International Journal of Hyperthermia
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ihyt20
Mild magnetic hyperthermia is synergistic with
an antibiotic treatment against dual species
biofilms consisting of S. aureus and P. aeruginosa
by enhancing metabolic activity
Layla A. Almutairi, Bing Yu, Eric Dyne, Alhussain A. Ojaym & Min-Ho Kim
To cite this article: Layla A. Almutairi, Bing Yu, Eric Dyne, Alhussain A. Ojaym & Min-Ho Kim
(2023) Mild magnetic hyperthermia is synergistic with an antibiotic treatment against dual
species biofilms consisting of S. aureus and P. aeruginosa by enhancing metabolic activity,
International Journal of Hyperthermia, 40:1, 2226845, DOI: 10.1080/02656736.2023.2226845
To link to this article: https://doi.org/10.1080/02656736.2023.2226845
© 2023 The Author(s). Published with
license by Taylor & Francis Group, LLC.
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INTERNATIONAL JOURNAL OF HYPERTHERMIA
2023, VOL. 40, NO. 1, 2226845
https://doi.org/10.1080/02656736.2023.2226845
Mild magnetic hyperthermia is synergistic with an antibiotic treatment against
dual species biofilms consisting of S. aureus and P. aeruginosa by enhancing
metabolic activity
Layla A. Almutairia,b, Bing Yuc, Eric Dynea, Alhussain A. Ojayma and Min-Ho Kima,c
a
School of Biomedical Sciences, Kent State University, Kent, OH, USA; bDepartment of Biology, Princess Nourah bint Abdulrahman
University, Riyadh, Saudi Arabia; cDepartment of Biological Sciences, Kent State University, Kent, OH, USA
ABSTRACT
ARTICLE HISTORY
Objective: The wound biofilm infections that develop tolerance to standard-of-care antimicrobial
treatment has been increasing. The objective of this study was to demonstrate a proof-of-concept of
mild magnetic nanoparticle (MNP)/alternating magnetic field (AMF) hyperthermia as an anti-biofilm
therapy against multispecies biofilm infections.
Methods: Using both an in vitro cell culture and in vivo murine model of wound infection, we investigated whether MNP/AMF hyperthermia applied at a mild thermal dosage would be synergistically
effective against dual species biofilm infection consisting of S. aureus and P. aeruginosa when combined with a broad-spectrum antibiotic, ciprofloxacin (CIP).
Results: The combined treatment of MNP/AMF hyperthermia and CIP to the wounds of diabetic mice
(db/db mice) significantly reduced the CFU number of S. aureus and P. aeruginosa by 2-log and 3-log,
respectively, compared to the untreated control group, whereas either mild MNP/AMF hyperthermia or CIP
treatment alone had little effect on the eradication of both bacteria. Our gene microarray data obtained
from the culture of S. aureus biofilm suggest that mild MNP/AMF could shift the expression of genes for
cellular respiration from anaerobic fermentation to an aerobic glycolytic/tricarboxylic acid cycle (TCA) pathway, implicating that the beneficial effect of mild MNP/AMF hyperthermia on the increased susceptibility
of biofilm bacteria to an antibiotic treatment is associated with an increased metabolic activity.
Conclusion: Our results support the translational potential of mild MNP/AMF as an adjunctive therapy
that can be combined with a broad-spectrum antibiotic treatment for the management of wound biofilm infections associated with multispecies bacteria.
Received 20 April 2023
Revised 8 June 2023
Accepted 13 June 2023
Introduction
The wound biofilm infections that develop a tolerance to
standard-of-care antimicrobial treatment has been increasing
[1–7]. We previously reported magnetic nanoparticle (MNP)
and alternating magnetic field (AMF)-induced hyperthermia
as a non-pharmacological strategy to combat biofilm infections [8], in which highly localized heat was generated on
the surface of MNPs that are delivered to bacterial pathogens upon the application of external AMF. In the study, we
demonstrated the capacity of MNP/AMF hyperthermia to target and inhibit the growth of bacterial pathogen using both
in vitro and in vivo models of Staphylococcus aureus (S. aureus) biofilm infection. However, the complete eradication of
bacterial pathogens required the application of MNP/AMF
hyperthermia at a higher thermal dose, which could elicit
nonspecific thermal damage in the host tissue.
To address this, we recently demonstrated that MNP/AMF
hyperthermia applied at a mild thermal dose, which is nontoxic
KEYWORDS
Magnetic nanoparticle
hyperthermia; antibiotics;
dual species biofilm;
S. aureus; P. aeruginosa
to host mammalian cells, could be effective in enhancing the
antibiofilm efficacy of conventional antibiotics in an in vitro
model of S. aureus biofilm [9]. This finding is in line with previously published studies showing that general hyperthermia
therapy could render biofilm bacteria to be susceptible to conventional antibiotics [10,11]. However, the detailed mechanism
by which mild MNP/AMF hyperthermia sensitizes the antibiofilm effect of antibiotics has yet to be elucidated.
A critical challenge in the treatment of chronic wounds lies
in the nature of biofilm infection formed by multispecies bacteria in a wound [7,12,13]. The common bacteria involved in
chronic wounds include S. aureus, Enterococcus faecalis,
Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae,
and Acinetobacter baumanii [14–16]. The multispecies biofilms
composed of those pathogens exhibited a higher level of antimicrobial tolerance than the biofilms formed by single species
bacteria [17–19]. However, it has not been shown whether
mild MNP/AMF hyperthermia could be effective against a
wound biofilm infection associated with multispecies bacteria.
CONTACT Min-Ho Kim
mkim15@kent.edu
Department of Biological Sciences, Kent State University, Kent, OH, 44242, USA.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/02656736.2023.2226845.
ß 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow
the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
2
L. A. ALMUTAIRI ET AL.
The goal of this study was to demonstrate a proof-of-concept of mild MNP/AMF hyperthermia in combination with a
broad-spectrum antibiotic as an anti-biofilm therapy against
multispecies biofilm infections. S. aureus and P. aeruginosa
are the prevalent types of bacterial pathogens found in polymicrobial infections in chronic wounds including diabetic
ulcers [5,20]. In this study, we employed a wound biofilm
model composed of dual species of bacteria, S. aureus and P.
aeruginosa, in diabetic mice. Additionally, in order to gain
more insight into the mechanism by which mild MNP/AMF
hyperthermia sensitizes the antibiofilm effect of antibiotics,
we examined the effect of mild MNP/AMF hyperthermia on
the global transcriptional profiling in the biofilm phase of
bacteria by using a full-genome microarray analysis in S. aureus biofilm. Our results showed that the application of mild
MNP/AMF to the wounds of diabetic mice infected with dual
spices biofilm formed by S. aureus and P. aeruginosa synergistically enhanced the antimicrobial efficacy of ciprofloxacin
(CIP), a broad-spectrum of antibiotics. Additionally, our fullgenome microarray analysis revealed that mild MNP/AMF
hyperthermia altered the transcriptional profiling of biofilm
phase of bacteria, especially in the genes associated with cellular metabolism.
Materials and methods
and incubated at 37 C for 48 h. After the culture, for subsequent CFU counting on selective agar media, the biofilm
matrix was disrupted through vortexing (at 900 rpm) followed by sonication in a sonication bath (Branson Ultrasonic
bath) for 5 min. The Mannitol salt agar and Triclosan agar
were used as a selective agent for S. aureus and P. aeruginosa, respectively (Figure S1A).
The susceptibility of single species and dual species
biofilm to ciprofloxacin
The susceptibility of either single species or dual species biofilm to a CIP treatment was determined using a broth
macro-dilution method [23]. CIP is a broad-spectrum fluoroquinolone antibiotic used clinically to treat both S. aureus
and P. aeruginosa infections. Either single species biofilm
formed by S. aureus or P. aeruginosa alone or dual species
biofilm formed by the co-culture of S. aureus or P. aeruginosa
was prepared as described above. The biofilms formed on
6 mm diameter polycarbonate membrane filters were transferred to sterile 96-well flat-bottom polystyrene tissue culture
plates (BD Falcon) and they were treated with CIP (SigmaAldrich) at concentrations ranging from 0.1 mg/mL to
3 mg/mL for 18 h at 37 C. The cells were then plated on
Mannitol salt agar for S. aureus CFU counting and Triclosan
agar for P. aeruginosa CFU counting.
Preparations of S. aureus and P. aeruginosa
S. aureus (ATCC 6538 strain) and P. aeruginosa (PAO1, ATCC
BAA-47) were obtained from the American Type Culture
Collection (ATCC). Both bacterial strains were streaked onto
the Tryptic soy agar (TSA) (BD Biosciences) and the streaked
plates were incubated overnight at 37 C. A single colony of
bacteria was inoculated to the 5 ml of Tryptic soy broth (TSB)
for S. aureus and Luria-Bertani (LB) broth (BD Biosciences) for
P. aeruginosa, respectively. They were then incubated under
aerobic conditions at 37 C in a shaker at 180 rpm overnight
until they reached a stationary phase. For the preparation of
planktonic phase of S. aureus and P. aeruginosa, the cells
were pelleted by centrifugation at 3,500 g at 4 C for 7 min
and diluted with TSB and LB, respectively, to obtain the
desired concentrations of bacteria (1 103 - 1 106
CFU/mL).
In vitro model of dual species biofilm formed by S.
aureus and P. aeruginosa
The dual species biofilm formed by S. aureus and P. aeruginosa were established using a static model described previously with some modifications [21,22]. Bacteria suspensions
of stationary phase growth of P. aeruginosa and S. aureus
were diluted in sterile phosphate buffer solution (PBS) at
final concentrations (1x103 - 1x106 CFU/mL) in each tube.
The diluted solution was then mixed at a varying density
between P. aeruginosa and S. aureus (1:1, 1:10, 1:100, and
1:1000) and 2 lL of the mixed suspension was inoculated on
to a 6 mm diameter of polycarbonate membrane filter
(0.2 lm pore size, GE). The filter was incubated on TSA plate
Biofilm mass assay
The reduction of biofilm mass to a CIP treatment was conducted by crystal violet (CV) staining method. Briefly, 96-well
polystyrene flat-bottomed microplate containing either single
species or dual species biofilms were washed with PBS three
times to remove any non-adherent bacteria. The biofilms
were then fixed with 200 mL of 99% (v/v) methanol for
15 min and left to air-dry for 30 min in a laminar flow. The
fixed biofilm cells were stained using filtered 0.5% crystal
violet (Sigma-Aldrich) for 5 min at room temperature. The
excess stain was removed by rinsing with running tap water,
then air dried, and crystal violet bound cells were solubilized
with 200 mL of 33% (v/v) of acetic acid (Merck). The biofilm
mass was quantified through the measurement of optical
density of the sample at 570 nm using a spectrophotometer
R M4 Multi-Mode Microplate Reader, Molecular
(SpectraMaxV
Devices).
Measurements of temperature increase (DT) during the
exposure of MNP/AMF hyperthermia
The procedure for the application of MNP/AMF hyperthermia
to the biofilm was performed as described in our previous
study [8]. In brief, varying concentrations of MNPs
(Ferumoxytol, AMAG) ranging from 1 to 3 mg/mL were
added to the wells of S. aureus biofilm (in 200 mL solution)
pre-formed on a polystyrene 8-well chamber slide and incubated for 2 h. For the application of AMF, the chamber slide
was positioned to the water-cooled coil chamber of the AMF
generator (5 kW power operated at 300–450kHz, MSI
INTERNATIONAL JOURNAL OF HYPERTHERMIA
Automation) and applied with AMF for 10 min at the field
strength of 60 kA/m and frequency of 375 kHz. The real-time
monitoring of temperature in the solution of biofilm during
the AMF exposure was conducted using a fiber optic temperature probe (Neoptix) placed in the solution.
Determination of cumulative equivalent minutes at
43 C (CEM43)
The CEM43 was calculated from equation as described [24],
P
CEM43 ¼
ti R(43T), where T is the average temperature
during i-th time interval ti (min), and R is a factor to compensate for temperature change, which is set at 0.25 for T43 C
and 0.5 for T > 43 C. The average temperature (T) was determined from the plot of temperature vs time at a given time
interval during the application of MNP/AMF hyperthermia.
Antibiotic susceptibility of dual species biofilm to
MNP/AMF hyperthermia
The dual species biofilm containing S. aureus and P. aeruginosa was formed on an 8 well-chamber slide for 48 h. The
biofilm was treated with MNPs (1 or 2 mg/mL, Ferumoxytol,
AMAG) for 2 h at 37 C and then applied with AMF for
10 min at the field strength of 60 kA/m and frequency of
375 kHz. Following the application of MNP/AMF, the biofilms
were treated with CIP at a concentration of 50 mg/mL for
18 h at 37 C and the CFU numbers of S. aureus and P. aeruginosa were counted as described above.
In vivo model of dual species biofilm infection in
wounds of diabetic mice
Diabetic db/db mice (BKS.Cg-Dock7m þ/þ Leprdb/J, male mice
at age of 8–12 weeks old) were obtained from Jackson
laboratory (Bar Harbor, ME). Mice were anesthetized with
15% isoflurane gas inhalation and one full-thickness, circular
wound was made on the dorsal surface of a mouse using a
6 mm sterile biopsy punch (Acuderm Inc.). The wound of
each mouse was inoculated with 20 lL of bacteria solution
mixed with P. aeruginosa (1x103 CFU/mL) and S. aureus
(1x106 CFU/mL) and covered with a transparent, semipermeable Tegaderm dressing (3 M). At day 2 post infection, either
20 lL of sterile saline, CIP (50 mg in 20 lL of sterile saline)
only, MNPs (40 mg of Ferumoxytol in 20 lL saline) only, or CIP
(50 mg) mixed with MNPs (40 mg of Ferumoxytol in 20 lL
saline) was topically injected to the area of wound infection.
The mice treated with MNPs were placed in a magnetic coil
chamber and AMF was applied for 10 min at the frequency
of 375 kHz and field strength of 60 kA/m. At day 3 post-infection, the wounded skin was excised and homogenized using
a tissue homogenizer (OMNI tissue homogenizer, OMNI Inc.),
and the homogenates were serially diluted and plated on
the agar with selective agent for the CFU counting of S. aureus and P. aeruginosa. The CFU number in the wound was
standardized with respect to the weight of excised wound.
Three mice were used for each experimental group. The
experimental protocol was reviewed and approved by the
3
Institutional Animal Care and Use Committee of Kent State
University.
Scanning electron microscopy (SEM) imaging of wound
biofilm
Wounded skins were harvested from db/db mouse at day 2
post-infection and fixed with 3% (v/v) glutaraldehyde for
10 min at room temperature, followed by overnight incubation at 4 C. The samples were treated with a series of ethanol washes (30, 50, 70, 95 and 100%, 10 min for each
concentration) and then subsequently placed in a critical
point dryer to replace ethanol with CO2. The dried samples
were then mounted to an aluminum stub and sputter-coated
with gold-palladium for 90 s and examined in a scanning
electron microscope (Hitachi S-4700 SEM, Hitachi High
Technologies America, Inc.) at x10,000 magnification.
Gene microarray analysis
The microarray analysis was run in the genomics shared
resource-comprehensive cancer center in Ohio State
University (Columbus, OH). RNA samples were processed
according to the Affymetrix SensationPlus FFPE Amplification
and 30 IVT Labeling Kit protocol and also as described previously [25]. Briefly, 50 ng of total RNA was reverse transcribed
to produce sense RNA via in vitro transcription (IVT), followed
by single-strand cDNA synthesis. The cDNA was fragmented,
end-labelled and hybridized to the Affymetrix GeneChip S.
aureus Genome Array (Affymetrix, Cat. No. 900514), which
contained probe sets to over 3,300 S. aureus open reading
frames. GeneChip arrays were scanned using an Affymetrix
3000 7 G scanner. The resultant data (CHP files) were submitR Transcriptome Analysis Console (TAC)
ted to the AffymetrixV
software for Gene Level Differential Expression Analysis. The
gene expression analysis was performed from three independent mRNA samples for each group. Genes with a p
value < 0.05 and threshold values of 2 and 2-fold
change between the MNP/AMF-treated S. aures biofilms and
the untreated control was defined as significantly differentially expressed.
Determination of metabolic activity
S. aureus biofilm was prepared on 96-well strip plate (SPL
Strip Immunoplate) and the metabolic activity of the biofilm
in response to MNP/AMF hyperthermia was determined
using a resazurin-based assay. In brief, either MNP/AMFtreated (at the thermal dose of CEM43 ¼ 6 min) or untreated
control S. aureus biofilm was rinsed and 120 ll of a resazurin
solution (Promega) was added to each well. The plate was
incubated for 20 min at 37 C and fluorescence was measured (at excitation wavelength of 560 nm and emission
wavelength
of
590 nm)
using
spectrophotometer
R M4 Multi-Mode Microplate Reader, Molecular
(SpectraMaxV
Devices). The values were expressed after normalizing the
fluorescence intensity with respect to the number of viable
bacteria in the well.
4
L. A. ALMUTAIRI ET AL.
Statistical analysis
Statistical analysis was performed using GraphPad Prism version 8.0 software. A two-tailed unpaired t-test was used to
determine statistical significance between two groups.
Statistical tests among multiple groups were analyzed using
one-way ANOVA followed by Turkey’s posttest for secondary
analysis for comparison. For all analyses, p-values of less than
0.05 was considered to be statistically significant. Data were
presented as mean ± standard deviation (SD). The in vitro
studies were run with at least three biological replicates, and
each biological replicate had three technical replicates.
Results
The susceptibility of dual species biofilm consisting of S.
aureus and P. aeruginosa
Since S. aureus and P. aeruginosa are major pathogens found
growing together in biofilms in chronic wounds including
diabetic wounds [5,20], we first sought to establish an
in vitro model of polymicrobial biofilms formed by the balanced growth of both organisms. Since P. aeruginosa was
shown to outcompete the growth of S. aureus under co-culture condition with an equal inoculation density [21], we reasoned that co-inoculating with increasing densities of S.
aureus over P. aeruginosa would result in the balanced
growth of both bacteria in the biofilm. To ascertain this, P.
aeruginosa and S. aureus were inoculated on to a membrane
filter at varying density ratios ranging from 1:1, 1:10, 1:100,
and 1:1000 and co-cultured to form a stable biofilm. For the
biofilm formed at the 1:1 inoculation ratio between P. aeruginosa and S. aureus, the CFU number of P. aeruginosa was significantly higher than that of S. aureus by 3 log (Figure
S1B). Increasing the density of S. aureus could significantly
increase the viable number of S. aureus in the biofilm, resulting in the difference between S. aureus and P. aeruginosa by
2-log at the 1:100 inoculation ratio (p < 0.05) and by less
than 1-log at 1:1,000 inoculation (p > 0.05). Thus, in the subsequent studies, dual species biofilm was formed by seeding
P. aeruginosa and S. aureus at 1:1,000 density.
By establishing the protocol for dual species biofilm with a
balanced growth of S. aureus and P. aeruginosa, we next
determined the susceptibility of dual species biofilm to an
antibiotic. We chose CIP as an antibiotic to be tested since it
exhibits a broad-spectrum antibacterial efficacy against both
S. aureus and P. aeruginosa in the planktonic phase. The minimum inhibitory concentration (MIC) values of CIP against
planktonic phase of S. aureus and P. aeruginosa were measured to be between 0.1 to 0.5 mg/mL. However, when each
strain of bacteria formed a biofilm by either S. aureus or P.
aeruginosa, the use of CIP at 100 mg/mL was required to
achieve a growth inhibitory effect on biofilm bacteria,
200 1,000 times higher concentration of CIP than the ones
for planktonic phase of bacteria (Figure 1(A)). Importantly, it
was even more difficult to eradicate the biofilm when both
bacteria co-existed by forming a dual species biofilm. This
was also evidenced by the incomplete eradication of biofilm
mass by CIP for dual species biofilm, compared to the biofilm
Figure 1. The susceptibility of dual species biofilm consisting of S. aureus and
P. aeruginosa to ciprofloxacin in vitro. (A) Log (CFU/mL) reduction of S. aureus
and P. aeruginosa in single species biofilms (Single-S. aureus and Single-P. aeruginosa) and dual species biofilms (Dual-S. aureus and Dual-P. aeruginosa) following the treatment of increasing concentrations of ciprofloxacin. (B) Biomass
reduction in single species biofilms (Single-S. aureus and Single-P. aeruginosa)
and dual species biofilms (Dual-S. aureus þ P. aeruginosa) following the treatment of increasing concentrations of ciprofloxacin. The dual biofilms data is
analysis of S. aureus or P. aeruginosa from the dual biofilm. N ¼ 3 per group.
p < 0.05 and #p < 0.01.
formed by single species bacteria (Figure 1(B)). Our result is
consistent with previous reports that showed a reduced susceptibility of multispecies biofilm to antibiotics [21,26], supporting the validity of our dual species biofilm model.
Determination of CEM43
Based on our recent study showing the synergistic efficacy
of mild MNP/AMF hyperthermia with conventional antibiotics
against S. aureus biofilm in vitro [9], we sought to determine
whether the mild MNP/AMF hyperthermia therapy could also
be effective against biofilm formed by multispecies bacteria.
As a type of MNP for this study, we used Ferumoxytol
(Feraheme, AMAG Pharmaceuticals), which is an FDAapproved superparamagnetic iron oxide nanoparticle (SPION)
to treat anemia associated with chronic kidney disease. We
first determined the hyperthermia characteristic of
Ferumoxytol by measuring the temperature increase (DT) for
varying concentrations of Ferumoxytol solutions at the fixed
AMF strength (60 kA/m) and frequency (375 kHz) (Figure
2(A)). The application of MNP/AMF at 1 mg/mL of
Ferumoxytol for 10 min resulted in DT of 5.3 C and the DT
value was further increased to 11.6 C and 16.1 C for
INTERNATIONAL JOURNAL OF HYPERTHERMIA
5
Figure 2. Effects of MNP/AMF hyperthermia on the CEM43 thermal dose. (A) An experimental setup for the measurment of temperature in MNP (Ferumoxytol) solution during the application of AMF (B) Increase of temperature (䉭T) during the application of AMF as a function of MNP concentration (1–3 mg/mL). N ¼ 3 per
group. (C) Estimation of MNP/AMF-induced CEM43 (min) as a function of MNP concentration (1–3 mg/mL). the dotted red line indicates the reported threshold of
safe thermal dose for the application of hyperthermia to skin tissue24. N ¼ 3 per group.
Figure 3. Effects of MNP/AMF hyperthermia on the killing of S. aureus and P. aeruginosa in dual species biofilm in combination with ciprofloxacin for varying thermal dose. (A) An experimental setup for the application of MNP/AMF hyperthermia to the culture of dual species biofilm consisting of S. aureus and P. aeruginosa.
(B) The effect of MNP/AMF hyperthermia alone or in combination with ciprofloxacin (CIP, 50 mg/mL) for varying CEM43 thermal doses (CEM43¼1 and 20 min) on the
log reduction of S. aureus and P. aeruginosa numbers (CFU/mL) in dual species biofilm. N ¼ 3 per group. p < 0.01.
2 mg/mL and 3 mg/mL of Ferumoxytol, respectively (Figure
2(B)), confirming the heating capacity of Ferumoxytol-MNPs.
Since CEM43 has been widely used as an accepted metric
for thermal damage assessment in human tissues in response
to various hyperthermia treatments, we next determined the
concentration of Ferumoxytol that could be applied to skin
tissue within the safety margin of thermal dose by correlating the measured values of DT with CEM43. The calculated
values of CEM43 ranged from 1 min for 1 mg/mL, 20 min for
2 mg/mL, and up to 7,450 min for 3 mg/mL of FerumoxytolMNPs (Figure 3(C)). Given the reported value of CEM43 of
41 min as a threshold thermal dose for the application of
hyperthermia to a skin tissue [24], treatment conditions of
Ferumoxytol-MNPs up to 2 mg/mL for 10 min AMF, equivalent to CEM43 of 20 min, was determined to be an acceptable
range of thermal dose for skin wounding experiments.
Mild MNP/AMF hyperthermia synergistically enhanced
the susceptibility of dual species biofilms to
ciprofloxacin in vitro
By observing an antibiotic tolerance of dual species biofilm
formed by S. aureus and P. aeruginosa to CIP, we next examined
whether mild MNP/AMF hyperthermia would enhance the antibacterial efficacy of CIP against the biofilm. For this, the culture
of dual species biofilm by S. aureus and P. aeruginosa was pretreated with MNP/AMF at the thermal dose of CEM43¼1 min
and CEM43¼20 min (Figure 3(A)) in the absence and presence of
CIP (50 mg/mL). The CIP alone had little effect on the killing of
both S. aureus and P. aeruginosa when they were grown
together in the biofilm phase (Figure 3(B)). The application of
MNP/AMF alone at the CEM43 thermal dosage of 1 min had also
little effect on the killing of both bacteria, while the combined
treatment of MNP/AMF and CIP resulted in about 1-log reduction of S. aureus and 0.6-log reduction of P. aeruginosa in the
biofilm. Increasing the level of CEM43 thermal dose to 20 min
resulted in a 2-log reduction of S. aureus and 1.4-log reduction
of P. aeruginosa bacteria in the presence of CIP, while the application of MNP/AMF alone resulted in only a 0.6-log and 0.3-log
reduction of S. aureus and P. aeruginosa, respectively (Figure
3(B)). Taken together, these results suggest that the application
of mild MNP/AMF hyperthermia is synergistic with CIP against
dual species biofilm.
In vivo validation of mild MNP/AMF hyperthermia in
wound of diabetic mice infected by S. aureus and P.
aeruginosa
By observing the synergistic efficacy of mild MNP/AMF with
CIP against dual species biofilm consisting of S. aureus and
6
L. A. ALMUTAIRI ET AL.
P. aeruginosa, we next engaged in a study to validate its efficacy using an in vivo model by employing a murine model
of wound infection in type 2 diabetic mice (db/db mice).
Mice were randomly divided into 4 groups based on treatment conditions, which include untreated control, MNP/AMF
only, CIP only, and MNP/AMF þ CIP. Our in vitro protocol for
inoculating a higher density of S. aureus over P. aeruginosa
could establish the balanced growth of S. aureus and P. aeruginosa in the biofilm (Figure 1(B)). Thus, in this study, db/db
mice were inoculated with the mixture of P. aeruginosa and
S. aureus prepared at 1:1,000 ratio (1 103 CFU of P. aeruginosa to 1 106 CFU of S. aureus) onto the wounds at day 0
(Figure S2A). As observed in the in vitro culture experiment,
the growth rate of P. aeruginosa was greater than S. aureus
in wounds of db/db mice and the number of both bacteria
in the wounds reached at a similar portion at day 2 postinfection (Figure S2B). The formation of dual species biofilm
by S. aureus and P. aeruginosa in the wound was confirmed
by SEM imaging of wound samples (Figure 4(A)).
At day 2 post-infection, the wounds of db/db mice were
topically applied with either a solution of Ferumoxytol-MNPs
(40 mg in 20 mL saline, equivalent to 2 mg/mL MNPs) only, CIP
(50 mg per wound) only, or a mixture of Ferumoxytol-MNPs
and CIP (40 mg MNPs þ 50 mg CIP in 20 mL saline) and the
mice were subsequently applied with an AMF (375 kHz and
60 kA/m) for 10 min (Figure 4(A)). The application of
MNP/AMF resulted in the increase of wound surface temperature by 4 C over the duration of 10 min (Figure 4(B)).
Since the application of high frequency AMF has been associated with the generation of eddy currents that leads to
nonspecific body heating, changes in rectal temperatures
were monitored as a measure of systemic temperature
changes during the application of AMF. The increase of rectal
temperature over 10 min by the application of AMF was
measured to be about 1 C, suggesting that the application
of mild MNP/AMF did not cause an adverse increase of systemic temperature. The CFU numbers of S. aureus and P. aeruginosa were quantified from the wounded skin harvested
after 24 h post-MNP/AMF application (i.e., day 3 post-infection). The treatment of CIP alone reduced the CFU number
of S. aureus and P. aeruginosa in wounds by 0.5-log and 1.5log, respectively, compared to the untreated control group,
while the application of MNP/AMF alone slightly reduced
P. aeruginosa by 0.4-log but had little effect on the viability
of S. aureus (Figure 4(C)). The combined treatment of
MNP/AMF and CIP to the wounds of mice significantly
reduced the CFU number of S. aureus and P. aeruginosa by
2-log and 3-log, respectively, compared to untreated control
group (Figure 4(C)).
The application of mild MNP/AMF hyperthermia altered
the global transcriptional profiling in S. aureus biofilm
Our results clearly support the beneficial effect of mild
MNP/AMF hyperthermia on the eradication of biofilm when
combined with conventional antibiotics. However, the
detailed mechanism by which MNP/AMF hyperthermia sensitizes the antibiofilm effect of antibiotics has yet to be elucidated. As a first step to address this question, we examined
the impact of MNP/AMF on the global transcriptional
Figure 4. The in vivo efficacy of combined treatment of mild MNP/AMF hyperthermia with ciprofloxacin in wounds of db/db mice infected with dual species biofilm. (A) An experimental procedure for the application of mild MNP/AMF and CIP to the wounds of db/db mice infected with dual species biofilm consisting of S.
aureus and P. aeruginosa. The SEM image shows the presence of S. aureus (spherical shape, white arrow) and P. aeruginosa (rod shape, yellow arrow) in the biofilm
collected from wounds of db/db mice day 2 post-infection. (B) Temperature increases in wound surface and rectum of db/db mice measured during the application
of MNP/AMF measured by fiber optic temperature probe. (C) The effect of combined mild MNP/AMF hyperthermia and CIP treatment on the reduction in CFU numbers of S. aureus and P. aeruginosa in wounds of db/db mice at day 3 post-infection. N ¼ 3 per group. p < 0.05 and p < 0.01.
INTERNATIONAL JOURNAL OF HYPERTHERMIA
profiling of biofilm bacteria by using a full-genome microarray analysis in an in vitro culture of S. aureus biofilm. The
microarray analysis revealed 170 genes whose expressions
were significantly altered (fold change 2 and 2,
p < 0.05) following the application of MNP/AMF hyperthermia
at a mild thermal dose (CEM43¼6 min). Among them, 74
genes were up-regulated while 96 genes were down-regulated (Figure 5(A)). The genes were further categorized into
functional clusters, which include genes associated with cell
wall remodeling, oxidative stress, efflux pump, stress
responses, DNA repair, protein synthesis, heat shock protein
(HSP), metabolic pathways and hypothetical proteins
(Supplementary Table 1).
Among the set of genes differentially expressed to mild
MNP/AMF in S. aureus biofilm, we particularly focused on
functional clusters that were either robustly increased or
decreased. The genes for cell wall stress stimulon and heatshock proteins were observed to be increased to mild a
MNP/AMF. For example, the expression of genes for cell wall
stress stimulon including murA, msrA1, tagH, bioD, lytH, and
yidC were upregulated by 3 to 4-fold following MNP/AMF
compared to an untreated control group. The expression of
HSP related genes (ctsR, mcsA, mcsB, grpE, clpL, hrcA, and
ctsR) were also upregulated by 4 to 11-fold (Supplementary
Table 1), which is in line with previous studies of heat-shock
responses in S. aureus [27]. In contrast, the expression of
genes associated with protein synthesis and cell division
were downregulated following the application of MNP/AMF,
albeit modestly (2 to 4-fold). The transcription of ribosomal
protein genes including, rplX, rplE, rpsN, rpsH, rplF, and rplR,
was shown to be downregulated in S. aureus biofilm, compared to its planktonic phase [28]. Our result showed that
the application of a mild MNP/AMF could increase the
expression of those genes by 2-fold, compared to an
untreated biofilm control group (Supplementary Table 1).
7
Of particular observation from the gene microarray data
was the shift in the expression of metabolism-related genes.
For example, the expression of genes for fermentation such
as ldhA, pflA and pflB were significantly downregulated by 8to 10-fold following the application of MNP/AMF hyperthermia (Figure 5(B)). The ldhA gene is involved in the synthesis
of fermentation enzymes like lactate dehydrogenases. The
pflB and pflA genes play a role in acetate and ethanol formation, which were shown to be upregulated under anoxic conditions within biofilms [29]. In contrast, the expression of
genes for glycolytic and tricarboxylic acid cycle (TCA) pathways such as Pyr, fda, odhA were significantly upregulated by
4 to 10-fold following the application of MNP/AMF hyperthermia (Figure 5(B)). The Pyr and fda are genes for key
enzymes in the glycolysis pathway under aerobic respiration
[30] and odhA is a gene for the TCA cycle [31].
The application of mild MNP/AMF hyperthermia was
associated with an increased metabolic activity in S.
aureus biofilm
Our gene microarray results imply that the MNP/AMF hyperthermia might shift cellular respiration of the biofilm phase
of bacteria from an anaerobic fermentation to an aerobic
glycolytic/TCA pathway. In our recent study, we demonstrated that the synergistic effect of mild MNP/AMF on the
antibiotic efficacy in S. aureus biofilm was associated with an
increased uptake of antibiotics [9]. It has been shown that
the bacteria within biofilms exhibited reduced metabolic
activity due to the limited oxygen and nutrients, and this has
been associated with a low susceptibility to antibiotics [32–
34]. These findings prompted us to examine whether the
altered expression of genes for cellular respiration was linked
to a change in metabolic activity in S. aureus biofilm
Figure 5. The effects of mild MNP/AMF hyperthermia on the metabolism of S. aureus biofilm. (A) Volcano plot showing the gene level analysis to select differentially expressed genes after treatment with mild MNP/AMF (CEM43¼6 min). the data for all genes are plotted as fold change versus log10 of the adjusted p-value.
Green, gray and red correspond to genes with < 2-fold, 2 to 2-fold and > 2-fold differential expression, respectively. (B) Changes in the expression of the
genes for anaerobic fermentation (ldhA, adhE, pflA and pflB) and genes for aerobic glycolysis/TCA pathway (Pyr, pckA, fda, and odhA) in response to mild MNP/AMF
hyperthermia (CEM43¼6 min) in S. aureus biofilm. The values listed in the figures are the calculated means of fold change from three parallel microarrays. (C)
Changes in metabolic activity in S. aureus biofilm in response to MNP/AMF (CEM43¼6 min) quantified by resazurin assay. N ¼ 8 per group. p < 0.05.
8
L. A. ALMUTAIRI ET AL.
following MNP/AMF. For this, the metabolic activity of S. aureus biofilm in the absence and presence of mild MNP/AMF
hyperthermia (CEM43¼6 min) was assessed by resazurin
assay, which has been used to assess the metabolic activity
of bacteria residing in biofilms [35,36]. The metabolic activity
of S. aureus was significantly increased for biofilm treated
with MNP/AMF hyperthermia compared to the untreated
control group of biofilms at both 1 and 4 h post-MNP/AMF
(p < 0.05) and the activity reverted back to baseline at 18 h
(Figure 5(C)).
Discussion
We recently reported that the application of mild MNP/AMF
hyperthermia could be synergistic with antibiotics in eradicating single species biofilm formed by S. aureus [9]. Here,
using both in vitro and in vivo murine models of biofilm
infection, we demonstrate that mild MNP/AMF hyperthermia
is also effective against dual species biofilm infection consisting of S. aureus and P. aeruginosa when combined with a
broad-spectrum antibiotic, which alone became ineffective
otherwise. Our protocol for inoculating the high density of S.
aureus over P. aeruginosa enabled the formation of dual species biofilm with a balanced growth of both bacteria. CIP is a
broad-spectrum antibiotic drug that has shown to be highly
effective for both S. aureus and P. aeruginosa with low MIC
values [37]. The dual species biofilm exhibited a higher tolerance to CIP than single species biofilm formed by either S.
aureus or P. aeruginosa alone. In the current study, we
employed a murine model of dual species biofilm in wounds
of db/db mice, a genetic mouse model of type 2 diabetes
associated with impaired wound healing and persistent infection [38,39]. In consistent with in vitro results, our in vivo
study validated that the combined treatment of mild
MNP/AMF and CIP could synergistically reduce the number
of viable bacteria of both S. aureus and P. aeruginosa in
wounds of the mice, compared to either CIP or MNP/AMF
treatment alone.
What is the potential mechanism by which mild
MNP/AMF hyperthermia can sensitize biofilm bacteria to be
susceptible to antibiotics? We speculate that this is associated with heat shock-induced alteration of the metabolic
state of bacteria in the biofilm phase. This is supported by
our result showing that the S. aureus biofilm treated with
mild MNP/AMF hyperthermia was sufficient to increase metabolic activity in the biofilm. We recently demonstrated that
MNP/AMF-induced heat shock to the culture of S. aureus biofilm could significantly enhance the uptake of antibiotic to
the biofilm phase of bacteria, with the response that is not
dependent on antibiotic penetration through the biofilm
matrix [9]. In an earlier study for testing P. aeruginosa biofilm
susceptibility to ciprofloxacin, oxygen limitation and the
resulting low metabolic activity, not the limited antibiotic
penetration, was attributed to antibiotic tolerance in the biofilm [34]. The bacterial cells embedded in biofilms exhibit a
suppressed metabolic activity and as such, an altered metabolic state can influence antibiotic susceptibility by altering
antibiotic uptake [32,40]. It is interesting to note that, for
gram-negative swarming bacteria including Proteus mirabilis,
hyperthermia-induced enhancement of antibiotic susceptibility was observed to be through the alteration of a cell wall
structure that resulted in the inhibition of penicillin-binding
protein-2 [41].
Our gene microarray data suggest that mild MNP/AMF
can shift the expression of genes for cellular respiration from
anaerobic fermentation to aerobic glycolytic/TCA pathways
in S. aureus biofilm. We speculate that this might contribute
to an increased antibiotic uptake by bacteria in a biofilm
phase. This speculation is supported by previous studies for
targeting TCA metabolic pathways as an antimicrobial strategy. For example, the metabolites from the TCA cycle could
improve the susceptibility of resistant bacteria to bactericidal
antibiotics [42,43] whereas alterations in metabolism involving a decreased TCA cycle activity was associated with the
tolerance of S. aureus to antibiotics including daptomycin
[44]. The role of TCA cycle activity in antibiotic tolerance was
also evidenced in a recent study using a polymicrobial biofilm model [19], in which decreased metabolic activity associated with decreased TCA cycle activity was found to be a
mechanism for increased antibiotic tolerance within polymicrobial cultures formed by S. aureus and Candida albicans,
compared to monocultures of S. aureus. Future study should
be directed toward determining whether a shift in metabolic
activity is functionally linked to the beneficial effect of mild
MNP/AMF hyperthermia against biofilm infections.
This study has some limitations. Firstly, in this study, we
used standard strains of bacteria and it is necessary to assess
the therapeutic efficacy of MNP/AMF hyperthermia using
clinical bacterial isolates for future clinical translation.
Additionally, the dose of CIP used in this study (50 mg/mL)
for combined therapy with MNP/AMF hyperthermia is relatively high for systemic treatment and it needs to be further
assessed using standard dose of antibiotics. Secondly, future
study should be directed toward optimizing treatment conditions such as a therapeutic window and frequency of
MNP/AMF application and examining long-term effects of
the therapy. A therapeutic window of MNP/AMF treatment
on the biofilm needs to be carefully determined whether
simultaneous or post exposure of antibiotic would be beneficial in the combination therapy. Additionally, our result of
metabolic activity showed that single session treatment of
MNP/AMF hyperthermia may not be sufficient to induce a
sustained increase in metabolic activity. It would be interesting to further examine whether repeated application at a
lower thermal dose would be effective in stimulating a sustained metabolic activity and thereby suppressing the potential regrowth of biofilm.
In summary, our results support the translational potential
of mild MNP/AMF as an adjunctive therapy that can be combined with a conventional antibiotic treatment for the management of wound biofilm infections associated with
multispecies bacteria. Additionally, our findings imply that
the beneficial effect of mild MNP/AMF hyperthermia on the
increased susceptibility of biofilm to an antibiotic treatment
is associated with an increased metabolic activity, which in
INTERNATIONAL JOURNAL OF HYPERTHERMIA
turn facilitates an increased antibiotic uptake by bacteria in
biofilm phase.
[10]
[11]
Author contributions
MK conducted the conception and design of the research. LA and MK
wrote the first draft of the manuscript. LA, BY, ED, and AO contributed
to the acquisition of data. LA, BY, and MK carried out the analysis and
interpretation of data. All authors read and approved the final version of
the manuscript.
[12]
[13]
[14]
Acknowledgments
The authors thank Dr. Min Gao and Dr. Lu Zou (Advanced Materials and
Liquid Crystal Institute at Kent State University, Kent, Ohio) for training
and assistance with SEM. This publication was made possible in part by
support from the Kent State University Open Access Publishing Fund.
Disclosure statement
No potential conflict of interest was reported by the author(s).
[15]
[16]
[17]
Funding
This research was supported by the National Institute of Health under
R01 NR015674 (to MK).
[18]
[19]
Data availability statement
The data that support the findings of this study are available from the
corresponding author (MK) upon reasonable request.
[20]
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