Revista de Ciencias Tecnológicas (RECIT). Volumen 3 (1): 10-22
Revista de Ciencias Tecnológicas (RECIT). Universidad Autónoma de Baja California ISSN 2594-1925
Volumen 4 (3): 157-170. Julio-Septiembre 2021 https://doi.org/10.37636/recit.v43157170.
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ISSN: 2594-1925
Layered double hydroxides: application in the inhibition of
coliforms
Hidróxidos dobles estratificados: aplicación en la inhibición de
coliformes
Roberto Guerra-González
1
, Martha Angélica Lemus-Solorio
2
, José Luis Rivera-Rojas
2
, Alfonso Lemus-
Solorio
1
, América Abisay Mondragón-Herrera
1
, Marco Antonio
Martínez-Cinco
1
1
Facultad de Ingeniería Química; Universidad Michoacana de San Nicolás de Hidalgo
2
Facultad de Ciencias Físico-Matemáticas; Universidad Michoacana de San Nicolás de Hidalgo
Ciudad Universitaria; Avenida Francisco J. Múgica S/N Ciudad Universitaria, Edificio “E”, Planta Alta. Laboratorio de
Investigación. Morelia, Michoacán; México.
Corresponding autor: Alfonso Lemus-Solorio, Facultad de Ingeniería Química; Universidad Michoacana de San Nicolás de
Hidalgo, Ciudad Universitaria; Avenida Francisco J. Múgica S/N Ciudad Universitaria, Edificio “E”, Planta Alta.
Laboratorio de Investigación. Morelia, Michoacán; México. E-mail: 1209689x@umich.mx. ORCID: 0000-0003-2736-5600.
Recibido: 24 de Marzo del 2021 Aceptado: 24 de Julio del 2021 Publicado: 31 de Agosto del 2021
Abstract. - In this work, the preparation of different organic/inorganic hybrid materials and their evaluation as bactericides against
Escherichia coli (E. coli) and Salmonella typhi (S. typhi) was studied. The main objective of the present investigation was to synthesize
and characterize biocompatible hybrid materials that immobilize molecules with antibacterial activity in inorganic lamellar double
hydroxides based inorganic lamellar matrices and to evaluate their antibacterial activity against Escherichia coli (E. coli) and
Salmonella typhi (S. typhi). The hybrid materials consist of the association of an inorganic lamellar double hydroxide, or hydrotalcite-
type compounds, with organic molecules with antibacterial activity, hosted in solids. Lamellar double hydroxides (LDH) are synthetic
structures formed by positively charged metal hydroxide films that are stabilized with interlamellar anions. Different hybrid materials
have been studied from hydrotalcite-type compounds, such as MgAl, ZnAl and MgFeAl, containing organic species of sodium
cephalexin and nalidixic and pipemidic acids. The intercalation of the different anions was performed by one of the different existing
methods: coprecipitation of the hydrotalcite-type compounds in the presence of the molecule of interest, and by the memory effect.
The characterization of the materials was carried out by X-ray diffraction, IR and solid nuclear magnetic resonance spectroscopy,
specifically analyzing the
27
Al and
13
C nuclei, and thermogravimetric analysis. The evaluation of the antibacterial activity of these
materials was evaluated on cultures of Escherichia coli (E. coli) and Salmonella typhi (S. typhi) strains. The antibacterial activity of
the tested hybrid systems is not always a direct function of the amount of antibiotic intercalated. It was obtained that the LDH ZnAl-
NADmem presents a controlled release, since when the material was exposed three times against Escherichia coli (E. coli) bacteria,
it continued eliminating bacteria, presenting a bacteriostatic effect in the third exposure, since it did not eliminate bacteria.
Keywords: inhibition, hybrid materials, antibacterial.
Resumen. - En este trabajo se estudió la preparación de diferentes materiales híbridos orgánicos / inorgánicos y su evaluación como
bactericidas frente a Escherichia coli (E. coli) y Salmonella typhi (S. typhi). El objetivo principal de la presente investigación fue
sintetizar y caracterizar materiales híbridos biocompatibles que inmovilizan moléculas con actividad antibacteriana en matrices
lamelares inorgánicas basadas en dobles hidróxidos lamelares inorgánicos y evaluar su actividad antibacteriana frente a Escherichia
coli (E. coli) y Salmonella typhi (S. typhi). Los materiales híbridos consisten en la asociación de un doble hidróxido laminar
inorgánico, o compuestos tipo hidrotalcita, con moléculas orgánicas con actividad antibacteriana, alojadas en sólidos. Los hidróxidos
dobles lamelares (LDH) son estructuras sintéticas formadas por películas de hidróxido metálico con carga positiva que se estabilizan
con aniones interlaminares. Se han estudiado diferentes materiales híbridos a partir de compuestos tipo hidrotalcita, como MgAl,
ZnAl y MgFeAl, que contienen especies orgánicas de cefalexina sódica y ácidos nalidíxico y pipemídico. La intercalación de los
diferentes aniones se realizó mediante uno de los diferentes métodos existentes: la coprecipitación de los compuestos tipo hidrotalcita
en presencia de la molécula de interés y por el efecto memoria. La caracterización de los materiales se realizó mediante difracción
de rayos X, espectroscopia de IR y resonancia magnética nuclear sólida, analizando específicamente los núcleos
27
Al y
13
C, y análisis
termogravimétrico. La evaluación de la actividad antibacteriana de estos materiales se evaluó en cultivos de cepas de Escherichia
coli (E. coli) y Salmonella typhi (S. typhi). La actividad antibacteriana de los sistemas híbridos probados no siempre es una función
directa de la cantidad de antibiótico intercalado. Se obtuvo que el LDH ZnAl-NADmem presenta una liberación controlada, ya que
cuando el material fue expuesto tres veces contra la bacteria Escherichia coli (E. coli), continuó eliminando bacterias, presentando
un efecto bacteriostático en la tercera exposición, ya que no eliminar las bacterias.
Palabras clave: inhibición, materiales híbridos, antibacteriano.
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1. Introduction
There are many disadvantages associated with
the use of certain drugs. Drugs are distributed in
the body according to their physical properties,
such as solubility, partition coefficient and
charge. Consequently, drugs can reach a wide
variety of sites where they may be outside their
therapeutic range, may be inactive, or their action
may be undesirable or harmful, and therefore
have negative side effects. There are currently
two methods to enhance drug action [1]:
Controlled release, which attempts to eliminate
or reduce side effects by producing a therapeutic
concentration of the drug that is stable in the
organism. It attempts to achieve zero-order
release kinetics and there are usually no changes
in the concentration of the drug in the body
(compared to intermittent concentration changes
in conventional dosing), and site-directed
release, which tries to ensure that the drug is
released at the required site, while keeping the
drug inactive elsewhere in the body.
Today there is an incessant demand for advances
in the field of controlled release of biologically
or chemically active and environmentally
sensitive molecules. The incorporation or
immobilization of biologically active molecules
into lamellar inorganic matrices allows their
isolation from the environment while improving
their stability and long-term storage. Thus, the
stabilization of active molecules in
biocompatible inorganic materials constitutes an
interesting route for the preparation of hybrid
materials that possess both the advantages of the
properties of the inorganic host material and
those of the organic host, in the same material.
Subsequent release of the active species, if
desired, is carried out by simple processes of
bipolar or anion exchange interactions with ions
present in the medium with which the hybrid
material is contacted.
Apart from the problem of storage and stability
of the active species we can find one more
problem linked to the release process of the
active species. An inefficient release system can
result in high concentrations of the drug where it
is not needed causing possible side effects; or in
a rapid drop of the drug concentration below the
desired levels. These problems can be solved by
designing new systems for the administration and
controlled release of active ingredients. These
systems should provide kinetic profiles in which
the concentration of the molecule remains at the
appropriate concentration levels and for an
adequate period of time [2].
Technologies to minimize the presence of
polluting chemical species in effluents or
watercourses have been studied and developed
for many decades. Among the many existing
possibilities, the immobilization of pollutants,
i.e., their passage from the liquid phase to a solid
phase, is widely used. Transport between these
phases can be driven by various physicochemical
phenomena. The processes involved can be as
varied as adsorption, dissolution-reprecipitation,
co-precipitation, occlusion and ion exchange.
The strategies used to date have been based
mainly on adsorption or
precipitation/arrestration. However, given that
the choice of substrates to be used depends not
only on the type of pollutant, but also on its
chemical, structural and textural characteristics,
which offer a universe of possibilities, these
systems are still under study. In addition to the
various immobilization alternatives, both anions
and cations can be fixed by ion exchange
reactions with suitable substrates. However, this
process has not been massively applied on a real
or plant scale due to the high cost of exchange
resins of synthetic origin. As a counterpart, the
use of inorganic exchangers seems promising.
Most of the available information deals with
cation exchange in natural clays. To a large
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extent, this is due to their implications on the
mobility and bioavailability of cations
(contaminants, or not) in soils and water
reservoir beds} Complementing the behavior of
clays, LDH-type structures allow the possibility
of exchanging the anions occupying their
interlaminar spaces. LDHs are easily
synthesized, in a wide range of compositions, and
at low cost. If the LDH components are properly
chosen, we will have an inorganic anion
exchanger [3];
Although the ability of LDHs to exchange anions
has been known for decades, few works have
focused on their study, and of all of them, only a
few discuss the application of these processes in
a real problem [4]. It is interesting then, to
evaluate the factors that control the
immobilization of ecotoxic anions in synthetic
Mg-Al LDHs, and to explore the possible
application of these solids as retention agents in
effluent treatments or remediation processes. The
use of layered double hydroxides (LDHs) as
anion immobilization agents also requires
knowledge of their stability under the operating
conditions in which they are planned to be used.
Interestingly, the thermodynamic and kinetic
stability of these phases is practically unknown.
Although it is known that these phases can be
prepared by coprecipitation, which implies that,
under certain conditions, they are more insoluble
than their component hydroxides, their solubility
products are still an unknown.
There is only one paper on this subject [5].
Nothing is known about their dissolution kinetics
either. Some authors only mention that they have
observed the leaching of the most soluble cation
[6]. Others suggest that this process is slower
than the dissolution of pure hydroxide [7]. Still
others simply ignore this possibility, and subject
LDH to conditions where dissolution may be
total [8].
In the case of nanopharmaceutics, the efficacy of
active ingredients depends on their intrinsic
physical and chemical properties, in addition to
their ability to be properly administered into the
body. In this sense, we are currently seeking to
overcome the limitations of therapeutics and to
minimize toxic side effects in order to carry out
good drug delivery. Thus, work has been done,
for example, on the development of mechanisms
to increase the half-life of the drug in plasma,
increase the stability of the active ingredient or
maximize its therapeutic activity.
The ideal objective of systems for the
administration and controlled release of
biologically active molecules contemplates two
important aspects: spatial localization and
temporal or controlled release of the active
molecule. Spatial localization is related to the
fact that the molecule can reach a specific organ
or tissue. Controlled release refers to the control
of the rate of release of the active species at the
site where it is required. These two aspects
cannot always be achieved and, therefore, in
many cases, advances in research are still needed
to propose new systems for the administration
and controlled release of active molecules. For
this, both the vehicle and the route of
administration, as well as the target (organ or
tissue) must always be taken into consideration
in order to propose a strategy that allows
increasing therapeutic efficiency and, in many
cases, decreasing the effects.
In recent research (2012) Martinez D.R. &
Carbajal G.G. on the subject of LDHs, were
dedicated to review the chemical and structural
characteristics of these compounds, the methods
of synthesis, their intercalation or
functionalization products and reaffirm the
various areas in which they can be applied.
In 2019 Aristizabal D. managed to optimize the
conditions to synthesize nano LDH with the
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appropriate properties to be used as drug
nanocarriers and describe the interaction of
nanocarriers with biological fluids, starting with
the incorporation of a protein, up to more
complex fluids, such as fetal bovine serum.
2. Methodology
The method of preparation of the LDH ZnAl is
described, as well as the preparation of the hybrid
materials from the LDH and the organic anion of
nalidixic acid. The techniques used for the
characterization of the different materials are
also described.
2.1 Homogeneous coprecipitation method with
urea
The ZnAl-NO
3
solid was synthesized by the urea
hydrolysis method, traditionally, due to the
products of urea hydrolysis (A. Inayat et al.,
2011), this method leads to the formation of LDH
with carbonate anions in the interlamellar region.
This result is independent of the type of metal
salts used in the syntheses (chlorides or nitrates).
Thus, Zn
+2
and Al
+3
cations precipitate in the
form of LDH due to the controlled hydrolysis of
urea at 90°C from a solution of Zn and Al
nitrates.
During the synthesis, the pH of the solution is
gradually increased as the hydrolysis of urea
proceeds, while achieving homogeneous local
concentrations resulting in the formation of
solids of higher crystallinity, larger crystal size
(on the order of µm) and homogeneous crystal
size distribution, compared to solids synthesized
by the coprecipitation method under high or low
supersaturation conditions. To avoid the
intercalation of CO
2
, in the form of CO
3
-2
, from
the hydrolysis of urea, an excess of NH
4
NO
3
was
added to provide NO
3
-
ions in solution.
For the synthesis of the solid ZnAl-NO
3
, 0.335
mole of Zn (NO
3
)
2
-
6•H
2
O and 0.165 mole of Al
(NO
3
)
3
-
9•H
2
O were dissolved in 500 ml of CO
2
-
free deionized water at room temperature.
Subsequently, 1.65 mole urea and 1 mole
NH
4
NO
3
were added and the resulting solution
was placed in a 500 ml three-hole ball flask
equipped with a reflux system. The system was
purged by bubbling argon gas for 1 h and the
temperature was increased to 90°C using a
thermostated bath with sand.
After 10 h at this temperature, the white
precipitate obtained was centrifuged for 15 min,
washed several times with hot CO
2
-
free
deionized water. Finally, the solid was dried at
120°C for 12 h in an oven. The prepared solid has
a Zn
+2
/ Al
+3
= 2 ratio.
2.2 Synthesis of Hybrid Materials
An intercalation reaction in LDH can be carried
out by several pathways, or by only one
depending on the LDH/anion system studied. In
this work, a strategy similar to that reported in the
literature was approached in order to perform the
intercalation reactions for each LDH/anion
system (U. Costantino et al., 2008).
2.3 Collation by Memory Effect
This is an indirect intercalation method in which
the mixed oxide obtained after heat treatment of
the corresponding LDH is brought into contact
with a solution containing the anion of interest.
After a certain time, the LDH will regain its
original lamellar structure and the anions
contained in the solution will reside in the
interlamellar region. This property is very useful
when you want to intercalate an anion different
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from the original one, especially in the case of
large organic anions.
First, 0.5 g of the LDH was subjected to a heat
treatment at 500°C (5°/min) for 5 h in a flow of
N
2
. The mixed oxide obtained was put in contact
with 30 ml of a solution, previously bubbled with
argon, containing the anion of interest (4.8
mmol) and adjusted the pH to a value of 9 with
NaOH (0.1 M). The obtained suspension was left
in agitation for 7 days. After this time, the solid
was separated from the solution by centrifugation
and washed with CO
2
-
free deionized H
2
O to
finally dry it at 50 °C for 48 h. In this way the
solids ZnAl-PIPmem, MgAl-PIPmem, MgAlFe-
PIPmem, ZnAl-NADmem, MgAl-NADmem and
MgAlFe-NADmem were obtained.
2.4 Experimental characterization techniques
The methodology of the techniques was followed
as reported by (Alejandra Santana Cruz, 2014).
X-Ray Diffraction
X-ray diffractograms (XRD) of the powder
samples were obtained on a Philips X'PERT PRO
diffractometer; the samples were analyzed in the
range 3.4-80 (2θ) and with wavelength CuKα1
=1.5418 Å. Voltage-amperage of 45 kV and 40
mA were used respectively.
Fourier transform infrared spectroscopy.
Infrared spectroscopy is used for the
identification and study of the functional groups
of the molecules that make up the material to be
analyzed. Measurements are performed with a
Perkin-Elmer FTIR Spectrum Two™
spectrometer, with which powdered solids, rigid
solids, plastics, elastic materials and liquids can
be analyzed. The advantage of this equipment is
that it is not necessary to prepare the sample for
measurement; the material to be analyzed is
deposited directly on the lens.
The infrared spectra were obtained using a
NICOLET MAGNA IR 750 spectrophotometer.
The analyzed region was 4000-400 cm
-1
. The
methodology used to obtain the spectra was by
forming a pellet by mixing the sample with KBr
in a sample:KBr ratio of 1:100 by weight in a
manual press. The IR spectra were obtained in
the Transmittance mode.
Thermogravimetric Analysis (TGA)
Thermogravimetry is part of a set of thermal
analyses that have been developed to identify and
measure the physical and chemical changes that
materials undergo when exposed to controlled
temperature variations (Conesa Ferrer, 2000).
Specifically, thermogravimetric analyses have
been used to study the primary reactions in the
decomposition of solid and liquid materials. With
thermogravimetry, desorption, adsorption and
decomposition reactions are analyzed in an inert
gas environment or in the presence of oxygen
(Fraga Grueiro, 2001).
The thermograms were obtained from the
thermogravimetric analysis equipment SDT Ǫ
60. A mass of between 5 and 20 mg of the
samples was used and analyzed in a temperature
range of 25 to 850 °C at a rate of 10 °C/min, in
nitrogen atmosphere.
2.5 Evaluation techniques for the inhibition of
the growth of microorganisms.
Bacterial strains:
Purity tests
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Gram staining was performed to verify the purity
of the E. coli strains, observing their morphology
under an optical microscope at 100x.
The corresponding smear was made on the strain
to be analyzed with crystal violet and an iodine
solution, all of them were stained with a purplish
color. Subsequently, they were treated with a
decolorizing solution (alcohol-ketone), the gram-
positive bacteria retained the dye, due to the
composition of their wall, while the gram-
negative bacteria did not retain it, the dye being
eliminated. Next, they were treated with a
contrast dye (diluted fuchsin), the gram-negative
bacteria were stained, which ensures that they are
gram-negative bacteria.
Turbidity standard for inoculum preparation
To standardize inoculum density, a barium
sulfate suspension was used as a turbidity
standard (0.5 on the McFarland scale). Turbidity
tests were performed on a Varian Cary 4000 UV-
Vis spectrophotometer. The absorbance was
measured at 625 nm and incubated under the
same conditions until growth was achieved in the
range between 0.7 and 0.8 optical density. The
standards were stored at room temperature away
from light.
Preparation of inoculum
For inoculum preparation, 3 isolated colonies of
the same type of morphology of the strains
maintained on tryptic-casein soy agar wedges
were taken and grown in tubes containing 5 ml of
tryptic-casein soy broth at 37 °C until standard
turbidity was reached. This suspension contained
approximately 1x10
8
Colony Forming Unit
(CFU)/ml of E. coli. The inoculum was reseeded
every 12 h for 5 days to confirm the exponential
phase of growth. These strains were stored at 4
°C in order to maintain viability.
2.6 Bacterial growth in the presence of hybrid
materials.
Bacterial growth in the presence of the biocidal
materials was determined. For this purpose, the
bactericidal capacity of the materials was
evaluated in relation to time and average CMB.
A standardized inoculum was challenged at fixed
concentrations of antimicrobial in a broth. A 0.5
ml sample of the liquid systems was inoculated
with bacteria (E. coli) in 10 ml of tryptic-casein
soy broth contained in screw-capped test tubes.
An amount of biocidal material (amount
determined as average CMB) was added to each
tube and incubated at 37°C with agitation at 30
rpm. Samples were taken at different times (0, 5,
15, 30, 60, 90 and 120 min). The sample taken
was seeded in Petri dishes with 20 ml of
MacConkey agar by the streak-plate technique.
As a control, one plate was inoculated with
culture without bactericidal material, at the
beginning and at the end. The plates were
incubated inverted at 37 °C for 24 h in an aerobic
atmosphere and colony counting was performed.
3. Results and Discussions
In order to facilitate the identification of lamellar
double hydroxides and hybrid materials, the
following nomenclature was adopted for LDH:
ZnAl-X, where X indicates the resident anion in
the interlaminar region. In our specific case, X
can be NO
3
-
, Cl
-
or CO
3
2-
.
3.1 Characterization of ZnAl-X materials
Zn- and Al-based lamellar double hydroxides
were synthesized with different anions by
different methods to obtain the solids ZnAl-CO
3
,
ZnAl-Cl and ZnAl-NO
3
. The X-ray
diffractograms of these solids and the interplanar
distances d
003
are shown in Figure 1. All the
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diffractograms show peaks associated to the
characteristic planes of hydrotalcite, no other
phases foreign to this mineral are observed; that
is to say that pure LDH were obtained.
Figure 1. X-ray diffractogram of LDH ZnAl-X.
On the other hand, the infrared spectra shown in
Figure 2 (for reasons of clarity the infrared
spectrum for the ZnAl-Cl solid is not shown) are
congruent with those reported in the literature.
On the one hand, both ZnAl-NO
3
and ZnAl-CO
3
solids show a broad and very intense absorption
band, centered around 3445 cm
-1
, which is
attributed to the vibrational frequency of the νOH
stretching mode of the O-H groups forming the
brucite-like films, so it can be stated that up to
this point three different types of ZnAl-X solids
are available and were used as starting materials
for the subsequent preparation of hybrid
materials.
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Figure 2. Infrared spectra of fresh LDH ZnAl-X. a) ZnAl-CO
3
and b) ZnAl-NO
3
.
Figure 3. Characterization of the Nalidixic acid molecule. a) FTIR spectrum, b) XRD
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3.2 Characterization of hybrid materials
In order to better understand the characterization
of the hybrid materials, it is convenient here to
present both the infrared spectrum and the X-ray
diffraction pattern of the molecules that were
studied; either of the starting acids or of the
sodium salts generated from them. This is with
the purpose of keeping them in mind as reference
analysis results. Thus, Figure 3 shows the IR
spectrum and the XRD pattern of nalidixic acid.
From the general formula of the LDH and the
charges of the incoming and outgoing anions, the
necessary amount of anions that can enter the
interlaminar space is known.
Taking into account the stoichiometry of the
exchange; for example, a NO
3
-
ion would be
replaced by a Cl
-
ion and vice versa; however, a
single CO
3
2-
anion would have to be replaced by
two NO
3
-
ions or by two Cl
-
ions in order not to
create a charge imbalance in the interlaminar
region. Thus, the charges of both the anion
initially present in the interlaminar region and the
anion to be introduced must be taken into account
to determine the minimum stoichiometric
amount of the incoming anion necessary to
achieve 100% replacement.
However, the intercalation process does not
ensure that the anion will occupy all of that
interlamellar space or achieve a 100%
replacement rate. Because of the above, when
only the minimum stoichiometric amount of
incoming anion is placed in contact with an LDH,
the exchange is likely to be only partial (Y. T.
Kameda et al., 2006).
3.3 Nalidixic Acid CMBs and CMIs
In order to verify that Nalidixic Acid has a better
bactericidal effect against E. coli with respect to
other drugs, MIC and BMC tests were carried out
on the most commonly used drugs for this
purpose. Tables 1 and 2 show the MIC and BMC
values, respectively, of the drugs as inhibitors of
E. coli, using the method of dilutions and sowing
on agar. Reproducibility was done in triplicate
for each bacterial strain. The values reported are
the average of the MICs and BMCs for each test,
in broth and MH agar, respectively.
Table 1. CMIs of different antibiotics.
CMI (mg/mL)
Drug
0.11
0.22
0.33
0.38
Cloxacillin
Sodium
+
+
+
-
Cephalexin
Sodium
+
+
-
-
Sodium
Ampicillin
+
+
-
-
Mg-Al
Ampicillin
+
+
-
-
Nalidixic
Acid
+
+
-
-
Pipemidic
Acid
+
+
-
-
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Table 2. CMBs of different antibiotics.
Turbidity in the inoculated tubes allowed
determining the MIC after 24 hours of exposure
of the bacteria to the different materials. The
quantification of bacterial colonies that grew on
plates with MH agar was performed after 18 and
24 hours of incubation at 37
o
C.
The exposed samples that did not show turbidity
were sown in petri dishes with Müller-Hinton
agar, showing that bacterial growth was null.
This shows that the materials have a bactericidal
effect on E. coli. It should be noted that those
translucent tubes whose turbidity was very low
(less than 50 NTU) or close to zero were
considered as systems in which there was no
growth. Those tubes with apparent turbidity and
values greater than 50 NTU were considered
systems with microbial growth.
With this descriptive turbidimetric test method,
some answers can be obtained about the behavior
of bacteria in the presence of bactericidal agents,
such as suppression in the level of growth in the
stationary phase, decrease in the growth rate and
lethality (Davidson and Parish, 1989).
Microbiological tests to evaluate inhibition times
Before evaluating the bactericidal character of
the materials, quality controls were performed on
the working strains by microdilutions. The
results of the quality controls (viability and
purity) of E. coli, used in the bactericidal
evaluation, met the acceptance criteria by
obtaining viability values in the order of 1x10
8
,
so that the purity results comply with the
condition of cultures free of microbial
contamination.
Table 3 and Figure 4 report the average values of
E. coli colonies that survived in each of the three
trials, for each of the exposure times to the
different drugs evaluated as biocides. The
microbial growth and colony count of the E. coli
strains in the presence of the different
bactericidal materials were calculated taking into
account the number of initial colonies in the
suspension of inoculated microorganisms and the
colonies that grew or were eliminated during the
exposure time.
The bactericidal effect of the materials was
assessed by measuring cell viability at 0, 5, 30,
60, 90 and 120 minutes after exposure and
incubation of the bacteria with the different
materials. The tests were performed in triplicate
on McConkey agar plates. These tests made it
possible to define the moment at which the
biocidal agent acts on the bacterial replication
cycle, in such a way that each drug presents a
different graph according to its mechanism of
action.
CMB (mg/mL)
Drug
0.11
0.22
0.27
Cloxacillin
Sodium
+
+
-
Cephalexin
Sodium
+
+
+
Sodium
Ampicillin
+
+
+
Mg-Al
Ampicillin
+
+
+
Nalidixic
Acid
+
+
-
Pipemidic
Acid
+
+
-
167
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Revista de Ciencias Tecnológicas (RECIT). Volumen 4 (3): 157-170
Table 3. Colonies of E. coli that survived against drugs.
Amoxicillin Sodium(C
16
H
19
N
3
O
5
S)
MgAl -ampicillin(C
16
H
18
N
3
NaO
4
S)
Time (min)
E1
E2
E
Average
Uncertainty
E1
E2
E3
Average
Uncertainty
0
170
170
174
171
1
172
171
170
171
1
5
153
150
155
152
2
71
70
71
70
0
15
117
120
118
118
1
40
42
42
41
1
30
86
88
88
87
1
31
30
33
31
1
60
68
71
67
68
1
18
19
21
19
1
90
44
48
45
45
1
11
12
14
12
1
120
16
15
14
15
1
0
1
2
1
1
Nalidixic Acid (C
12
H
12
N
2
O
3
)
Pipemidic Acid(C
14
H
17
N
5
O
3
)
Time (min)
E1
E2
E3
Average
Uncertainty
E1
E2
E3
Average
Uncertainty
0
171
170
168
169
1
169
170
170
169
0
5
109
110
111
110
1
82
80
81
81
1
15
63
62
61
62
1
52
51
51
51
1
30
11
10
12
11
1
5
6
5
5
1
60
1
0
0
0
0
0
0
0
0
0
90
0
0
0
0
0
0
0
0
0
0
120
0
0
0
0
0
169
170
170
169
0
Cephalexin Sodium(C
16
H
17
N
3
O
4
S)
Cloxacillin Sodium(C
19
H
18
N
3
ClO
5
S)
Time (min)
E1
E2
E3
Average
Uncertainty
E1
E2
E3
Average
Uncertainty
0
172
171
175
172
1
172
170
172
171
1
5
124
121
131
125
4
121
120
123
121
1
15
91
83
85
86
3
90
85
87
87
2
30
54
52
53
53
1
43
42
45
43
1
60
33
30
31
31
1
27
29
32
29
2
90
27
26
28
27
1
15
17
18
16
1
120
8
7
8
7
0
7
8
6
7
1
168
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Revista de Ciencias Tecnológicas (RECIT). Volumen 4 (3): 157-170
Figure 4 shows the results of the tests performed
with different antibiotics as inhibitors of E. coli,
showing that NAD and Pimemidic acid (PIP)
were the ones that eliminated the bacteria in the
shortest time; however, NAD is more economical
than PIP, so it was decided to perform this work
with NAD (Nalidixic acid).
The prolonged release of the biocide from
biocidal LDHs must be attributed to dipolar
interactions between the organic biocide and
LDHs and occurs in other similar organic-LDH
hybrid compounds [14,15].
In similar studies by Santana Cruz et al. in 2018
refer (Although S. typhi is more resistant than E.
coli to both nalidixic acid and pipemidic acid
incorporated into LDHs, it is clear that LDH-
biocide interactions are favorable for killing S.
typhi) (Santana A.; Flores J.L.; Guerra R.; &
Martínez M.J.et al., 2016). In results of
Antibacterial activity of pipemidic acid-hybrid
ions. MgFeAl-Cl allows the rapid development
of colonies of S. typhi. In contrast, the MgFeAl-
PIP hybrid material showed good activity to kill
bacteria, as only 12% of colonies survived after
90 min of exposure. MgFeAl-PIP is less active in
killing S. typhi than E. coli pathogens.
Figure 4. Colonies of E. coli that survived against drugs.
169
ISSN: 2594-1925
Revista de Ciencias Tecnológicas (RECIT). Volumen 4 (3): 157-170
4. Conclusions
According to the results obtained, the LDH
ZnAl-NADmem presents a controlled release,
since when the material was exposed three times
to E. coli bacteria, it continued to eliminate
bacteria, presenting a bacteriostatic effect in the
third exposure, since it did not eliminate bacteria.
The ZnAl-NADmem material obtained by
memory effect, is a laminar and very crystalline
material but with a very low degree of
intercalation, which to some extent is an
advantage because it does not require much
antibiotic to present good antibacterial activity,
this is due to a synergistic effect between the
inorganic matrix and the antibiotic. LDH
MgFeAl-NADmem also shows good
antibacterial activity, however, its synthesis
represents a higher cost with respect to ZnAl-
NADmem. In general, on the hybrid systems
analyzed, the antibacterial activity is not always
a direct function of the amount of antibiotic
intercalated. It seems that the activity depends
precisely on the inorganic matrix-antibiotic
system studied. From the above arises the idea
that there is, in some systems, a synergistic effect
between the inorganic matrix and the antibiotic
that results in an efficient inhibition system.
5. Author acknowledgement
Roberto Guerra-González: Research;
Methodology; Supervision; Acquisition of funds;
Resources; Writing original draft. Martha
Angélica Lemus-Solorio: Research;
Methodology; Formal analysis, Visualization;
Writing: revision and editing. José Luis Rivera-
Rojas: Research; Data Curation; Methodology;
Software; Formal Analysis. Alfonso Lemus-
Solorio: Methodology, Conceptualization,
Original draft writing, Project management.
América Abisay Mondragon-Herrera:
Conceptualization; Research, Methodology.
Marco Antonio Martínez-Cinco: Visualization;
Validation; Formal Analysis; Data Curation.
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