size fractions. By
mechanism of CsAg02 growth inhibition is unique and, more
differentially centrifuging the mineral particles, we concentrated
importantly, is unrelated to the mode of action of b-lactam,
most of the non-clay minerals in the coarse size fraction (1–
macrolide and fluoroquinolone antibiotics. These results areof significant clinical relevance considering the serious healthimplications of MRSA infections.
To assess the effect of the CsAr02 and CsAg02 clay minerals
on the growth of non-pathogenic and pathogenic mycobacterialstrains, susceptibility testing of M. smegmatis ATCC 19420 andM. marinum ATCC 927 was performed. Initial mycobacterialcultures (107 bacteria/400 mL) were incubated with 200 mg ofbulk clay at 378C on a rotating drum for 48 h (M. smegmatis) or5 days (M. marinum). After incubation, the bacteria – clay mix-tures were diluted and plated onto supplemented Middlebrook7H10 agar to determine the number of viable bacteria. Similarto the bactericidal effects demonstrated upon Gram-negativebacteria (Figure 1), incubation with CsAg02 resulted in com-plete growth inhibition of M. marinum (Figure 3b). Unlike thebactericidal effect upon M. marinum, M. smegmatis growth inthe presence of CsAg02 was reduced 1000-fold in comparisonwith cultures grown without minerals for 48 h (Figure 3a).
Figure 4. Effects of CsAg02 fractionation, Kþ-exchange and thermal
Incubation with CsAr02 clay did not significantly affect M.
treatment on E. coli growth. E. coli ATCC 25922 was grown in the absence
smegmatis growth, while M. marinum growth was decreased
of clay minerals (white bars) for 24 h, in the presence of bulk CsAr02 (black
50-fold in the presence of CsAr02 (Figure 3). Notably, the
bar) and in the presence of fractionated, Kþ-exchanged or thermally treated
CsAg02 clay was able to completely kill the pathogenic myco-
CsAg02 (grey bars) for 24 h. The reported values represent the average and
bacterial strain, M. marinum, most closely related to M.
SD of at least three independent experiments.
Haydel et al.
2 mm), while the smaller size fractions were progressively enriched
antidiarrhoeal agents and to soothe the digestive tract.4 In
in pure clay minerals. To determine if the relative surface area of
addition, kaolinite and smectite clay minerals are hallmark addi-
mineral crystals was an important parameter for antibacterial
tives used by the cosmetic industry in topical applications to
activity, various crystal size fractions of CsAg02 (,0.2, 0.2 – 1.0
serve as skin protectants and to absorb skin secretions.4 The data
and 1.0 – 2.0 mm) were tested against E. coli (Figure 4). Results
obtained in this study, together with biosafety studies with animal
indicate that the finest size fraction (,0.2 mm), which makes up
and controlled human trials, will be essential in supporting and
the greatest percentage of the bulk sample, is bactericidal, whereas
validating the use of specific minerals or derived products as
the larger CsAg02 size fractions showed no statistically significant
inexpensive antibacterial agents. Analyses of the chemical inter-
effect on E. coli growth (Figure 4).
action at the mineral – bacterial interface are ongoing and will be
Standard cation exchange was performed on CsAg02 where the
pertinent in understanding the mechanism by which bacterial
natural exchange sites ( primarily interlayer ions in the expandable
growth inhibition occurs.
smectite mineral) were replaced by Kþ.17 Incubation of E. coli
Buruli ulcer is recognized by the WHO as a global health
with Kþ-exchanged CsAg02 resulted in complete loss of bacteri-
threat, and recent observations showing that the clay minerals
cidal activity (Figure 4). These results indicate that a chemical
used in this study were effective at healing necrotic Buruli ulcer
exchange from CsAg02 is responsible for the antibacterial activity
disease11 prompted our investigations of these geological nano-
and that surface properties of the clay have no direct effect on bac-
materials. The use of clay minerals in the treatment and healing
terial growth. Similar to CsAr02, Kþ-exchanged CsAg02
of Buruli ulcer lesions represents great promise for the develop-
enhanced the growth of E. coli (Figure 4).
ment of an inexpensive cure for many skin diseases and topical
Clay minerals can be heated to gradually remove or destroy
infections. Our aim is to evaluate the effect of these minerals
different bonds in the mineral structure.20 By progressively
on M. ulcerans, but initial investigations have been directed
destroying different bonds and vaporizing mineral elements in
towards assessing the broad-spectrum antibacterial properties of
thermal reactions, we may determine whether variable clay
specific minerals against several bacterial pathogens.
structures and/or chemical characteristics of the mineral play a
CsAg02 and CsAr02 are geological materials, primarily com-
role in the antibacterial effectiveness of CsAg02. Heating the
posed of the minerals smectite and illite, which contain variable
clay to 2008C sterilizes the sample, dehydrates the minerals and
elemental constituents and adsorbed ions. The CsAg02 and
collapses the mineral interlayers, while mineral dehydroxylation
CsAr02 minerals have been used previously to treat children
and decomposition and combustion of organic matter occurs
with Buruli ulcer.11 The CsAg02 mineral exhibits bactericidal
after heating to 5508C.20,21 Heating the clay to 9008C destroys
activity against E. coli, ESBL E. coli, S. enterica serovar
its silicate structure, oxidizes the clay components and releases
Typhimurium, P. aeruginosa and M. marinum, and significantly
many elements that are volatile at lower temperatures.20 Since
reduces growth of S. aureus, PRSA, MRSA and non-pathogenic
autoclaving the CsAg02 clay to achieve sterilization does not
M. smegmatis 1000-fold compared with cultures grown without
affect its antibacterial activity, it was not unexpected to deter-
added mineral products. We have demonstrated that the CsAg02
mine that heating the CsAg02 ,0.2 mm fraction to 2008C was
inhibitory effect occurs in liquid culture medium, while a similar
not detrimental to its bactericidal properties (Figure 4). Heating
mineral, CsAr02, displays enhanced or no effect on bacterial
the CsAg02 ,0.2 mm fraction to 5508C also resulted in reten-
viability in liquid culture. To our knowledge, these experiments
tion of its bactericidal activity on E. coli (Figure 4), indicating
represent precedential investigations of a novel geological nano-
that CsAg02 organic compounds and elements bound to the
material that displays broad-spectrum antibacterial effects against
hydroxyls are not associated with antibacterial activity. Upon
human pathogens, including antibiotic-resistant strains.
exposure to 9008C-heated CsAg02, E. coli growth was unaf-
Clay minerals are layered substances comprised of sheets of
fected and was similar to control cultures lacking clay minerals
silicate tetrahedra (SiO4) and octahedra (containing Al, Mg and
(Figure 4). Since CsAg02 no longer kills after heating to 9008C,
Fe). Smectite and illite, the primary minerals in CsAg02 and
the remaining oxides, non-volatile elements in their oxidized
CsAr02, are similar in structure (2:1 layer clays), but the silicate
forms and increased surface area are not associated with the
layers of the smectite are separated by an expandable interlayer
region containing water, cations and molecules. If the interlayerregion is collapsed due to a high attraction between the silicatesheets with cations ( primarily Kþ) balancing the charge, then
the mineral is illite. The interlayer surfaces of smectite are nega-tively charged due to substitution of Al3þ for Si4þ in some of
Worldwide, the number of antibiotic-resistant pathogenic bac-
the tetrahedral sites, and Mg2þ for Al3þ (for example) in octa-
teria has substantially increased within the past 50 years.22
hedral sites. Therefore, cations are most commonly attracted to
These alarming trends, reduced drug discovery and development
the interlayer, with Kþ, Naþ, Ca2þ and NHþ
productivity,23 and the emergence and increasing incidence of
and forming weak surface bonds. The edges of the crystals can
antibiotic-resistant infections, such as S. aureus, Streptococcus
have a positive charge and attract anions such as hydroxyls,
pneumoniae, Enterococcus faecalis and Mycobacterium tubercu-
phosphates or sulphates.29 Water can enter the interlayer and
losis,24 – 28 indicate a progressive need to identify and analyse
expand the clay structure to accommodate additional com-
new antibacterial agents.
pounds, including neutral and negatively charged species. Illite
Numerous mineral products are currently used for therapeutic
has the same structure as smectite, but has a higher layer charge,
purposes in the pharmaceutical and cosmetic industries. For
which attracts and holds interlayer cations ( particularly Kþ)
example, smectite minerals adhere to the gastrointestinal mucosa
tightly. Such interlayer cations are not easily exchanged with
to adsorb and rid the body of dissolved toxins, bacteria and
solutions, but are ‘fixed-cations'.17 Because of the special
viruses, whereas kaolinite and palygorskite are primarily used as
quality of smectite for incorporating various ions, the surface
Antibacterial activities of clay minerals
can be either hydrophilic or hydrophobic depending on the
element components, exchanged ions and surface properties of
charge and available solutes. A hydrophobic surface is inher-
the clays are in progress. However, there is not a single com-
ently organophyllic and could harbour an organic substance that
ponent of the CsAg02 clay (e.g. transition metals) that stands
is lethal to bacteria. If the mineral surface is hydrophilic, it might
out as an obvious antibacterial agent, so it may be a fortuitous
compete with bacteria for cations that are essential nutrients for
combination of factors (multiple components) responsible for
metabolic function or release a toxic inorganic substance that
the inhibitory property. Further work is in progress to identify
either inhibits a particular metabolic function or precipitates on
additional antibacterial nanominerals, to isolate chemicals
the cell wall.
released from the antimicrobial clays and to simulate the effect
Enhanced growth of bacteria upon incubation with clay min-
using synthetic materials.
erals, as demonstrated with CsAr02, is not an uncommon occur-
Clay minerals can affect bacterial metabolism indirectly by
rence, as microorganisms inherently require numerous trace
altering the physicochemical properties of a specific environ-
elements to facilitate growth. Montmorillonite, a variety of 2:1
ment or directly through surface interactions.40 Although we
layer smectite and kaolinite, a 1:1 layer clay composed of one
demonstrate that the nanoparticle-sized fraction (,200 nm) of
tetrahedral layer and one octahedral layer, can stimulate bacterial
CsAg02 retains bactericidal activities against E. coli, cation-
growth and promote biofilm formation to serve as important
exchanged CsAg02 completely loses its antibacterial effectiveness.
bioremediation substances.30 For example, Shewanella oneiden-
Therefore, we hypothesize that the physicochemical properties
sis respires structural ferric iron bound by smectite and uses it as
of hydrated CsAg02 indirectly kill bacteria by generating an
the sole electron acceptor for bacterial growth.31 Additionally,
unfavourable environment. Since the physicochemical properties
clay minerals may serve to protect environmental bacteria from
of iron-rich smectite can be greatly affected by the structural Fe
UV irradiation or toxic substances.32 By providing structural
oxidation state,41 determining the oxidation state of Fe in the
support and organic or inorganic nutrient acquisition via its high
CsAg02 nanoparticle-sized fraction, which contains primarily
cationic exchange capacity, clay minerals, particularly montmor-
iron-rich illite and smectite minerals,9 is important. Thermal inac-
illonite, may protect bacteria and serve as a minimal nutritional
tivation of the antibacterial properties when CsAg02 is heated to
sphere for bacterial proliferation.33
9008C could indicate that the change in oxidation state of inherent
Since cation exchange eliminated the antibacterial activity
elements, including Fe which is highly oxidized at 9008C, or the
of CsAg02, we have analysed the exchange solution by induc-
loss of vaporized elements removes the critical components of the
tively coupled plasma mass spectrometry (ICP-MS) to assess the
abundance of elements in the exchange solution relative to the
Reactive oxygen species, including oxygen ions, free radicals
total amount in the clay (L. B. Williams and S. E. Haydel,
and peroxides, are by-products of aerobic bacterial metabolism
unpublished results). The most significantly abundant elements
with demonstrated toxic effects on bacteria.42 Mediated by the
removed by cation exchange are silicon (30% of total), barium
Fenton reaction, hydroxyl radicals are formed by the reduction
(35% of total) and strontium (45% of total) (L. B. Williams and
of hydrogen peroxide and ferrous iron,42,43 and cause oxidative
S. E. Haydel, unpublished results). Since a silicon concentration
damage to bacterial DNA, proteins and lipids.44 – 48 Furthermore,
of .5000 mg/L (178 mM) is toxic for E. coli,34 the levels of
numerous transition metals, which are inherent in clay minerals,
silicon in the CsAg02 exchange solution (96 mg/L; 3.42 mM)
can also participate in Fenton-like reactions to produce hydroxyl
are not likely to be important for bactericidal activity. High
radicals.49 The combination of elevated levels of reduced iron in
levels of barium (144 mg/L; 1.05 mM) and strontium (154 mg/
CsAg02 and excessive free radical production in the presence of
L; 1.76 mM) in the CsAg02 exchange solution could be of inter-
oxygen could cause oxidative stress and damage to bacterial
est since these elements potentially could be acquired by cation
cells, resulting in death.50,51 Most importantly, Kohanski et al.52
uptake systems, inhibit transport mechanisms or substitute as
recently demonstrated that three major classes of bactericidal
enzymatic cofactors in bacterial cells to interfere with normal
antibiotics stimulate production of hydroxyl radicals via Fenton
physiological processes.35 – 39 Although the CsAr02 exchange
chemistry to contribute to bacterial cell death. However, regard-
solution contains slightly lower levels of barium (119 mg/L)
ing CsAg02, additional experiments are necessary to assess the
(L. B. Williams and S. E. Haydel, unpublished results), E. coli
effect that varying redox environments and chemical speciation
cultures incubated in the presence of high levels of barium
changes have on microbial viability.
(144 mg/L) grew normally (data not shown). Thus far, no single
During the past 25 years, 70% of newly discovered drugs
element identified in the CsAg02 cation exchange solution was
introduced in the USA have been derived from natural pro-
greatly different than the concentration identified in the CsAr02
ducts.23 Our discovery that natural geological minerals harbour
exchange solution or was determined to be solely responsible for
antibacterial properties should provide impetus for exploring ter-
E. coli toxicity (L. B. Williams and S. E. Haydel, unpublished
restrial sources for the presence of novel therapeutic compounds.
results). Therefore, the possibility exists that the antibacterial
Combining the availability of natural bioactive resources with
activity could be attributed to a combination of elements and/or
powerful combinatorial chemistry optimization methodologies
chemical compounds that work in concert to mediate toxicity.
could result in the development of new antibacterial agents to
The uniqueness of the French clay minerals is evident consid-
fight existing antibiotic-resistant infections and diseases for
ering that it has previously been used to treat children afflicted
which there are no known therapeutic agents, such as advanced
with Buruli ulcer,9,11 and shown to kill or significantly decrease
M. ulcerans infections.
the growth of both antibiotic-susceptible and antibiotic-resistanthuman bacterial pathogens. Moreover, of the six independentclay samples collected from the French supplier and testedagainst various bacteria (data not shown), only CsAg02 dis-played antibacterial effects. Analyses of the various trace
Haydel et al.
14. Edelstein H. Mycobacterium marinum skin infections. Report of
31 cases and review of the literature. Arch Intern Med 1994; 154:
We thank the ASU College of Liberal Arts and Sciences for sup-
1359 – 64.
porting travel to France for sample collection and geologic field
investigations, Sonora Quest Laboratories (Tempe, AZ, USA)
Performance Standards for Antimicrobial Susceptibility Testing—
for providing quality control bacterial strains and for performing
Fourteenth Edition: Approved Standard M100-S15. NCCLS, Wayne,
the antimicrobial susceptibility panels and Dr Thierry Ferrand
PA, USA, 2002.
for supplying additional clay samples. Thierry Brunet de
16. Aubry A, Jarlier V, Escolano S et al. Antibiotic susceptibility
Courssou is responsible for alerting us to the humanitarian
pattern of Mycobacterium marinum. Antimicrob Agents Chemother
efforts and clinical observations of his mother, Line Brunet de
2000; 44: 3133 –6.
Courssou, who passed away in 2006. We appreciate her hard
work and careful observations of the effect of clay minerals on
Madison, WI: University of Wisconsin, 1974.
the wounds of Buruli ulcer patients in western Africa.
18. Moore DM, Reynolds RC. X-ray Diffraction and the Identification
and Analysis of Clay Minerals, 2nd edn. New York, NY: OxfordUniversity Press, 1997.
19. Frost RL, Ruan H, Kloprogge JT et al. Dehydration and dehy-
droxylation of nontronites and ferruginous smectite. Thermochim Acta2000; 346: 63– 72.
This research was supported by Public Health Service grant
20. Giese RF. Differential scanning calorimetry of clay minerals and
AT003618 from the National Institutes of Health to L. B. W.
their intercalates. In: Stucki JW, Bish DL, eds. Thermal Analysis in
and S. E. H., and an ASU research initiatives grant to S. E. H.
Clay Science. Chantilly, VA: Clay Minerals Society, 1990; 10– 48.
21. Guggenheim S, Koster van Groos AF. Baseline studies of the
clay minerals society source clays: thermal analysis. Clay Clay Miner2001; 49: 433 – 43.
22. Andersson DI, Levin BR. The biological cost of antibiotic resist-
ance. Curr Opin Microbiol 1999; 2: 489 – 93.
None to declare.
23. Newman DJ, Cragg GM. Natural products as sources of new
drugs over the last 25 years. J Nat Prod 2007; 70: 461 – 77.
24. Diederen BM, Kluytmans JA. The emergence of infections with
community-associated methicillin resistant Staphylococcus aureus.
J Infect 2006; 52: 157 – 68.
1. Abrahams PW, Parsons JA. Geophagy in the tropics: a literature
25. Menichetti F. Current and emerging serious Gram-positive infec-
review. Geogr J 1996; 162: 63– 72.
tions. Clin Microbiol Infect 2005; 11: 22 –8.
2. Aufreiter S, Hancock RGV, Mahaney WC et al. Geochemistry
26. Shah PM. The need for new therapeutic agents: what is the
and mineralogy of soils eaten by humans. Int J Food Sci Nutr 1997;
pipeline? Clin Microbiol Infect 2005; 11: 36– 42.
48: 293 – 305.
27. Sharma R, Sharma CL, Kapoor B. Antibacterial resistance:
3. Hunter JM. Geophagy in Africa and in the United States. Geogr
current problems and possible solutions. Indian J Med Sci 2005; 59:
Rev 1973; 63: 170 – 95.
4. Carretero MI. Clay minerals and their beneficial effects upon
28. Zetola N, Francis JS, Nuermberger EL et al. Community-
human health. A review. Appl Clay Sci 2002; 21: 155 – 63.
acquired methicillin-resistant Staphylococcus aureus: an emerging
5. Viseras C, Lopez-Galindo A. Pharmaceutical applications of
threat. Lancet Infect Dis 2005; 5: 275 – 86.
some spanish clays (sepiolite, palygorskite, bentonite): some preformu-
29. Brindley GW, Brown GC. Crystal Structures of Clay Minerals
lation studies. Appl Clay Sci 1999; 14: 69 – 82.
and their X-ray Identification. London: Mineralogical Society, 1980.
6. Herrera P, Burghardt RC, Phillips TD. Adsorption of Salmonella
30. Chaerun SK, Tazaki K, Asada R et al. Interaction between clay
enteritidis by cetylpyridinium-exchanged montmorillonite clays. Vet
minerals and hydrocarbon-utilizing indigenous microorganisms in high
Microbiol 2000; 74: 259 – 72.
concentrations of heavy oil: implications for bioremediation. Clay Miner
7. Hu CH, Xu ZR, Xia MS. Antibacterial effect of Cu2þ-exchanged
2005; 40: 105 – 14.
montmorillonite on Aeromonas hydrophila and discussion on its mech-
31. Kostka JE, Dalton DD, Skelton H et al. Growth of iron(III)-
anism. Vet Microbiol 2005; 109: 83 – 8.
reducing bacteria on clay minerals as the sole electron acceptor and
8. Tong G, Yulong M, Peng G et al. Antibacterial effects of the
comparison of growth yields on a variety of oxidized iron forms. Appl
Environ Microbiol 2002; 68: 6256 – 62.
Salmonella choleraesuis. Vet Microbiol 2005; 105: 113 –22.
32. Bitton G, Henis Y, Lahav N. Effect of several clay minerals and
9. Williams LB, Holland M, Eberl DD et al. Killer clays! Natural anti-
humic acid on the survival of Klebsiella aerogenes exposed to ultra-
bacterial clay minerals. Mineral Soc Bull 2004; 139: 3 –8.
violet irradiation. Appl Microbiol 1972; 23: 870 – 4.
10. Wilson MJ. Clay mineralogical and related characteristics of geo-
33. Lu¨nsdorf H, Erb RW, Abraham WR et al. ‘Clay hutches': a novel
phagic materials. J Chem Ecol 2003; 29: 1525 –47.
interaction between bacteria and clay minerals. Environ Microbiol 2000;
11. Brunet de Courrsou L. Study Group Report on Buruli Ulcer
2: 161 – 8.
Treatment with Clay. Fifth WHO Advisory Group Meeting on Buruli
34. Adams LK, Lyon DY, McIntosh A et al. Comparative toxicity of
Ulcer, Geneva, Switzerland, 2002. Geneva, Switzerland: WHO.
nano-scale TiO2, SiO2 and ZnO water suspensions. Water Sci
12. van der Werf TS, van der Graaf WT, Tappero JW et al.
Technol 2006; 54: 327 –34.
Mycobacterium ulcerans infection. Lancet 1999; 354: 1013– 8.
35. Cuzic S, Hartmann RK. Studies on Escherichia coli RNase P
13. Weir E. Buruli ulcer: the third most common mycobacterial infec-
RNA with Zn2þ as the catalytic cofactor. Nucleic Acids Res 2005; 33:
tion. Can Med Assoc J 2002; 166: 1691.
2464 – 74.
Antibacterial activities of clay minerals
36. Estrela C, Sydney GB, Bammann LL et al. Mechanism of action
44. Beauchamp C, Fridovich I. A mechanism for the production of
of calcium and hydroxyl ions of calcium hydroxide on tissue and bac-
ethylene from methional. The generation of the hydroxyl radical by
teria. Braz Dent J 1994; 6: 85– 90.
xanthine oxidase. J Biol Chem 1970; 245: 4641 – 6.
37. Snijder HJ, Dijkstra BW. Bacterial phospholipase A: structure
45. McCord JM, Day ED Jr. Superoxide-dependent production of
and function of an integral membrane phospholipase. Biochim Biophys
hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett 1978; 86:
Acta 2000; 1488: 91 –101.
38. Tsuchiya T, Rosen BP. Characterization of an active transport
46. Moody CS, Hassan HM. Mutagenicity of oxygen free radicals.
system for calcium in inverted membrane vesicles of Escherichia coli.
Proc Natl Acad Sci USA 1982; 79: 2855– 9.
J Biol Chem 1975; 250: 7687– 92.
39. Wackett LP, Dodge AG, Ellis LB. Microbial genomics and the
scavenging enzymes in Escherichia coli suppresses spontaneous
periodic table. Appl Environ Microbiol 2004; 70: 647 –55.
mutagenesis and sensitivity to redox-cycling agents in oxyR – mutants.
EMBO J 1988; 7: 2611 – 7.
40. Stotzky G. Influence of soil mineral colloids on metabolic pro-
cesses, growth, adhesion, and ecology of microbes and viruses. In:
48. Storz G, Christman MF, Sies H et al. Spontaneous mutagenesis
Huang PM, Schnitzer M, eds. Interactions of Soil Minerals with Natural
and oxidative damage to DNA in Salmonella typhimurium. Proc Natl
Organics and Microbes. Wisconsin: Soil Society of America, Inc.,
Acad Sci USA 1987; 84: 8917 – 21.
1986; 305 – 427.
49. HaMai D, Bondy SC, Becaria A et al. The chemistry of transition
metals in relation to their potential role in neurodegenerative pro-
41. Stucki JW, Bailey GW, Gan H. Oxidation-reduction mechanisms
cesses. Curr Top Med Chem 2001; 1: 541 – 51.
of iron-bearing phyllosilicates. Appl Clay Sci 1996; 10: 417 –30.
50. Keyer K, Imlay JA. Superoxide accelerates DNA damage by ele-
42. Imlay JA, Linn S. DNA damage and oxygen radical toxicity.
vating free-iron levels. Proc Natl Acad Sci USA 1996; 93: 13635 –40.
Science 1988; 240: 1302 – 9.
51. Touati D. Iron and oxidative stress in bacteria. Arch Biochem
43. Imlay JA, Chin SM, Linn S. Toxic DNA damage by hydrogen
Biophys 2000; 373: 1 – 6.
peroxide through the Fenton reaction in vivo and in vitro. Science
52. Kohanski MA, Dwyer DJ, Hayete B et al. A common mechanism
1988; 240: 640 – 2.
of cellular death induced by bactericidal antibiotics. Cell 2007; 130:797 –810.
Hypothalamic Proopiomelanocortin Neurons Are Glucose Responsive and Express KATP ChannelsNurhadi IbrahimDepartment of Physiology and Pharmacology Martha A. BoschDepartment of Physiology and Pharmacology James L. SmartGeorge Fox University, [email protected] Jian QiuDepartment of Physiology and Pharmacology Marcelo RubinsteinInstituto de Investigaciones en Ingenierıa Genetica y Biologia Molecular
PROCESSUS STRATEGIQUES : DES ELEMENTS CLES POUR COMPRENDRE L'APRES FUSION : LE CAS SANOFI AVENTIS Philippe REBIERE Professeur Associé (ICN Business School) Université Nancy 2 CEREFIGE Cahier de Recherche n°2010-02 Université Nancy 2 13 rue Maréchal Ney Téléphone : 03 54 50 35 80