Methamphetamine Alters the Antimicrobial Efficacy of Phagocytic Cells during Methicillin-Resistant Staphylococcus aureus Skin Infection

Abstract

Unlabelled:
Methamphetamine (METH) is a major drug of abuse in the United States and worldwide. Furthermore, Staphylococcus aureus infections and METH use are coemerging public health problems. S. aureus is the single most important bacterial pathogen in infections among injection drug users, with skin and soft tissue infections (SSTI) being extremely common. Notably, the incidence of SSTI, especially in drug users, is difficult to estimate because such infections are often self-treated. Although there is substantial information on the behavioral and cognitive defects caused by METH in drug users, there is a dearth of knowledge regarding its impact on bacterial infections and immunity. Therefore, we hypothesized that METH exacerbates S. aureus skin infection. Using a murine model of METH administration and wound infection, we demonstrated that METH reduces wound healing and facilitates host-mediated collagen degradation by increased expression and production of matrix metalloproteinase-2 (MMP-2). Additionally, we found that METH induces S. aureus biofilm formation and leads to detrimental effects on the functions of human and murine phagocytic cells, enhancing susceptibility to S. aureus infection. Our findings provide empirical evidence of the adverse impact of METH use on the antimicrobial efficacy of the cells that comprise innate immunity, the initial host response to combat microbial infection.

Importance:
METH is an extremely addictive central nervous system stimulant that is frequently administered by injection. SSTI, common problems among injection drug users, result in serious morbidity for patients and costly hospitalizations for treatment of superficial wounds and incision and drainage of abscesses; however, there has been little etiologic or preventive epidemiological research on this problem. In addition, the evasive nature of injection drug users toward medical care complicates our ability to accurately predict the prevalence of these infections. Hence, this study investigated the impact of METH use on S. aureus skin infection. Our findings demonstrate that this drug of abuse promotes biofilm formation and negatively impacts the wound healing process and innate immune function, exacerbating susceptibility to S. aureus infection. The findings may translate into new knowledge and development of therapeutic and public health strategies to deal with the devastating complications of METH abuse.

Full text

Methamphetamine Alters the Antimicrobial Efficacy of Phagocytic
Cells during Methicillin-Resistant Staphylococcus aureus Skin Infection
Mircea Radu Mihu,
a
Jessica Roman-Sosa,
b,c
Avanish K. Varshney,
b,c
Eliseo A. Eugenin,
d,e
Bhavikkumar P. Shah,
f
Hiu Ham Lee,
g
Long N. Nguyen,
h
Allan J. Guimaraes,
i
Bettina C. Fries,
j,k
Joshua D. Nosanchuk,
b,c
Luis R. Martinez
b,c,g
Division of Infectious Diseases, Montefiore Medical Center, Bronx, New York, USA
a
; Division of Infectious Diseases, Department of Medicine,
b
and Department of
Microbiology and Immunology,
c
Albert Einstein College of Medicine, Bronx, New York, USA; Public Health Research Institute
d
and Department of Microbiology and
Molecular Genetics,
e
New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, USA; Department of Biomedical Sciences, Long Island
University—Post, Brookville, New York, USA
f
; College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, New York, USA
g
; Signature Research
Program in Cardiovascular and Metabolic Disorders, Duke—NUS Graduate Medical School, Singapore
h
; Instituto Biomédico, Universidade Federal Fluminense, Rio de
Janeiro, Brazil
i
; Division of Infectious Diseases, Department of Medicine,
j
and Department of Molecular Genetics and Microbiology,
k
Stony Brook University, Stony Brook,
New York, USA
ABSTRACT Methamphetamine (METH) is a major drug of abuse in the United States and worldwide. Furthermore, Staphylococ-
cus aureus infections and METH use are coemerging public health problems. S. aureus is the single most important bacterial
pathogen in infections among injection drug users, with skin and soft tissue infections (SSTI) being extremely common. Nota-
bly, the incidence of SSTI, especially in drug users, is difficult to estimate because such infections are often self-treated. Although
there is substantial information on the behavioral and cognitive defects caused by METH in drug users, there is a dearth of
knowledge regarding its impact on bacterial infections and immunity. Therefore, we hypothesized that METH exacerbates S.
aureus skin infection. Using a murine model of METH administration and wound infection, we demonstrated that METH re-
duces wound healing and facilitates host-mediated collagen degradation by increased expression and production of matrix
metalloproteinase-2 (MMP-2). Additionally, we found that METH induces S. aureus biofilm formation and leads to detrimental
effects on the functions of human and murine phagocytic cells, enhancing susceptibility to S. aureus infection. Our findings pro-
vide empirical evidence of the adverse impact of METH use on the antimicrobial efficacy of the cells that comprise innate immu-
nity, the initial host response to combat microbial infection.
IMPORTANCE METH is an extremely addictive central nervous system stimulant that is frequently administered by injection.
SSTI, common problems among injection drug users, result in serious morbidity for patients and costly hospitalizations for
treatment of superficial wounds and incision and drainage of abscesses; however, there has been little etiologic or preventive
epidemiological research on this problem. In addition, the evasive nature of injection drug users toward medical care compli-
cates our ability to accurately predict the prevalence of these infections. Hence, this study investigated the impact of METH use
on S. aureus skin infection. Our findings demonstrate that this drug of abuse promotes biofilm formation and negatively im-
pacts the wound healing process and innate immune function, exacerbating susceptibility to S. aureus infection. The findings
may translate into new knowledge and development of therapeutic and public health strategies to deal with the devastating com-
plications of METH abuse.
Received 23 September 2015 Accepted 28 September 2015 Published 27 October 2015
Citation Mihu MR, Roman-Sosa J, Varshney AK, Eugenin EA, Shah BP, Ham Lee H, Nguyen LN, Guimaraes AJ, Fries BC, Nosanchuk JD, Martinez LR. 2015. Methamphetamine
alters the antimicrobial efficacy of phagocytic cells during methicillin-resistant Staphylococcus aureus skin infection. mBio 6(6):e01622-15. doi:10.1128/mBio.01622-15.
Editor Larry S. McDaniel, University of Mississippi Medical Center
Copyright © 2015 Mihu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license,
which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Address correspondence to Luis R. Martinez, lmarti13@nyit.edu.
Staphylococcus aureus is an immotile Gram-positive coccus that
frequently colonizes human nasal membranes and skin. It is
responsible for the majority of superficial and invasive skin infec-
tions, resulting in over 12,000,000 outpatient/emergency room
(ER) visits (1) and 400,000 hospital admissions annually in the
United States (2). Notably, in a study performed with multiple
ERs across the United States, methicillin-resistant S. aureus
(MRSA) strains were isolated from 61% of abscesses and 53% of
purulent wounds (3). Also, certain S. aureus clinical strains have
recently evolved with resistance to vancomycin, an antibiotic to
which staphylococci had previously been uniformly susceptible.
Although the vancomycin-resistant strains remain rare, MRSA
infections are increasingly common (4), and the incidence of
community-acquired MRSA strains has increased severalfold over
the past several years (5).
Methamphetamine (METH) is an extremely addictive central
nervous system stimulant abused by individuals worldwide, and
the drug is a major threat in many developed countries. The in-
toxicating effects of METH alter judgment and reduce inhibitions,
leading people to engage in unsafe activities that put them at risk
for acquiring transmissible microbes (6, 7). S. aureus, including
community-acquired MRSA, is the most important bacterial
RESEARCH ARTICLE crossmark
November/December 2015 Volume 6 Issue 6 e01622-15 ®mbio.asm.org 1

pathogen in skin and soft tissue infections (SSTI) among drug
users (8–10). SSTI incidence is difficult to assess among drug users
because such infections are often self-treated (11). However, crys-
tal METH injection is associated with frequent visits to the ER due
to abscesses, cellulitis, and other skin infections (12). Additionally,
MRSA, particularly USA300 strains, causes most SSTI in METH
users (13).
METH is associated with several socioeconomic and behav-
ioral risk factors that may predispose individuals to MRSA SSTI
(11). Notably, even noninjection use is associated with MRSA
SSTI, as smoking METH is an independent risk factor for MRSA,
including primary skin abscesses and invasive infections (13, 14).
In addition, skin picking is associated with MRSA SSTI. METH
use causes formication, a sensation of something crawling on the
body or under the skin, which can lead to skin-picking behavior
and skin breakdown (10, 15). Furthermore, METH injection is
also associated with the introduction of bacteria into the skin,
especially if the user fails to clean injection sites or shares drug
paraphernalia (16).
The effects of METH on host responses have not been exten-
sively described. However, limited studies about the effects of
METH on immune function show that its abuse has profound
implications for host immunity. The injection of 25 mg/kg of body
weight of METH into rats induces apoptotic death in thymic and
splenic lymphocytes (17). METH also reduces thymic and splenic
cellularity and alters peripheral T lymphocyte populations in
treated mice (18). METH exposure results in mitochondrial oxi-
dative damage and causes dysfunction of primary human T cells
(19). Also, METH is an immunosuppressive agent; it alkalizes
normally acidic organelles within immune cells, inhibits antigen
presentation, and impairs phagocytosis (20). Moreover, METH
negatively alters antibody and cytokine production (21).
Although there is a clear clinical association of METH use and
MRSA disease, there has not been a defined biological link be-
tween the immune response and wound healing capacity of a
METH user with an increased susceptibility to S. aureus. However,
animal studies demonstrate that METH suppresses both innate
and adaptive immunity (22, 23) and alters immune cell gene ex-
pression (24). Here, we investigated whether METH facilitates
MRSA skin infections and provide evidence that the drug has neg-
ative effects on human-derived phagocytic cells.
RESULTS
METH alters wound healing. METH administration decreased
the wound healing rate significantly compared with results in un-
treated animals (Fig. 1A). Moreover, METH administration and
FIG 1 METH decreases wound healing in mice infected with MRSA. (A) Wounds of BALB/c mice (n⫽5 per group) uninfected and untreated (untreated),
infected with MRSA, treated with methamphetamine (METH), or treated with METH and infected with MRSA (METH ⫹MRSA), at days 3, 5, 7, 9, 11, 13, 15,
and 17. Bar, 5 mm. (B) Wound size analysis of BALB/c mouse skin lesions. Wounds were uninfected and untreated, infected with MRSA, treated with METH,
or treated with METH and infected with MRSA. Symbols are the averages of the results for six measurements at each time interval, and error bars denote standard
deviations. Pvalues of ⬍0.05 were considered significant, calculated by ANOVA.
␾
,
␹
, and
␲
indicate significantly higher eschar sizes in METH ⫹MRSA mice
than sizes for untreated, MRSA-infected, or METH-treated groups, respectively. This experiment was performed twice and similar results were obtained. The
results shown are representative of an individual experiment.
Mihu et al.
2®mbio.asm.org November/December 2015 Volume 6 Issue 6 e01622-15

MRSA infection (METH-MRSA) induced visible inflammation
and decreased wound healing compared with all other conditions
(Fig. 1A). In fact, the wounds in infected or METH-treated mice
demonstrated a significant increase in size relative to the initial
wound, which did not occur in the untreated, uninfected animals.
At day 3, eschars in untreated wounds were ~4.4 mm in diameter,
whereas eschars of animals treated with METH alone or untreated
MRSA-infected or METH-MRSA wounds were ~7 mm (P⬍
0.001, compared with untreated). At day 7 (Fig. 1B), eschars in the
untreated group were ~2.4 mm, whereas the eschars of wounds in
the MRSA, METH, or METH-MRSA treatment groups were ~3.9
mm (P⬍0.05), ~6.5 mm (P⬍0.001), and ~6.9 mm (P⬍0.001),
respectively. At day 13 (Fig. 1B), the wounds of untreated mice
showed complete closure, whereas eschars of MRSA, METH, or
METH-MRSA wounds were ~1.7 mm (P⬍0.01), ~2.1 mm (P⬍
0.01), and ~4.9 mm (P⬍0.001), respectively. Complete wound
closure was reached by day 17 for MRSA- or METH-treated mice,
whereas complete wound healing in the METH-MRSA group
took 19 days. Mice treated with METH or MRSA alone had similar
resolution of wounds during days 9 to 17, although METH-
exposed mice had slower healing at day 7.
METH enhances MRSA burden. Qualitative histological ex-
aminations revealed that wounds of untreated, uninfected mice
and of METH-treated uninfected mice had less inflammation
along with increased fibrin deposition than MRSA-infected tis-
sues, and no evidence of bacteria (Fig. 2A). MRSA-infected mice
showed localized epidermal inflammation (Fig. 2A). However,
wounds of METH-treated MRSA-infected mice had intense in-
flammatory infiltrates in both the epidermal and dermal layers,
along with extensive cell necrosis (Fig. 2A). Tissue Gram stains of
MRSA-infected and of METH-treated MRSA-infected samples
displayed large numbers of Gram-positive cocci (Fig. 2A, insets),
with larger bacterial clusters in biofilm-like arrangements evident
in the sections from METH-treated animals.
MRSA-infected mice treated with METH had significantly
higher, by 2 logs, microbial burdens than untreated MRSA-
infected mice at day 7 postinfection (P⬍0.05) (Fig. 2B). Less than
30 bacterial CFU were detected in the skin of uninfected mice, and
MRSA was not identified.
METH impairs wound healing by mediating host matrix
metalloproteinase-2 collagen degradation. The mechanisms
through which METH impairs wound healing were explored by
examining whether METH decreased collagen deposition in
wound tissue (Fig. 3). Collagen content, in both the epidermal and
dermal layers, was lowest in METH-treated MRSA-infected
wounds (Fig. 3A). The lack of blue stain indicated lower tissue
collagen formation in METH-treated MRSA-infected wounds,
suggesting that METH administration augmented MRSA dermal
collagen degradation. Figure 3B presents the results of a morpho-
metric analysis of the data shown in Fig. 3A.
To determine whether METH affects collagen deposition dur-
ing wound healing, we assessed mRNA expression of collagen type
I and III in murine wound tissue (Fig. 3C). METH-treated MRSA-
infected samples had significantly increased mRNA collagen type
I and III expression, ~18-fold higher, compared to untreated, un-
infected control tissues (P⬍0.001). Notably, tissues from MRSA-
infected mice had an 8-fold increase in collagen type III expression
compared to tissues from untreated, uninfected animals (P⬍
0.001) (Fig. 3C), but there was no difference in collagen I expres-
sion.
S. aureus staphopains are cysteine proteases involved in tissue
destruction and collagen degradation. Therefore, we examined
whether METH enhances collagen degradation by staphopain A
(Fig. 3D). Surprisingly, increased concentrations of METH signif-
icantly reduced the degradation of collagen by staphopain A (P⬍
0.05). Similarly, untreated and METH-treated heat-inactivated
staphopain A showed lower collagen degradation than active pro-
tease.
FIG 2 METH enhances MRSA burden in superficial skin lesions. (A) Histological analysis of BALB/c mice uninfected and untreated (untreated), infected with
MRSA (MRSA), treated with METH (METH), or treated with METH and infected with MRSA (METH ⫹MRSA), at day 7. Mice were infected with 10
7
MRSA
bacterial cells. Representative H&E-stained sections of the skin lesions are shown. Bars, 25
␮
m. The insets depict Gram stain results for the boxed areas, and the
panels reveal MRSA cells (shown in purple). The arrows in these insets indicate bacterial clusters in biofilm-like arrangements. (B) Wound bacterial burdens
(CFU) in METH-treated mice infected with 10
7
MRSA cells were significantly higher than those in MRSA-infected, untreated mice at 7 days after infection (n⫽
10 per group). Each symbol represents one animal; dashed bars are the averages for each group, and error bars denote standard deviations. Asterisks denote
Pvalues of ⬍0.05, calculated by using Student’s ttest. These experiments were performed twice, and similar results were obtained. The results shown are
representative of an individual experiment.
Methamphetamine Exacerbates MRSA Skin Infection
November/December 2015 Volume 6 Issue 6 e01622-15 ®mbio.asm.org 3

To define the host’s contribution in collagen degradation after
METH administration and MRSA infection, we analyzed the ex-
pression of matrix metalloproteinase-2 (MMP-2) in mouse
wounded tissue (Fig. 3E). METH-treated or METH-treated,
MRSA-infected tissues had significantly increased mRNA MMP-2
expression levels, by ~4- and 6-fold, respectively, above those in
untreated, uninfected control tissues (P⬍0.001). Then, we inves-
tigated whether METH increased collagen degradation by MMP-2
(Fig. 3F). METH exacerbated collagen degradation by MMP-2
relative to that in untreated tissue (25
␮
M[P⬍0.05]; 50
␮
M[P⬍
0.001]). No differences were observed between untreated tissues
and those in the METH treatment group that received heat-
inactivated MMP-2. Western blot analysis was performed to con-
firm the upregulation of MMP-2 protein levels in METH-treated
tissues (Fig. 3G). We found that METH increased the expression
of MMP-2 in both uninfected and MRSA-infected wounds. To-
FIG 3 METH impairs wound healing by enhancing the host’s MMP-2-mediated collagen degradation. (A) Histological analysis of untreated, MRSA, METH,
or METH ⫹MRSA treatment groups of BALB/c mice at day 7 after wounding. The blue stain indicates collagen. Bar, 25
␮
m. (B) Quantitative measurement of
collagen intensity in tissues from untreated, MRSA, METH, and METH ⫹MRSA mice were measured using ImageJ software. Bars are the averages of the results,
and error bars denote standard deviations. *, #, and & indicate significantly lower collagen intensites than in untreated, MRSA-infected, or METH-treated groups,
respectively. (C) Gene expression analysis of collagen type I and III in cutaneous lesions.
␾
,
␹
, and
␲
indicate significantly higher fold changes than in untreated,
MRSA-infected, or METH-treated groups, respectively. (D) Collagen degradation by MRSA staphopain A. FITC-conjugated type I collagen was incubated with
10
␮
M staphopain A and METH (25 or 50
␮
M)for3hat37°C. The fluorescence of the supernatant was measured. Heat-inactivated staphopain A was utilized
as a control. Dashed lines represent the averages for three measurements per condition, and error bars denote standard deviations. *, #, and & indicate
significantly lower relative fluorescence than in the untreated or 25
␮
M METH groups after cells were incubated with active staphopain A, respectively. (E) Gene
expression analysis of MMP-2 in murine wounds.
␾
,
␹
, and
␲
indicate significantly higher fold changes than in untreated, MRSA-infected, or METH-treated
groups, respectively. (F) Collagen degradation by MMP-2. FITC-conjugated type I collagen was incubated with 10
␮
M MMP-2 and METH (25 or 50
␮
M) for 3 h
at 37°C.
␾
and
␲
indicate significantly higher relative fluorescence values than in the untreated or 25
␮
M METH groups, respectively. *, #, and & indicate
significantly lower relative fluorescence than in cells from untreated or the 25 or 50
␮
M METH groups incubated with active MMP-2, respectively. (G) Western
blot analysis of MMP-2 in wounded tissue (7 days) of untreated, MRSA, METH, or METH ⫹MRSA treatment in BALB/c mice. GAPDH was used as a loading
control. For panels B to F, Pvalues (with significance defined as P⬍0.05) were calculated by using an ANOVA. All the experiments in the figure were performed
twice, and similar results were obtained. The results shown are representative of an individual experiment.
Mihu et al.
4®mbio.asm.org November/December 2015 Volume 6 Issue 6 e01622-15

gether, these results suggest that wound healing in METH-treated
animals is reduced, at least in part, by an increase in the host’s
MMP-2 levels.
METH decreases the number of phagocytic cells in the blood
of treated BALB/c mice. We examined whether METH adminis-
tration depleted phagocytes in the blood of BALB/c mice by using
differential leukocyte staining. Light microscopy images showed
reduced numbers of phagocytic cells in the blood of METH-
treated mice compared with numbers in untreated mice (Fig. 4A).
Cell count analysis showed that METH-treated animals had sig-
nificantly lower numbers of circulating phagocytes in blood com-
pared to numbers in controls (P⬍0.05) (Fig. 4B).
METH does not inhibit S. aureus growth. MRSA growth with
and without exposure to METH was determined in real time for
24 h (Fig. 5). METH did not alter bacterial growth during a 24-h
coincubation with 25 or 50
␮
M METH compared with untreated
S. aureus controls.
METH enhances S. aureus biofilm formation. We evaluated
the effect of METH on biofilm formation by six clinical S. aureus
isolates to confirm the results obtained in the murine model. S. aureus
biofilms on 96-well plates were incubated with METH (25 or 50
␮
M)
or phosphate-buffered saline (PBS) for 24 h. Cell viability and meta-
bolic activity were determined by CFU and 2,3-bis(2-methoxy-4-
nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium
hydroxide (XTT) reduction assays, respectively (Fig. 6A and B). On
average, METH promoted S. aureus biofilm formation compared to
untreated controls. S. aureus biofilms incubated with METH dis-
played higher CFU counts (P⬍0.0001) (Fig. 6A), and cells within
biofilms were more metabolically active (P⬍0.0001) (Fig. 6B) than
untreated controls.
Confocal microscopic examination was used to correlate the
decreases in CFU and the XTT reduction assay results with the
visual effects on biofilm structure (Fig. 6C). In the figure, regions
of green fluorescence represent viable cells; the red fluorescence
indicates metabolically inactive or nonviable cells. Untreated
MRSA strain 6498 biofilms were ~21
␮
m thick, whereas biofilms
grown in the presence of 25
␮
M METH showed a more robust
architecture with a thickness of ~71
␮
m (Fig. 6C). The METH-
treated biofilm metabolic activity observed corresponded with the
XTT results.
The S. aureus gene ica encodes an intercellular adhesion pro-
tein, and disruption of this gene leads to decreased biofilm forma-
tion (25). All isolates in the present study were found to carry the
ica gene (data not shown). Hence, we investigated the impact of
METH on the expression of the ica gene by biofilm-associated
cells via quantitative reverse transcription-PCR (qRT-PCR)
(Fig. 6D). S. aureus biofilm-associated cells exhibited significantly
increased ica expression after exposure to METH (25
␮
M[P⬍
0.0001] and 50
␮
M[P⬍0.0001]).
METH affects murine neutrophil functions. We investigated
the effect of METH on murine neutrophil effector functions in
MRSA infection. METH significantly inhibited neutrophil migra-
tion (25
␮
M, P⬍0.05; 50
␮
M, P⬍0.01) in a concentration-
dependent manner compared to untreated or chloroquine
(Chlq)-treated cells (Fig. 7A). As expected, cytochalasin D
(CytD)-treated cells (25
␮
M) displayed a significant reduction in
migration (P⬍0.01) compared to untreated cells.
We examined whether METH interfered with neutrophil-
mediated killing (NMK) of MRSA strain 6498. When untreated
neutrophils and MRSA 6498 were coincubated, the majority of
bacteria were killed within 80 min (Fig. 7B). Similarly, Chlq-
treated neutrophils showed an increase in bacterial killing over
time, albeit to a lesser extent than untreated neutrophils. In con-
trast, METH more significantly impaired bacterial killing, in a
concentration-dependent manner (P⬍0.05, 60 and 80 min) by
stationary neutrophils, and likewise, CytD interfered with phago-
cytic killing (Fig. 7B).
Confocal microscopic examination was used to correlate the
killing assay results with the visual effects on the impact of METH
on neutrophil morphology (red fluorescence [actin]) and phago-
cytosis of MRSA (blue fluorescence [Alexa-labeled S. aureus])
(Fig. 7C). METH-treated neutrophils (25 and 50
␮
M; P⬍0.05)
and CytD-treated neutrophils (25
␮
M; P⬍0.05) significantly
FIG 4 METH-treated animals displayed fewer phagocytes in blood. (A) Light microscopy images of blood smears from untreated or METH-treated mice
preinfection. Arrows indicate phagocytic cells (purple). Bar, 20
␮
m. (B) Numbers of phagocytic cells per field in blood of untreated or METH-treated mice. Each
black circle or red square represents the number of neutrophils per individual field. Dashed lines and error bars denote averages and standard deviations of 40
counts, respectively. Asterisks denote significant Pvalues (P⬍0.05), calculated using Student’s ttest. The experiment was performed twice with similar results
obtained. The results shown are a combination of two independent experiments.
FIG 5 METH does not affect S. aureus viability in vitro. The effect of METH
on S. aureus growth kinetics was determined via Bioscreen C analysis. S. aureus
was grown in the absence (untreated) or presence of METH (25 or 50
␮
M).
Each point represents the average of three measurements. The experiment was
performed twice with similar results obtained. The results shown are represen-
tative of an individual experiment.
Methamphetamine Exacerbates MRSA Skin Infection
November/December 2015 Volume 6 Issue 6 e01622-15 ®mbio.asm.org 5

reduced phagocytosis of MRSA, compared to that of untreated or
Chlq-treated cells 30 min after coincubation (Fig. 7C). Untreated
neutrophils displayed multiple protrusions and actin polymeriza-
tion, engaged in phagocytic and killing interactions with S. aureus
(Fig. 7D), and contained an increased number of internalized bac-
teria. In contrast, METH- or CytD-treated neutrophils displayed a
large rounded morphology, fewer protrusions, and reduced inter-
actions with bacteria, and the cells were surrounded by multiple
bacterial cells (Fig. 7D).
Neutrophils treated with METH (50
␮
M; P⬍0.001) or CytD
(25
␮
M; P⬍0.001) showed significantly lower levels of NO pro-
duction after MRSA infection than did untreated neutrophils
(Fig. 7E). Both METH (25
␮
M) and Chlq (25
␮
M) showed a trend
toward a reduction in NO production. Similarly, the release of
myeloperoxidase (MPO) by neutrophils was significantly de-
creased by METH (25
␮
M; P⬍0.01; 50
␮
M, P⬍0.001) or CytD
(25
␮
M; P⬍0.001) (Fig. 7F).
METH impairs the effector functions of human neutrophils.
We analyzed the effects of achievable physiological METH doses
on phagocytosis and killing of six clinical S. aureus isolates by
human neutrophils via fluorescence-activated cell sorting (FACS)
analysis. METH significantly reduced phagocytosis of S. aureus by
neutrophils, compared with the untreated or Chlq᎑treated (25
␮
M
Chlq) cells (25
␮
M METH, P⬍0.01; 50
␮
M METH, P⬍0.01;
compared to untreated and Chlq groups) (Fig. 8A). Our results
showed 52.5% and 46% S. aureus phagocytosis by cells treated
with METH (25 or 50
␮
M) compared to untreated (77%) or 25
␮
M Chlq-treated cells (75.5%). As expected, CytD-treated cells
displayed a significant reduction in phagocytosis compared to
cells in all the other groups, with the exception of 50
␮
M METH
(P⬍0.001).
We again also examined whether METH interfered with
neutrophil-mediated killing of S. aureus cells. CytD and METH
significantly reduced bacterial killing by human neutrophils (P⬍
0.0001) compared to untreated cells (Fig. 8B). These phagocytic
cells mostly kill bacteria via NADPH oxidase-derived reactive ox-
ygen species (ROS). As a result, we evaluated the impact of METH
on the neutrophil oxidative burst by measuring luminol chemilu-
minescence intensity. METH significantly decreased ROS pro-
duction compared to untreated or Chlq-treated, S. aureus-
exposed neutrophils (Fig. 8C) (P⬍0.05; 25 to 60 min). Minimal
production of ROS was observed in untreated and unstimulated
controls or CytD-treated cells (P⬍0.05; 30 to 60 min).
METH alters macrophage function. We identified macrophage-
like infiltration in wound tissue by measuring the expression of
Iba-1, which is mostly expressed and upregulated during the acti-
vation of these cells. Tissue sections from murine wounds in the
METH-treated (uninfected) group showed only scattered
macrophage-like cells, whereas untreated or MRSA-infected mice
displayed massive macrophage-like cell infiltrations throughout
the wound area (Fig. 9A). Additionally, METH-treated MRSA-
FIG 6 METH promotes biofilm formation by S. aureus clinical isolates (n⫽6), based on CFU counts (A) and XTT reduction (B) after 24 h. Dashed lines and
error bars represent the averages and standard deviations of six measurements (each symbol represents a single strain) from three plates per well per strain. (C)
Confocal microscopy of MRSA 6498 biofilms after treatment with METH. Representative three-dimensional images of biofilms show viable (green [SYTO 9])
and dead (red [propidium iodide]) cells. The thickness and morphology of the bacterial biofilms can be observed in the Z-stack reconstruction. The pictures were
taken at a magnification of ⫻63. Bar, 50
␮
m. (D) Expression analysis of the ica gene in S. aureus strains after incubation with METH. For panels A, B, and D,
significance (P⬍0.05) was calculated by ANOVA.
␾
indicates significantly higher fold change than untreated group. These experiments were performed twice
with similar results obtained. The results shown are representative of an individual experiment.
Mihu et al.
6®mbio.asm.org November/December 2015 Volume 6 Issue 6 e01622-15

infected wounds demonstrated minimal macrophage-like cell in-
filtration.
METH (25
␮
M,P⬍0.05; 50
␮
M, P⬍0.01) significantly in-
hibited macrophage migration in a concentration-dependent
manner compared to untreated or Chlq-treated cells (Fig. 9B).
CytD-treated cells (25
␮
M; P⬍0.01) displayed a significant re-
duction compared to untreated cells.
METH (25 and 50
␮
M) significantly stimulated phagocytosis
of bacterial cells by murine macrophages, compared with the un-
treated, Chlq᎑treated (25
␮
M), or CytD-treated (25
␮
M) cells (P
⬍0.01 for 25
␮
M METH; P⬍0.001 for 50
␮
M METH) (Fig. 9C).
Furthermore, METH decreased the eradication of bacteria within
macrophages (Fig. 9D).
After phagocytosis, macrophages produce NO in response to
pathogens within the phagosome. Macrophages treated with
METH (50
␮
M; P⬍0.001) or CytD (25
␮
M; P⬍0.001) showed
significantly lower levels of NO production after infection with
MRSA strain 6498 than did untreated macrophages (Fig. 9E).
Both the METH (25
␮
M) and Chlq (25
␮
M) treatment groups
showed a trend toward a decrease in NO production.
Phagosome acidification was studied to elucidate the mecha-
nism by which METH interferes with MRSA 6498 killing after
phagocytosis. The acidification of phagosomes of primary macro-
phages containing MRSA labeled with pH-sensitive and pH-
insensitive probes was measured using a spectrofluorimeter. Stan-
dard curves were generated with fluorophores. METH induced
the alkalization of macrophage phagosomes that contained MRSA
6498 cells. The pH of phagosomes in macrophages infected with
FIG 7 METH modifies neutrophil effector functions. (A) Percentages of neutrophils (polymorphonuclear lymphocytes [PMNs]) migrating at various time
points (20, 40, and 60 min) after exposure of uninfected cells to the chemoattractant fMLP. Neutrophils were isolated from BALB/c mice and treated with PBS
(untreated), Chlq (25
␮
M), CytD (25
␮
M), or METH (25 or 50
␮
M) and coincubated with MRSA. (B and C) METH interferes with neutrophil-mediated killing
(NMK) (B) and phagocytosis of MRSA cells (C). For panel C, fluorescence intensity (in arbitrary units [A.U.]) refers to blue-labeled bacteria inside neutrophils
after their phagocytosis. (D) Confocal microscopy of untreated or CytD- or METH-treated neutrophils interacting with MRSA. Alexa Fluor 350 (blue) was used
to label bacterial cells. Actin-specific (red) staining was used to label cell bodies of neutrophils. 4=,6-Diamidino-2-phenylindole (blue) was used to label the nuclei.
Bar, 10
␮
m. (E) NO production in infected neutrophils was quantified using the Griess method. (F) MPO released by neutrophils was measured using the
EnzChek MPO activity assay. For panels A to C and E to F, the values presented are averages and standard deviations. All experiments were performed three times
with similar results obtained. Each point or bar represents the average of three measurements per condition. The results shown are representative of an individual
experiment. Significance (P⬍0.05) was calculated by an ANOVA. *, #, and & indicate results significantly lower than in untreated, Chlq, or 25
␮
M METH-
treated groups, respectively.
Methamphetamine Exacerbates MRSA Skin Infection
November/December 2015 Volume 6 Issue 6 e01622-15 ®mbio.asm.org 7

the bacteria for 2 h was analyzed. In macrophages treated with
METH (25 or 50
␮
M) or Chlq (25
␮
M), the phagosomal pH was
significantly elevated (pH 5.6, 6.4, and 6.8, respectively) compared
with the pH in untreated control or CytD-treated (25
␮
M) mac-
rophages (pH 4.8 and 4.4; P⬍0.001) (Fig. 9F).
METH-treated MRSA-infected mice displayed decrease an-
giogenesis. We investigated the effect of METH on angiogenesis
in wounded skin infected with MRSA by measuring the expression
of CD34 (Fig. 10). Tissue sections from untreated or METH-
treated uninfected murine wounds displayed dense vasculariza-
tion, whereas wounds of MRSA-infected mice showed localized
formation of blood vessels in dermal tissue. Moreover, METH-
treated MRSA-infected wounds demonstrated minimal vessel for-
mation in the epidermis and dermis. Therefore, the combination
of METH and MRSA infection visually reduces vascularization in
murine cutaneous wounds, and this qualitative observation is as-
sociated with decreased healing.
METH alters cytokine expression. Twenty-four hours after
infection, wound tissue of mice infected with MRSA contained
significantly higher quantities of gamma interferon (IFN-
␥
),
interleukin-1
␤
(IL-1
␤
), IL-12, monocyte chemoattractant pro-
tein 1 (MCP-1), and tumor necrosis factor alpha (TNF-
␣
) than
those under all other conditions (Table 1). Transforming growth
factor
␤
(TGF-
␤
) was significantly elevated in MRSA-infected
mice (MRSA and METH-MRSA) groups. There was a significant
increase in IL-6 and IL-10 production in METH-treated animals
(METH and METH-MRSA) compared to untreated conditions
(uninfected or MRSA groups). There were significant decreases in
vascular endothelial growth factor (VEGF) and TNF-
␣
for the
MRSA and METH-MRSA groups, respectively, compared to all
other conditions. IL-6 and TGF-
␤
levels were significantly re-
duced in untreated animals relative to the other groups. There
were no differences in IL-4 production between any of the exper-
imental groups.
At day 7 after infection, wound tissue of METH-treated mice
infected with MRSA contained significantly higher quantities of
IFN-
␥
, TNF-
␣
, IL-4, and TGF-
␤
than tissues of uninfected or
MRSA-treated mice (Table 2). There was a significant increase in
IL-12 and IL-10 production in MRSA-infected animals compared
to all the other treatment groups. Significant increases in IL-6 and
IL-1
␤
and decreases in VEGF were measured under all the other
conditions, compared to results in uninfected mice. MCP-1 and
VEGF levels were significantly reduced in MRSA-treated animals
relative to the other groups.
DISCUSSION
We used a murine model to investigate the effects of METH on
MRSA superficial skin infection. Our findings showed that
METH-treated mice displayed reduced wound healing in the
presence or absence of MRSA infection compared with untreated,
FIG 8 METH reduces human neutrophil phagocytosis, respiratory burst, and killing of S. aureus. (A) Phagocytosis (percentage) of FITC-labeled S. aureus
clinical strains (n⫽6) by human neutrophils was determined using FACS analysis after a 60-min incubation with METH. (B) Killing of S. aureus by neutrophils
was determined from CFU counts. For panels A and B, dashed lines and error bars represent the averages and standard deviation of six measurements (each
symbol represents a single strain). Significance (P⬍0.05) was calculated by an ANOVA and adjusted by use of the Bonferroni correction. For panel A, *, #, and
& indicate a lower phagocytosis percentage than in untreated, 25
␮
M Chlq, or 25
␮
M METH groups, respectively. For panel B,
␾
,
␹
, @, and $ indicate a higher
survival percentage than in untreated, 25
␮
M Chlq, 25
␮
M METH, and 50
␮
M METH groups, respectively. (C) Oxidative burst was quantified for 60 min based
on luminol chemiluminescence after untreated or Chlq-, CytD-, or METH-treated neutrophils were coincubated with MRSA strain 6498. Untreated and
uninfected neutrophils were also used as controls. Symbols and error bars denote means and standard deviations. Significance (P⬍0.05) was calculated by an
ANOVA and adjusted by use of the Bonferroni correction at each time point. This experiment was performed twice and similar results were obtained. The results
shown are representative of an individual experiment.
Mihu et al.
8®mbio.asm.org November/December 2015 Volume 6 Issue 6 e01622-15

uninfected mice, with the longest delay in wound closure occur-
ring in the METH-MRSA group. In this regard, our results from
the measurement of collagen deposition in wound tissue revealed
increased collagen degradation in the METH-MRSA treatment
group compared with the controls. Collagen plays a crucial role in
the proliferative phase of the wound healing process, along with
angiogenesis, granulation tissue formation, and epithelialization
(26). The amount of collagen present in the wound during the
healing process reflects a balance between new collagen produc-
tion by fibroblasts and degradation by collagenases and other fac-
tors. In the early phases of healing of simple, uninfected wounds,
synthesis exceeds degradation, so collagen levels in the wound rise;
later, production and degradation become equal (26). This bal-
ance was disrupted by METH and MRSA. The expression levels of
genes regulating collagen type I and III production were found to
be significantly increased in the METH-MRSA group as well;
however, the accelerated degradation process led to reduced col-
lagen content in the wound and thus impaired healing. Notably,
another contributing factor to the delayed wound closure was the
decreased angiogenesis under the influence of METH-MRSA, as
qualitatively demonstrated by the reduced expression of CD34, a
marker for blood vessel formation. For instance, histological anal-
yses of tissues from METH-MRSA mice showed high infiltration
of inflammatory cells confined to the dermal area of the cutaneous
tissue, but the cells were largely unable to reach the epidermal
layer, where most of the bacteria were located. This observation
FIG 9 METH alters macrophage effector functions. (A) Histological analysis of untreated, MRSA, METH, and METH-MRSA wounded BALB/c mice at day 7.
The brown staining indicates macrophage infiltration. Representative Iba1-immunostained sections of the skin lesions are shown. Bars, 25
␮
m. (B to F) Primary
macrophages were isolated from BALB/c mice and treated with PBS (untreated), Chlq (25
␮
M), CytD (25
␮
M), or METH (25 or 50
␮
M) and coincubated with
MRSA (C to F). (B) Uninfected macrophage migration (as a percentage) after exposure to the chemoattractant fMLP. Significance (P⬍0.05) was calculated by
an ANOVA. *, #, and & indicate a significantly lower percentage of macrophages than in untreated or Chlq- or 25
␮
M METH-treated groups, respectively. (C)
Phagocytosis (percentage of cells) was analyzed by FACS analysis after a 30-min incubation. (D) CFU determinations were performed after the 30-min
incubation. (E) NO production was quantified using the Griess method. Significance (P⬍0.05) was calculated by an ANOVA. Asterisks indicate results
significantly lower than in untreated groups. (F) The pH levels in phagosomes of primary macrophages that contained MRSA labeled with pH-sensitive and
pH-insensitive probes were measured using a spectrofluorimeter. For panels B to F, the values presented are averages and standard deviations. For panels C, D,
and F, Significance (P⬍0.05) was calculated by an ANOVA.
␾
,
␹
,
␲
, and
␭
indicate a significant increase compared to results in untreated or Chlq-, CytD-, or
25
␮
M METH-treated groups, respectively. All the experiments were performed twice with similar results obtained. Each bar represents the average of three
measurements per condition. The results shown are representative of an individual experiment.
Methamphetamine Exacerbates MRSA Skin Infection
November/December 2015 Volume 6 Issue 6 e01622-15 ®mbio.asm.org 9

may explain the 2-log-higher bacterial counts in the skin samples
of the METH-treated group relative to the untreated mice. Like-
wise, in vitro and in vivo studies demonstrated that METH impairs
phagocytic cell migration and antimicrobial activity as well as re-
duces phagocytes in the bloodstream. The prolonged presence of
an elevated number of macrophages and neutrophils in the skin
tissue may be associated with increased free radical accumulation
(27), which can inhibit wound healing.
We investigated whether the difference in collagen deposition
was due to METH-induced bacterial or host breakdown of colla-
gen. We originally thought that S. aureus staphopain collageno-
lytic activity might be implicated in cutaneous tissue destruction
in the setting of METH exposure (28). However, in vitro experi-
ments determined that these bacterial cysteine proteases did not
contribute to collagen breakdown. We cannot completely rule out
the possibility that other virulence factors produced by S. aureus
may be detrimental for the healing process. More research is
needed to better identify specific MRSA virulence factors involved
in dysregulating wound healing in the setting of drug abuse.
However, we successfully identified a host factor involved in
collagen degradation after METH administration and MRSA in-
fection. METH increased the expression of MMP-2, a metallopro-
tease that can interfere with wound healing and enhance suscep-
tibility to bacterial infections (29, 30). In HIV-associated
dementia, the HIV-1-encoded protein Tat and METH increase
MMP-2 levels in supernatants of human neuron/astrocyte cul-
tures (31). Previous studies have shown that MMP-2 plays crucial
roles in METH-induced behavioral sensitization and reward by
regulating METH-induced dopamine release and uptake via do-
pamine transporters in the brain (32). Dopamine has been re-
TABLE 1 Cytokine levels in wounds of BALB/c mice 24 h after wounding
Treatment
Cytokine level
a
(pg/ml) (avg ⫾SD)
IFN-
␥
IL-1
␤
IL-4 IL-6 IL-10 IL-12 MCP-1 TGF-
␤
TNF-
␣
VEGF
Untreated 1,951.74
⫾16.62
791.80
⫾11.57
81.25
⫾10.74
452.93
⫾8.91
398.26
⫾6.61
367.91
⫾6.45
374.08
⫾11.13
1,996.55
⫾5.02
3,852.01
⫾15.12
4,623.52
⫾15.09
MRSA 2,339.10
⫾10.65
␾
859.25
⫾6.47
␾
91.52⫾5.32 614.37
⫾4.63
␾
321.83
⫾7.36*
414.5
3⫾5.37
␾
421.54
⫾4.02
␾
2,459.32
⫾4.46
␾
4,157.19
⫾8.12
␾
2,071.90
⫾23.61*
METH 1,398.46
⫾5.68
*#
645.32&#
x00B1;6.04
*#
75.26⫾5.97 897.25
⫾7.98
␾␹
497.21
⫾6.54
␾␹
362.83
⫾8.81
#
200.87
⫾9.09*
#
2,085.31
⫾16.25
#
2,736.14
⫾11.95
*#
3,027.11
⫾6.72
*
␹
METH-MRSA 1,765.44
⫾4.21
*#
␲
701.33
⫾9.26
*#
␲
79.69
⫾5.97
811.85
⫾8.23
␾␹
&
505.21
⫾9.22
␾␹
388.24
⫾8.65
#
398.08
⫾7.12
#
␲
2,369.84
⫾6.35
␾
1,977.25
⫾12.81
␹
#&
2,545.15
⫾7.01
*
␹␲
a
␾
,
␹
, and
␲
indicate a significantly higher cytokine level (P⬍0.05) than in the untreated, MRSA, or METH group, respectively. *, #, and & indicate a significantly lower cytokine
level (P⬍0.05) than in the untreated, MRSA, or METH group, respectively.
TABLE 2 Cytokine levels in wounds of BALB/c mice 7 days after wounding
Treatment
Cytokine level
a
(pg/ml) (avg ⫾SD)
IFN-
␥
IL-1
␤
IL-4 IL-6 IL-10 IL-12 MCP-1 TGF-
␤
TNF-
␣
VEGF
Untreated 2,321.58
⫾15.84
2,598.36
⫾10.89
251.26
⫾10.28
2,245.35
⫾8.65
474.36
⫾13.47
936.32
⫾6.47
4,932.21
⫾10.58
10,365.9
⫾5.47
1,901.26
⫾14.74
1,5369.7
⫾16.56
MRSA 4,531.08
⫾9.45
␾
3,426.65
⫾7.21
␾
384.08
⫾5.05
5,057.15
⫾9.14
␾
4,197.85
⫾7.05
␾
1,682.09
⫾12.75
␾
1,173.07
⫾21.65*
16,658.95
⫾16.71
␾
1,383.62
⫾7.81*
6,312.28
⫾12.43*
METH 5,987.21
⫾6.01
␾␹
3,501.98
⫾5.97
␾
1,223.36
⫾11.71
␾
8,569.61
⫾12.58
␾␹
625.36
⫾6.99
␾
#
1,256.12
⫾8.58
␾
#
4,864.36
⫾9.71
␹
16,723.1
⫾13.89
␾
3,821.36
⫾11.32
␾␹
1,2154.7
⫾10.06
*
␹
METH-MRSA 15,806.1
⫾4.66
␾␹␲
3,568.59
⫾8.17
␾
1,462.59
⫾6.18
␾␹␲
9,841.99
⫾15.04
␾␹␲
592.4
⫾9.54
␾
#
1,145.13
⫾9.16
␾
#
5,215.42
⫾19.14
␹
32,454.7
⫾22.27
␾␹␲
5,121.88
⫾5.81
␾␹␲
1,0172.2
⫾9.28
*
␹
&
a
␾
,
␹
, and
␲
indicate a significantly higher cytokine level (P⬍0.05) than in the untreated, MRSA, or METH group, respectively. *, #, and & indicate a significantly lower cytokine
level (P⬍0.05) than in the untreated, MRSA, or METH group, respectively.
FIG 10 METH-treated MRSA-infected mice displayed decreased angiogenesis in wounded skin. Tissue sections are shown for untreated, MRSA, METH, or
METH-MRSA wounded BALB/c mice at day 7. Brown staining indicates vascularization. Representative CD34-immunostained sections of the skin lesions are
shown. Black arrows denote dense vascularization. Bars, 25
␮
m.
Mihu et al.
10 ®mbio.asm.org November/December 2015 Volume 6 Issue 6 e01622-15

ported to decrease mitosis of keratinocytes (33), the most abun-
dant cells in skin tissue, whereas D2-like receptors (D2, D3, and
D4) are involved in epidermal barrier homeostasis (34). For in-
stance, the D2 receptor is present in the basal epidermis, and the
D4 receptor is in the uppermost layer of the epidermis (34). These
receptors have been implicated in the pathogenesis of skin inflam-
matory diseases (35, 36) and regulation of wound healing (37, 38).
Although further studies are warranted, METH may induce do-
pamine production and D2-like receptor expression by keratino-
cytes, which could substantially reduce the wound healing rate.
Additionally, METH promoted the collagenolytic activity of
MMP-2 in a concentration-dependent manner, providing an-
other possible explanation for the negative impact of this drug in
wound healing. The late (day 7 postinjury) augmented produc-
tion of inflammatory cytokines like TNF-
␣
, IFN-
␥
, IL-6, and
TGF-
␤
observed in METH-treated groups promotes the activa-
tion of MMP-2 via NF-
␬
B signaling in dermal fibroblasts embed-
ded in collagen type I (39). Our study is important because self-
management of injection-related wounds is common among
injection drug users (IDUs) (11). Specifically, IDUs are more
likely to engage in potentially harmful self-management behav-
iors, increasing their susceptibility to MRSA infections due to a
combination of aggressive management of their wounds and an
underlying slow healing rate. In a previous study, IDUs who had
ever injected amphetamines were more likely to engage in poten-
tially harmful self-management behaviors (11). Furthermore, the
inability of phagocytic cells to be recruited and/or proactively fight
invading microbes at the site of infection could exacerbate disease
in IDUs.
Another important finding of this study is the increase in in-
flammation and cell necrosis in the wounds of METH-treated and
infected mice compared with control groups. For instance, CFU
analysis showed an increased bacterial burden in the wound tis-
sues in the METH-MRSA group. Chronic wounds on METH us-
ers are an ideal environment for S. aureus biofilm formation. The
necrotic tissue and debris allow bacterial attachment, and wounds
are susceptible to infection due to an impaired host immune re-
sponse. Evidence suggests that biofilms play a significant role in
the inability of chronic wounds to heal. Biofilms are present in
only 6% of acute wounds but over 90% of chronic wounds (40).
We found that METH enhanced biofilm formation by multiple
clinical S. aureus strains. The presence and persistence of biofilms
on chronic skin wounds can affect cellular function (leukocytes,
keratinocytes, endothelial cells, and fibroblasts), the inflamma-
tory cellular response, the cutaneous innate immune response,
and the repair phase of wound healing (angiogenesis and fibro-
genesis). For example, keratinocytes produce more MMPs in
chronic biofilm-challenged wounds (41). These infections de-
velop gradually and may be slow to produce explicit symptoms. In
addition, the vast majority of bacteria reside in the eschar above
the wound bed (42). Once established, however, biofilm infec-
tions often persist. Understanding the impact of METH on S.
aureus pathogenesis in IDUs warrants the pursuit of further stud-
ies addressing the influence of the drug on the multitude of viru-
lence factors expressed by this microbe during infection, includ-
ing its toxins.
Another possible explanation for S. aureus persistence in
wounded tissue of drug users is that METH alters the effector
functions of neutrophils and macrophages. The initial and most
obvious function of inflammatory cells at the site of injury is to
provide specific and nonspecific defenses against pathogens. The
first cells that infiltrate a wound are neutrophils, which remove
foreign particles and bacteria. In the skin, neutrophils appear in
the wound bed within minutes after injury. We demonstrated that
METH inhibits neutrophil chemotaxis and the production of NO
and MPO in a concentration-dependent fashion, thus reducing
the antimicrobial armamentarium of activated murine neutro-
phils. Notably, METH compromised S. aureus phagocytosis, the
respiratory burst, and killing by human neutrophils, suggesting
that the detrimental effects of the drug observed in murine-
derived cells can be similarly achieved in human cells, which val-
idates the mouse as a good model for these types of studies.
METH-mediated phagocytosis dysfunction by neutrophils may
be associated with reduced expression of GTPase-RhoA, a key
regulator of the actin polymerization signaling cascade, given that
CytD-treated cells displayed a similar deficiency in engulfing the
microbe. Also, impaired phagocytosis may be associated with im-
mobilization of the complement receptor 3 (CR-3) on the surface
of neutrophils and deregulation of cytokines (21). Neutrophils are
nevertheless a major source of several inflammatory cytokines,
such as TNF-
␣
and IL-1
␤
, which stimulate attracted monocytes to
differentiate into M1 (classical activation) macrophages (43, 44).
We observed that METH reduces the early (24 h postinjury) pro-
duction of these two cytokines in METH-treated groups. More-
over, elevated levels of IL-10 in homogenates of METH-treated
groups during the early inflammatory response suggest an impor-
tant role of these factors in reducing the recruitment of phagocytic
cells to the site of injury and infection.
METH inhibited the migration of macrophages to the infec-
tion site as observed in vitro and in the histological analysis. Inter-
estingly, we found that primary macrophages exposed to METH
displayed increased MRSA phagocytosis; however, these macro-
phages showed impaired intracellular bacterial killing leading to
increased pathogen survival. As in our prior work, METH dis-
rupted macrophage effector functions (21), including alkalization
of the phagosomal pH and inhibition of NO and superoxide pro-
duction. One study using a mouse model of catheter-associated
biofilm infection showed that S. aureus biofilms caused changes in
macrophage function by suppressing microbicidal activity, alter-
ing gene expression toward M2 phenotype cells, decreasing migra-
tion, and increasing cell death (45). Those investigators showed
that macrophages were able to phagocytose planktonic cells but
not biofilm-associated cells, suggesting that the in vitro experi-
ments do not necessarily reflect in vivo situations. During the pro-
liferative phase, macrophages stimulate proliferation of connec-
tive, endothelial, and epithelial tissues directly and indirectly. In
particular, fibroblasts, keratinocytes, and endothelial cells are
stimulated by macrophages during this phase to induce and com-
plete extracellular matrix formation, reepithelialization, and neo-
vascularization. Once recruited to the wound, macrophages in-
duce angiogenesis. This is predominantly exerted by macrophages
releasing TNF-
␣
and VEGF. TNF-
␣
may in turn induce VEGF
expression by keratinocytes and fibroblasts (46). Depletion of
macrophages in the wound results in reduced vascularization (47)
and also leads to severe hemorrhage and fibrin and serum exu-
dates in macrophage-depleted granulation tissue (48). This has
been reported to be mainly caused by withdrawal of macrophage-
derived TGF-
␤
and VEGF (47, 48), supporting an important role
for macrophages in promoting angiogenesis via their secretory
products.
Methamphetamine Exacerbates MRSA Skin Infection
November/December 2015 Volume 6 Issue 6 e01622-15 ®mbio.asm.org 11

METH alters cytokine expression, as indicated by our mea-
surements of cytokine levels in the wounds. The late (day 7 postin-
jury) increased levels of inflammatory cytokines in the METH-
MRSA group correlated with our histological observations of
increased local inflammation in their wounds. Nevertheless, the
disabled immune machinery in the METH-treated mice failed to
contain the infection, and the initial inflammatory cascade led to
detrimental local outcomes with increased cell necrosis and im-
paired healing. METH has been linked to a shift in the immune
response from a dominant Th1 to a mixed Th1-Th2 phenotype
(49). In our infection model, we found high levels of endogenous
TNF-
␣
and IFN-
␥
in METH-treated mice. However, the in-
creased levels of the two Th1 cytokines were not sufficient to res-
cue mice from the lethal effects of MRSA. Thus, METH might play
a role in inducing a dominant nonprotective Th2 response to
MRSA.
In conclusion, this is the first study that has empirically ad-
dressed the effect of METH on immunity during infection by S.
aureus. These findings should raise awareness of the negative im-
pact of METH use on the overall community health status, as
alarming data in this regard have been published on the high in-
cidence of MRSA skin and soft tissue infections among METH
consumers (13). Although wound debridement remains a proven
cornerstone for wound management, perhaps in part because a
microbial biofilm is removed from the scab, it is plausible to think
that wounds in METH users become chronic and difficult to man-
age because of a combination of bacterial persistence, drug-related
immune deficiency, and the IDU behaviors, which include stereo-
typical formication and lack of hygiene during injections, making
these individuals highly susceptible for S. aureus reinfection. A
possible limitation of the study is that the in vitro and in vivo
METH treatments were not equivalent, since in vitro the phago-
cytes were exposed for a few hours to METH, whereas in vivo they
were exposed for 21 days. However, METH doses used in both
animals and tissue cultures were similar or closely related to those
measured in people that chronically abuse the drug. Further re-
search is needed to understand the biological and management
complexity of infected wounds in METH users and to best focus
prevention efforts to reduce morbidity and improve wound care.
MATERIALS AND METHODS
S. aureus.A total of six S. aureus clinical isolates were used in this study.
The characteristics of each strain are described in Table 3. The MRSA 6498
isolate used in the majority of the experiments in this report is a USA300
strain collected from a patient’s wound and has been extensively utilized
in wound healing studies (49, 50). The strains were stored at ⫺80°C in
brain heart infusion (BHI) broth with 40% glycerol until use.Test organ-
isms were grown in tryptic soy broth (TSB) overnight at 37°C on a rotary
shaker set at 150 rpm. Growth was monitored by measuring the optical
density at 600 nm and use of a microtiter plate reader.
METH. Most high-dose METH abusers initially use small amounts of
the drug intermittently before progressively increasing the dose (51). To
simulate this pattern, we used increasing daily doses (2.5, 5, and 10 mg/
kg/day on weeks 1, 2, and 3, respectively) of METH that were intraperi-
toneally (i.p.) administered to female BALB/c mice (6 to 8 weeks old) over
21 days, as described previously (21). As controls, mice received equiva-
lent volumes of PBS.
Wound and infection model. At day 21, METH- and PBS-treated
BALB/c mice were anesthetized (100 mg/kg ketamine, 10 mg/kg xylazine),
their back hair was removed, and skin was disinfected with iodine. Five-
millimeter-diameter full-thickness excision wounds in the center of the
backs were achieved using surgical punches. A suspension containing 10
7
S. aureus strain 6498 in PBS was inoculated onto each wound. Uninfected
METH- or PBS-treated mice were used as additional controls. Photo-
graphs of the wounds were taken on days 3, 5, 7, 9, 11, 13, 15, and 17. The
wound dimensions were measured every other day by two different oper-
ators using dial calipers in a blinded fashion. Although mice experience
formication after METH administration, this behavior did not influence
the results obtained, given that each animal was isolated in their own cage,
eliminating the possibility of mice scratching other mice; in addition, the
location of the wound made it difficult for the animals to scratch their own
wounds. For histology, CFU determinations, and gene expression analy-
sis, animals were euthanized at day 7 and wound tissues were excised.
Animals were otherwise euthanized at day 21, by which time all wounds
had healed.
Ethics statement. Animal experiments were performed according to
the guide published by the Institute of Laboratory Animal Resources of
the National Research Council. Animal care for this study was approved
by the Animal Welfare and Research Ethics Committee at the Albert Ein-
stein College of Medicine (protocol number 20110402).
Histology. Skin tissues were excised and fixed in 4% paraformalde-
hyde (PFA) for 24 h. Tissues were processed and embedded in paraffin,
and 4-
␮
m vertical sections were fixed to glass slides. Tissue sections were
stained with hematoxylin and eosin (H&E), Gram stain, and Gomori’s
trichrome, and for CD34 or Iba-1, to examine tissue morphology, bacte-
ria, collagen deposition, vascularization, and macrophage infiltration, re-
spectively. Slides were visualized using an Axiovert 40CFL inverted mi-
croscope (Carl Zeiss), and images were captured with an AxioCam MrC
digital camera using the Zen 2011 digital imaging software.
Collagen deposition determinations. Collagen deposition was mea-
sured based on intensity, using ImageJ software (National Institutes of
Health, USA) (52). The software possesses threshold filters to discrimi-
nate between the stain colors (white, red, and blue), providing an accurate
measurement of each color’s intensity. Ten 40⫻fields were evaluated per
section.
CFU determinations. Excised wound tissues were homogenized in
PBS and plated on tryptic soy agar (TSA) for CFU determinations. The
results were normalized by tissue weights.
Real-time (RT)-PCR. For RT-PCR analysis of host gene expression,
we analyzed col1,col3, and mmp2 expression. col1 encodes collagen type I,
and col3 encodes collagen type III, whereas mmp2 encodes matrix
metalloproteinase-2. At day 7, excised tissues were homogenized, cells
were collected and washed, and then RNA was isolated using an RNeasy
kit. col1,col3, and mmp2 expression levels were analyzed by RT-PCR as
described elsewhere (52).
In S. aureus,ica encodes the intercellular adhesion locus and is re-
quired for S. aureus biofilm formation. Twenty-four hours after incuba-
tion with METH, bacterial RNA was isolated and analyzed by RT-PCR as
described previously (53).
TABLE 3 Characteristics of S. aureus clinical strains tested in this
study
a
Strain Source mecA SCC ACME MLST Toxin genes
038 Blood ⫹IV ⫺CC 8 seb,sek,sep,seq,ser,seu
067 Wound ⫺MSSA ⫺CC 8 sea,seb,sek,sep,seq,ser,seu
085
b
Wound ⫹IV ⫹CC 8 pvl,sek,sep,ser
112 Wound ⫹IV ⫹CC 8 sek,ser,seu
132 Wound ⫹IV ⫺ND
c
seb,sek,ser
6498
b
Wound ⫹IV ⫹CC 8 pvl,sek,sep,ser
a
Toxin profiles were determined by PCR amplification with primer sets for 19 different
toxins. Abbreviations (corresponding gene): staphylococcal enterotoxin a (sea),B(seb),
K(sek),P(sep),Q(seq),R(ser),U(seu), and Panton-Valentine leukocidin (pvl).
ACME, arginine catabolic mobile element; MLST, multilocus sequence type; CC, clonal
complex; ND, not determined.
b
USA300 strain.
Mihu et al.
12 ®mbio.asm.org November/December 2015 Volume 6 Issue 6 e01622-15

For an internal mRNA control, we used primers specific for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Phagocytic cell counts in blood. Twenty-one days after METH ad-
ministration, phagocytic cell counts were performed by differential leu-
kocyte count for samples from all experimental animals by using a Hema
3 Stat Pack kit and light microscopy.
Rationale for METH doses used in in vitro studies. Controlled stud-
ies indicated that a single 260-mg dose peaks at a level of 7.5
␮
M (54).
Thus, a single dose of 260 mg/g would be expected to produce 7.5 to 28.8
␮
M blood METH levels. IDUs tend to self-administer METH in binges,
and as the drug exhibits a half-life of 11.4 to 12 h (55, 56), this can lead to
higher drug levels. Published studies modeling binge patterns of use in
individuals have shown that the fourth administration of 260 mg during a
single day produced blood levels of 17
␮
M and could reach 20
␮
Monthe
second day of such a binge (54). Thus, binge doses of 260 to 1,000 mg
produce 17 to 80
␮
M blood METH levels and levels in the micromolar
range of hundreds in organs, including the brain and the spleen (57).
Therefore, we selected ~25 to 50
␮
M METH to perform our in vitro
experiments.
Bacterial viability assay. To determine the impact of the METH on S.
aureus growth, 1 ml of TS broth (TSB) was inoculated with a 24-h colony
of the bacterium grown on TSA. One hundred microliters of S. aureus
broth suspension was inoculated in each well of a 200-well plate, contain-
ing 100
␮
l of TSB with either 25 or 50
␮
M METH. The inoculated plate
was then incubated for 24 h at 37°C. Controls included wells containing
bacterial suspension alone (untreated). Growth was assessed every 15 min
using a microplate reader set at an optical density (OD) of 600 nm.
Collagenolytic activity assay. Collagenolytic activity was measured
using murine fluorescein isothiocyanate (FITC)-conjugated type I colla-
gen (28). FITC-conjugated type I collagen was incubated with a 10 nM
concentration of either staphopain A or MMP-2 for3hat37°C. Then,
100
␮
l of the solution was mixed with 100
␮
l ethanol (70% ethanol–
0.17 M Tris-HCl buffer, 0.67 M NaCl) and centrifuged for 10 min at 4°C.
To assess collagen degradation, 70
␮
l of the supernatant was withdrawn,
and fluorescence was measured with a multilabel plate reader with emis-
sion at 520 nm and excitation at 495 nm.
Western blot analysis. To further characterize the role of METH in
wound healing, we assessed MMP-2 expression in the wounds of BALB/c
mice via Western blot analysis, as described previously (52).
Biofilm formation. For each strain, 100
␮
l of a suspension with bac-
terial cells in RPMI 1640 medium supplemented with 1% Casamino Acids
was added into individual wells of polystyrene 96-well plates, and the
plates were incubated at 37°C without shaking as previously described
(58). The biofilms grown with PBS or METH (25 and 50
␮
M) were al-
lowed to form for 24 h.
Quantitation of biofilms. Measurement of biofilm formation and vi-
ability was done by CFU determinations and assessment of the metabolic
activity of the attached cells in an XTT reduction assay, as previously
described (59).
Biofilm architecture visualization. The structural integrity of bio-
films formed in the absence or presence of METH was examined by using
the LIVE/DEAD biofilm viability kit and confocal microscopy, as previ-
ously described (60).
Isolation of peripheral blood neutrophils from mice and humans.
Retroorbital puncture was used to obtain blood from BALB/c mice,
whereas whole human blood was purchased from the Interstate Blood
Bank, Inc. (Memphis, TN). Erythrocytes were removed by hypotonic ly-
sis, and neutrophils were separated from the remaining cells by centrifu-
gation over discontinuous Percoll gradients. Neutrophils were ⬎95% vi-
able, as determined by Wright-Giemsa staining. Recovered neutrophils
(~98% as determined by FACS, using Ly-6G as a marker) were briefly
maintained (⬍30 min; 37°C, 10% CO
2
) in RPMI 1640 supplemented with
10 mM HEPES (pH 7.4) and 10% fetal calf serum (FCS) prior to use.
Controls. Chloroquine is a weak base and a well-known inhibitor of
endosomal acidification (61), whereas CytD is a potent inhibitor of actin
polymerization. Both reagents were used as controls in the in vitro studies
of S. aureus-phagocyte interactions.
Murine neutrophil killing assay. For killing assays (62), 3 ⫻10
5
neu-
trophils in 200
␮
l of RPMI 1640 with 10% FCS were incubated with
METH (25 or 50
␮
M), Chlq (25
␮
M), CytD (25
␮
M), or PBS in micro-
centrifuge tubes with 8 rpm rotation for2hat37°C, 5% CO
2
.S. aureus
6498 cultures were washed twice in PBS, diluted to a concentration of 4.5
⫻10
6
CFU in 100
␮
l RPMI 1640 plus 10% FCS (feeding medium), and
mixed with washed neutrophils in the same medium (ratio, 15 bacteria:1
neutrophil), centrifuged at 1,200 rpm for 5 min, and then incubated at
37°C in a 5% CO
2
incubator. Gentamicin (final concentration of
400
␮
g/ml for S. aureus) was added after 10 min to kill extracellular bac-
teria. At specified time points (20 to 80 min), the contents of sample wells
were withdrawn, centrifuged to pellet the neutrophils, and washed to
remove the antibiotic medium. Neutrophils were then lysed in 0.02%
Triton X-100, and CFU were calculated after plating on TSA.
Macrophages. Primary macrophages were isolated from untreated
BALB/c mice by lavage of the abdominal cavity with Hanks’ balanced salt
solution with 1 mM EGTA (21).
Phagocytosis. For neutrophils, phagocytosis was determined by using
confocal microscopy. Primary murine neutrophils were incubated with
METH (25 or 50
␮
M), Chlq (25
␮
M), CytD (25
␮
M), or PBS for 90 min
at 37°C and 5% CO
2
, prior to incubation for phagocytosis in the presence
of Alexa Fluor 350 (Alexa)-labeled S. aureus 6498 cells for 30 min. Genta-
micin (400
␮
g/ml) was added to kill extracellular bacteria. Similarly, ex-
tracellular bacteria were quenched with trypan blue to prevent interfer-
ence with the assay. Microscopic examinations of neutrophils were
performed with a Leica TCS SP5 confocal laser scanning microscope.
Z-stack images and measurements were corrected by utilizing Bio-Rad
LaserSharp 2000 software in deconvolution mode. The fluorescent inten-
sity of bacteria inside neutrophils was analyzed using Adobe Photoshop
software (63).
For macrophages, phagocytosis was determined by using FACS anal-
ysis. Primary macrophages were incubated with METH (25 or 50
␮
M),
Chlq (25
␮
M), CytD (25
␮
M),orPBSfor2hat37°C and 5% CO
2
, prior
to incubation for phagocytosis in the presence of Alexa-labeled S. aureus
6498 for 30 min. Gentamicin (400
␮
g/ml) was added to kill extracellular
bacteria. Similarly, extracellular bacteria were quenched with trypan blue
to prevent interference with the assay. Samples were processed (10,000
events per condition) on an LSRII flow cytometer (BD) and were analyzed
using FlowJo software.
Macrophage killing assay. Primary macrophages were first allowed to
phagocytize S. aureus 6498 cells for 30 min. Each well containing interact-
ing cells was gently washed with feeding medium and incubated with
feeding medium supplemented with gentamicin (400
␮
g/ml) and either
PBS, METH (25 or 50
␮
M), Chlq (25
␮
M), or CytD (25
␮
M) for 30 to
120 min. Quantification of viable S. aureus cells was determined by mea-
suring CFU after lysis of macrophages (64).
Phagosomal pH. The pH of S. aureus 6498-containing phagosomes in
primary macrophages was determined as described elsewhere (21).
Nitric oxide and myeloperoxidase production. NO production was
quantified using a Griess method kit (Promega). Similarly, MPO pro-
duced by N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated
neutrophils in culture after coincubation with S. aureus was measured
using the myeloperoxidase assay kit.
Chemotaxis. Chemotaxis was measured using a transwell chamber
with 6.5-mm-diameter polycarbonate filters (3-
␮
m pore size). Immedi-
ately after isolation, cells were incubated in the absence or presence of
METH (25 or 50
␮
M) for 2 h. Chlq (25
␮
M) and CytD (25
␮
M) were used
as positive controls. Cells were transferred to RPMI 1640 with heat-
inactivated FCS, cultivated on filters, and allowed to migrate toward the
chemoattractant fMLP or cultured in medium alone at 37°C, 5% CO
2
.
After various time points (20, 40, and 60 min), the filters were removed,
and the cells that migrated through the membrane were fixed, stained, and
Methamphetamine Exacerbates MRSA Skin Infection
November/December 2015 Volume 6 Issue 6 e01622-15 ®mbio.asm.org 13

counted by light microscopy (400⫻) as described elsewhere (65). Ten
nonoverlapping and randomized fields were counted by one scorer.
Human neutrophil phagocytic and killing assays. Phagocytosis and
killing assays were performed as previously described (66). For the phago-
cytosis assay, FACS analysis was employed. Human neutrophils (10
6
cells)
were incubated on six-well plates with feeding medium supplemented
with METH (25 or 12.5
␮
M)orPBSfor2hat37°C and 5% CO
2
. FITC-
labeled S. aureus cells were incubated with 25% human serum for 30 min
to allow complement proteins to opsonize S. aureus cells.Bacterial cells
were washed and then 10
7
were added to the 10
6
neutrophils for 60 min.
Similarly, extracellular bacteria were quenched with trypan blue to pre-
vent interference with the assay. Samples were processed on an LSRII flow
cytometer and were analyzed using FlowJo software.
Since METH reduces S. aureus phagocytosis by neutrophils, for the
killing assays leukocytes were first allowed to phagocytize S. aureus cells
for 0.5 h to determine initial uptake. Each well containing interacting cells
was gently washed with feeding medium and incubated with feeding me-
dium supplemented with gentamicin (400
␮
g/ml, to kill extracellular bac-
teria) and either PBS or METH (25 or 50
␮
M) for 4 h. Viable bacteria were
released from neutrophils following 0.5 or4hofhost-cell interaction by
forcibly subjecting cultures to 27-gauge needle passage 5 to 7 times for
efficient lysis. Four microtiter wells per condition were used to ascertain
CFU. For each well, serial dilutions were plated in triplicates onto TSA
plates, which were incubated at 37°C for 24 h prior to CFU tallying.
Luminol chemiluminescence assay. ROS signals were made chemilu-
minescent (CL) by using luminol (1 mM). CL was monitored for 30 min
with an automatic luminescence analyzer (SpectraMax L; Molecular De-
vices) at 37°C.
Cytokine determinations. Cutaneous tissues from mice were excised
at 24 h and 7 days after wounding and homogenized in PBS with protease
inhibitors. Cell debris was removed from homogenates by centrifugation
at 6,000 ⫻gfor 10 min. Samples were stored at ⫺80°C until tested.
Supernatants were tested for IFN-
␥
, IL-1
␤
, IL-4, IL-6, IL-10, IL-12p70,
MCP-1, TGF-
␤
, TNF-
␣
, and VEGF in an enzyme-linked immunosorbent
assay (OptEIA kits; Becton, Dickinson Biosciences).
Statistical analysis. All data were analyzed using Prism (GraphPad, La
Jolla, CA). Analyses of CFU (Fig. 2B) and phagocytic cell count (Fig. 4B)
data were performed using Student’s ttest. Analysis of all the other data in
the study was performed using analysis of variance (ANOVA) and ad-
justed by use of the Bonferroni correction. Pvalues of ⬍0.05 were con-
sidered significant.
ACKNOWLEDGMENTS
E.A.E. is supported by the National Institutes of Health (NIH MH096625)
and PHRI internal awards. A.J.G. is supported by Fundação de Apoio a
Pesquisa do Estado do Rio de Janeiro and Conselho Nacional de Pesquisa
e Desenvolvimento (CNPq). B.C.F. is supported by NIH
2R01AI059681-06 and R21AI087564-01. J.D.N. is supported in part by an
Irma T. Hirschl/Monique Weill-Caulier Trust Research award. L.R.M.
was supported by NIH NIAID award 5K22A1087817-02 and NYIT COM
start-up funds.
M.R.M. and J.R.-S. performed CFU, cytokine, and histological analy-
ses. B.P.S. performed the phagocytic cell counts and MMP-2-related ex-
periments. A.K.V. and B.C.F. performed the in vivo wound healing exper-
iments. L.N.N. performed the collagen gene expression analysis. A.J.G.
and J.D.N. performed chemotaxis, phagocytosis, nitric oxide production,
and killing assays. E.A.E. performed the confocal microscopy analysis.
J.D.N. assisted in data analysis and editing of the manuscript. H.H.L.
performed the experiments involving human neutrophils. L.R.M. di-
rected the overall design of the experiments, analysis of the data, and
writing of the manuscript.
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