Accepted Manuscript

N-acetylcysteine inhibits growth, adhesion and biofilm formation of Gram-positive skin

pathogens

Daria Eroshenko, Tatyana Polyudova, Vladimir Korobov

PII: S0882-4010(17)30050-5
DOI: 10.1016/j.micpath.2017.02.030
Reference: YMPAT 2139

To appear in: Microbial Pathogenesis

Received Date: 20 January 2017

Revised Date: 17 February 2017
Accepted Date: 20 February 2017

Please cite this article as: Eroshenko D, Polyudova T, Korobov V, N-acetylcysteine inhibits growth,
adhesion and biofilm formation of Gram-positive skin pathogens, Microbial Pathogenesis (2017), doi:
10.1016/j.micpath.2017.02.030.

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 ACCEPTED MANUSCRIPT
N-acetylcysteine inhibits growth, adhesion and biofilm formation of Gram-

positive skin pathogens

Daria Eroshenko1* , Tatyana Polyudova1 , Vladimir Korobov 1, 2, 3

1
Institute of Ecology and Genetics of Microorganisms, Ural Branch of Russian

Academy of Sciences, Goleva, 13, Perm, Russia, 614000,

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2
Perm State National Research University, Bukireva Str., 15, Perm, Russia, 614990

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3
Perm National Research Polytechnic University, Prosp. Komsomolsky, 29, Perm,

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Russia, 614990,

[email protected], [email protected], [email protected]

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* corresponding author, [email protected] +7 (961) 7599718
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Abstract
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N-Acetylcysteine (NAC) is a non-antibiotic drug with antimicrobial properties against

biofilm phenotypes of Gram-positive and Gram-negative bacteria. Our aim was to
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assess the effects of NAC on the growth of Gram-positive human skin and mucous
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membrane pathogens in the planktonic and biofilm phases. The minimum inhibitory
concentrations (MICs) of NAC against Enterococcus faecalis, Corynebacterium
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ammoniagenes, Mycobacterium smegmatis, Propionibacterium acnes, Staphylococcus

aureus, S. epidermidis, Streptococcus pyogenes, and 14 clinical strains of coagulase-
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negative staphylococci (CNS) ranged from 0.098 to 25 mg/ml. We found that at sub-
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MICs of NAC the adherence of E. faecalis, S. epidermidis, and nine CNS strains
significantly reduced. However, biofilm formation of E. faecalis, S.aureus and two
CNS strains increased at sub-MICs of NAC. Furthermore, a dose-related decrease in
biofilm formation of C. ammoniagenes, M. smegmatis, P. acnes, S. pyogenes, and S.
epidermidis was observed. The effect of NAC on planktonic growth and biofilm
formation of the M. smegmatis cell was also time-dependent. We have selected P.
acnes VKM Ac-1450 Rifr strain with total resistance to rifampicin and used this
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microorganism for multispecies P. acnes – S. epidermidis biofilm model. The biofilm
formation and growth of mixed culture of P. acnes and S. epidermidis was
significantly slowed at 12.5 mg/ml of NAC. NAC also has a higher disruptive effect on
both mature M. smegmatis and mixed P. acnes – S. epidermidis biofilm. Thus, NAC
appears to be a promising, non-antibiotic alternative to prevent biofilm-associated
infections.

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Keywords: adhesion, biofilm, skin, bacteria, N-acetylcysteine, rifampicin
Abbreviations:

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CNS – coagulase-negative staphylococci
FOV – field of view

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GSH – glutathione
LB – Luria-Bertani medium
NAC – N-acetylcysteine
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ROS – Reactive Oxygen Species
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1. Introduction
The human skin microbiome is dominated by members of abundant genera
include Propionibacterium, Corynebacterium, Staphylococcus, Micrococcus,
Streptococcus, and Brevibacterium [1]. Coagulase-negative staphylococci (CNS) are
major human skin commensals, and S. epidermidis is one of the most frequently
isolated species from human epithelia with a ubiquitous presence on the skin [2].

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Bacteria S. aureus also can be considered as part of normal human microbiota because
more than 30% of people are colonized by it without any associated disease [3]. In

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addition, Mycobacterium smegmatis can be found in normal human-genital secretions
and human skin [4].

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However, most of these skin bacteria can be a pathogenic and cause of infections
related to indwelling medical devices such as intravascular catheters, heart valves,

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implantable neurological stimulators, prosthetic joints, as well as most contact lenses
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[5, 6]. Medical devices related infections are usually difficult to be treated with
antibiotics due to the formation of biofilms, which comprise bacterial cells embedded
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within an extracellular polysaccharide matrix on the device surface [7]. The biofilm
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infections can be caused both single species and mixed-microbial communities, for
example, it has been reported that acne lesions contain P. acnes and S. epidermidis [8].
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Hence, we evaluated monospecies as well as multispecies biofilm in our experimental

studies.
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In recent years, with increased concerns over antibiotic resistance [9], there is
renewed interest in drugs belonging to different pharmacological classes for possible
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antimicrobial and, especially, anti-biofilm activity. Among these drugs, the potential of
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which has not yet been completely exhausted, N-acetylcysteine (NAC) holds a special
place. Initially, NAC was widely used as mucolytic agent in medical treatments of
chronic bronchitis and acetaminophen overdose [10]. Mucolytic activity of NAC due
to its ability to break down the disulfide bonds in the molecules of mucins is the basis
of its use to reduce the viscosity of secretions for subsequent microscopic detection of
acid-fast Mycobacterium [11]. Recently, new mechanisms of antioxidant, glutathione
replacement, and detoxifying action of NAC have been identified [12]. In addition,
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NAC has been actively studied by clinical microbiologists due to its high antibacterial
activity, including against M. tuberculosis [13], and its ability to enhance the effects of
conventional antibiotics [14, 15]. In addition to the role of NAC as a ROS scavenger,
NAC has also been shown to effectively inhibit the adhesion of bacteria to the
epithelial cells and polymeric materials and subsequent biofilm formation of variety of
medically important Gram-positive bacteria, including Enterococcus faecalis,

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S. epidermidis, S. aureus and Lactobacillus salivarius [15, 16, 17, 18]. The suppression
of extracellular matrix synthesis is suggested a one of the possible mechanisms of

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inhibiting of biofilm formation by NAC [19].
However, there is a lack of data about the influence of NAC on skin bacteria and

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their biofilms, and a better understanding of bacterial responses to NAC may
determine strategies for optimal antibacterial therapy of infections caused by

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opportunistic bacteria. The aims of this study were to evaluate effect of NAC on
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adhesion and biofilm formation of Gram-positive skin pathogens, including
M. smegmatis and multispecies biofilm.
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2. Materials and methods
2.1 Bacterial strains and cell growth
Twenty four bacterial strains: Enterococcus faecalis NCIMB 13280,
Corynebacterium ammoniagenes IEGM 1862, Mycobacterium smegmatis mc2 155,
Propionibacterium acnes VKM Aс-1450 (ATCC 6919) and their rifampicin-resistant
derivatives, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC

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33591, Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis ATCC
29887, Streptococcus pyogenes NCIMB 1475, and 14 clinical strains of coagulase

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negative staphylococci were used.
Bacteria were cultured to exponential phase in Luria-Bertani (LB) broth (10 g

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tryptone, 5 g yeast extract, 6.4 g KCl and Milli-Q water to 1 l; Sigma–Aldrich, USA)
at 37°C and shaking 160 rpm. Following growth for 16 - 18 h (for M. smegmatis - 48

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h) the cells were pelleted by centrifugation (13000 rpm, 5 min), the supernatant
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medium discarded, and the cells were washed twice with 0.85% NаCl and were diluted
to 107 CFU/ml in LB medium.
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2.2 Selection of rifampicin-resistant derivatives of P. acnes

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For progressive rifampicin resistance selection an adapted protocol from

Furustrand Tafin et al. was used [20]. Briefly, bacteria were exposed to serial 2-fold
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increasing concentrations of rifampicin in LB broth for a total of 5 cycles, each cycle

being 72 h. A series of tubes containing 2-fold increasing concentrations of rifampicin
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were inoculated with 108 CFU/ml (final concentration). Following incubation, the
increased resistance to rifampicin was evaluated with a disk diffusion test (disk
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concentration of rifampicin 5 µg/ml) and 0.1 mL samples from the tubes containing the
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highest antibiotic concentration, and still showing turbidity, were used to inoculate a
new series of tubes containing antibiotic dilutions. The experiments were performed
twice. The stability of rifampicin-resistant isolates was confirmed by subculture on
rifampicin-free agar three times. The resistance to rifampicin was then retested by a
disk diffusion test. All resistant isolates were identified by biochemical tests.
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2.3 Determination of minimum inhibitory concentration of NAC
The solution (Fluimucil; Zambón, Madrid, Spain) with concentration of 100
mg/mL of NAC, containing 25 mg/mL of NaOH and 1 mg/mL of EDTA for adjusting
of pH to 6.5 was used. The antibacterial activity of NAC against the 24 skin bacteria
was examined by determining minimum inhibitory concentrations (MICs) in
accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines

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[21]. Briefly, each bacterial suspension was adjusted to the turbidity in LB broth to a
final inoculum concentration of approximately 105 CFU/ml, as confirmed by viable

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counts. A 96-well round bottom microtiter plate containing 100 µL of serially diluted
NAC in LB broth (0.098 – 50 mg/ml) was inoculated with 10 µl of exponential phase

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cells of studied bacteria. The blank control comprised LB medium without inoculum.
An inoculated LB medium without NAC was the positive control. The plates were

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incubated under aerobic condition at 37°C for 24 h. MIC was defined as the lowest
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concentration of NAC that allowed no visible growth.
2.4 Adhesion assay
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Bacterial adhesion was studied in static conditions [22]. Briefly, two ml of fresh
suspensions bacterial cells (107 CFU/ml) in LB broth containing 0.5xMIC of NAC
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were added to 40-mm polystyrene Petri dishes. Bacterial suspensions without NAC
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were used as control. After incubation for 30 min in static conditions at 37°C dishes
were rinsed three times with two ml of sodium-phosphate buffer (10 mM, pH 7.2) to
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remove any unbound bacteria. The adherent cells were fixed by heating at 60°C for 30
min, stained by two ml of crystal violet (0.1% (w/v) in water) for 2 min and excess
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stain was rinsed off with running tap water. After the drying the dishes were directly
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imaged using the digital optical microscope (µViso-103; Lomo, Russia). In order to
determine the mean of the number of adherent bacteria the adsorb cells were counted at
least in 10 field-of-view (FOV) in each dish at magnification ×1000.
2.5 Biofilm formation assay
Quantification of biofilm formation was performed using a previously described
microtiter plate assay [23], with modifications. In brief, doubling dilutions of NAC
(0.098 – 25 mg/ml) were prepared in volumes 100 µl in a 96-well flat-bottom
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microtiter plates in LB broth. An each well was inoculated with 100 µl of exponential
phase cells of studied bacteria, approximately 107 CFU/ml and plates were incubated at
37°C. The medium alone served as negative control. After 24, 48, 96 and 120 h for M.
smegmatis, 48 h of incubation for S. epidermidis ATCC 29887, rifampicin resistance
derivate of P. acnes VKM Ac-1450 both mono- and multispecies biofilm, and after 24
h of incubation for other bacteria the total growth yield the bacterial community was

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measured. For this purpose, the absorbance wells containing both planktonic and
biofilm cells was measured at 600 nm in microtiter plate reader (Benchmark Plus

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Microplate Spectrophotometer System; Bio-Rad, USA). Then the medium and
unbounded cells were removed, followed by gentle washing (three times) with 200

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µl/well of sodium-phosphate buffer (10 mM, pH 7.2). The remaining biofilm was fixed
by heating at 60°C for 30 min and then stained in 200 µl/well of crystal violet (0.1%

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(w/v) in water) for 20 min. Excess stain was rinsed off with running tap water and the
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plates were dried. The dye was extracted with 200 µl/well of ethanol for 30 min
without shaking. The absorbance was measured at 570 nm using a microtiter plate
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reader (Benchmark Plus Microplate Spectrophotometer System, Bio-Rad, USA).

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To assess the effect of medium composition on the NAC action against M.

smegmatis in half of experiments of LB broth was replaced with a M63 medium (2 g
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(NH4)2SO4, 13.6 g KH2PO4, 0.5 g FeSO4·7H2O, and MilliQ water to 1 l supplemented

with 0.2% glycerol and 1 mM MgSO4; Sigma–Aldrich, USA)
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To prepare multispecies suspensions, bacterial cultures of the two species,

S. epidermidis ATCC 29887 and rifampicin resistance derivates of P. acnes VKM Ac-
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1450, containing of 107 CFU/ml were mixed in equal proportions. The mixed bacterial
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culture suspension was then dispensed into wells of a polystyrene 96-well flat-bottom
microtiter plate (100 µl/well) containing various concentrations of NAC in LB broth
(100 µl/well). The following steps were performed similarly as in the case
monospecific biofilms.
2.6 Biofilm eradication assay
We formed monospecies biofilms of S. epidermidis ATCC 29887, rifampicin
resistance derivates of P. acnes VKM Ac-1450 and M. smegmatis mc2 155 and
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multispecies biofilm of S. epidermidis ATCC 29887 and P. acnes VKM Ac-1450 Rifr.
For this, 200 µl aliquots of a bacterial suspension of 107 CFU/ml in LB broth were
inoculated into each well of a 96-well flat-bottom microtiter plate and incubated at
37°C. After 48 hours of incubation for S. epidermidis and P. acnes and 120 hours of
incubation for M. smegmatis, each well was washed three times with 10 mM sodium-
phosphate buffer (pH 7.2) under aseptic conditions to eliminate unbound bacteria, and

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200 µl of NAC dilution in 12.5 mg/ml or sterile saline as control were added. After 4 h
exposure, drug solutions were discarded and each well was washed once with the

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sodium-phosphate buffer (10 mM, pH 7.2). Then the biomass of the remaining biofilm
was determined as described above in 2.5 Biofilm formation assay section.

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2.7 Statictics
Statistical analyses were performed in accordance with the results of the Shapiro-

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Wilk test of normal distribution. Data were further analyzed using a one-way ANOVA,
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followed by post-hoc 2-sided Dunnett’s tests. The results reflect the average (±standard
deviations [SD]) of three independent experiments.
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3. Results
3.1 Selection of rifampicin-resistant derivatives of P. acnes
P. acnes had been considered as susceptible to rifampicin until recently but
Furustrand Tafin et al. demonstrated that rifampicin resistance in P. acnes can easily
be selected in vitro [20]. We also have selected P. acnes VKM Ac-1450 Rifr strain
with resistance to rifampicin, as evidenced by the complete absence of zone of growth

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inhibition around the disc in disk diffusion test. The obtaining of rifampicin-resistance
to entailed an increase in the biofilm formation and a changing of the sensitivity of P.

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acnes VKM Ac-1450 Rifr to other antibiotics. So, the sensitivity to gentamicin
decreased almost twice. We also observed a significant reduction in the diameter of

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zone of inhibition in case cephalexin and oxacillin (data not shown). The alteration of
biofilm formation, cross-resistance, and the stability of resistance at cultivation of P.

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acnes VKM Ac-1450 Rifr on rifampicin-free medium may be connected with fact that
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the resistance P. acnes to rifampicin is usually due to point mutations in the rpoB gene
[20].
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3.2 Determination of minimum inhibitory concentration of NAC

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Table shows the MIC of NAC against skin pathogens. MIC of NAC is defined as
the lowest concentration of NAC required to inhibit bacterial growth. We found that
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the MIC of NAC ranged from 0.098 to 25 mg/ml and the most sensitive
microorganism to NAC was M. smegmatis mc2 155. MICs for all studied
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staphylococcal strains were 25.0 mg/ml of NAC. Besides, the rifampicin-resistant

derivative of P. acnes VKM Ac-1450 was susceptible to NAC to the same extent as
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the parent strain.

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Table
Minimum inhibitory concentrations (MICs) of NAC against Gram-positive skin
pathogens
Microorganism MIC, mg/ml
Enterococcus faecalis NCIMB 13280 25.0
Corynebacterium ammoniagenes IEGM 1862 25.0
Mycobacterium smegmatis mc2 155 0.098

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Propionibacterium acnes VKM Aс-1450 6.25
Propionibacterium acnes VKM Aс-1450 Rifr 6.25

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Staphylococcus aureus ATCC 25923 25.0
Staphylococcus aureus ATCC 33591 25.0
Staphylococcus epidermidis ATCC 12228 25.0

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Staphylococcus epidermidis ATCC 29887 25.0
Streptococcus pyogenes NCIMB 1475 25.0
CNS 1 25.0

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CNS 5 25.0
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CNS 6 25.0
CNS 8 25.0
CNS 19 25.0
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CNS 26 25.0
CNS 27 25.0
CNS 33 25.0
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CNS 34 25.0
CNS 38 25.0
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CNS 42 25.0
CNS 44 25.0
CNS 110 25.0
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CNS 192 25.0

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3.3 Ability of NAC to inhibit adhesion and biofilm formation of skin pathogens.
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In order to assess the inhibitory effect of sub-inhibitory concentration of NAC on

adhesion and biofilm formation, the bacterial species were grown to exponential phase
in LB and then exposed to 0.5MIC of NAC during 30 min or 24 h. In the presence of
NAC the adhesion of C. ammoniagenes IEGM 1862, S. pyogenes NCIMB 1475, and
P. acnes VKM Aс-1450 did not change in contrast to E. faecalis NCIMB 13280 that
showed a significant reduction in the adhesion by 20%. NAC at concentrations of 12.5
mg/ml which is 0.5xMIC for all studied staphylococcal species significantly reduced
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the adherence of nine of fourteen clinical CNS strains and of two reference strains of
S. epidermidis (Fig. 1)

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Fig. 1 Effect of sub-MICs of NAC on adhesion to polystyrene of 14 strains of CNS,
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S. epidermidis ATCC 29887, and S. epidermidis ATCC 12228 in LB medium. The
error bars indicate the standard deviations of the means for three experiments. (1.5-
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column fitting image)

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We also examined the ability of sub-inhibitory concentrations of NAC to prevent

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the biofilm formation of CNS. The inhibitory effect of NAC weakened with increasing
the incubation time up to 24 h compared to 30 min adhesion. As shown in Fig 2 the
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biomass of 1-day old biofilm of all studied CNS strains in presence of 12.5 mg/ml of
NAC was similar to control (without NAC).
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Fig. 2 Biofilm formation of 14 strains of coagulase-negative staphylococci (CNS) in
the presence of NAC (0.5xMIC). The error bars indicate the standard deviations of the

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means for three experiments. (1.5-column fitting image)
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To further corroborate these results, we tested the bacterial growth and biofilm
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formation of two strong (CNS 19, CNS 34) and one moderate (CNS 33) biofilm
producers at range of concentration of NAC (0.78 – 25 mg/ml).
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3.4 Effect of NAC on bacterial growth and biofilm formation.

Different bacteria exhibited different abilities to grow in the presence of NAC, as
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shown in Fig. 3. Six of the bacteria used in this study were able to grow in the presence
of low concentrations of NAC (≤3.125 mg/ml), while the bacterial growth of the other
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bacteria tested (C. ammoniagenes, S. aureus ATCC 25923, and P. acnes) was slowed
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starting at 1.56 mg/ml of NAC (Fig. 3). Additionally the lowest tested concentration of
NAC (0.78 mg/ml) slightly stimulates the overall growth for all CNS. We found that at
a concentration of 25.0 mg/ml NAC diminished growth by 50% and more for all tested
bacteria.
А dose-dependent decrease in biofilm formation of C. ammoniagenes, S.
pyogenes, and P. acnes, was observed (Fig. 3). For example, for P. acnes, biofilm
biomass was significantly reduced to 50% of that of the control in the presence of
NAC as low as 1.56 mg/ml. However, biofilm formation of E. faecalis, CNS 19, and
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CNS 34 increased at sub-MICs of NAC (12.5 mg/ml). Treatment with 25 mg/ml NAC
completely inhibited biofilm formation of C. ammoniagenes, S. pyogenes, P. acnes,
CNS 34, and two strains of S. aureus.

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Fig. 3 Effect of NAC on the growth and biofilm formation of E. faecalis NCIMB
13280, C. ammoniagenes IEGM 1862, S. pyogenes NCIMB 1475, three strains of
coagulase negative staphylococci (CNS 19, CNS 33, CNS 34), S. aureus ATCC 25923,
S. aureus ATCC 33591 and P. acnes VKM Aс-1450. The error bars indicate the
standard deviations of the means for three experiments. (2-column fitting image)
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3.5 Effect of NAC on growth of M. smegmatis in different medium
The total growth (planktonic and biofilm) of the M. smegmatis cell in M63 and
LB medium was significantly inhibited by ≥1.563 mg/ml NAC starting from first day
of incubation (Fig. 4). Additionally, the lowest tested concentrations of NAC (0.098
and 0.391 mg/ml) impeded the growth of mycobacteria in the M63 medium in
comparison with LB medium. So, the cultivation of mycobacteria in rich medium (LB)

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led to that the inhibitory effect of NAC in 1xMIC (0.098 mg/ml) and 4xMIC (0.391
mg/ml) lasted only 1 day, and after 5 days of incubation the total bacterial growth

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without NAC and with 4x MIC of NAC was not much different. Measurement of the
amount of biofilm after 5 days of incubation by staining with crystal violet revealed

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that 1.563 mg/ml of NAC also caused a two and four-fold reduction in biofilm biomass
in LB and M63 media, respectively. As in the case of the total bacterial growth 1xMIC

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of NAC could not prevent the biofilm formation of M. smegmatis mc2 155 in LB
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medium.
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Fig. 4 Effect of NAC on growth of M. smegmatis mc2 155 cultured in M63 (A) and LB
(B) medium. Biofilm formation of M. smegmatis mc2 155 (C) in the presence of NAC
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on 5 day of incubation. The error bars indicate the standard deviations of the means for
three experiments. (2-column fitting image)
3.6 Examining the effect of NAC on bacterial ecology within mixed P. acnes and
S. epidermidis biofilms
Then bacteria grown without NAC, the total growth of S. epidermidis and P.
acnes was almost twice differed (Fig. 5, A). The planktonic growth of the multispecies

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culture in NAC-free medium was approximately in the middle of OD600 values for
monocultures. While, in the absence of NAC S. epidermidis demonstrated an expected

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growth pattern, and as expected, P. acnes and mixed culture showed little biofilm
formation (Fig. 5, B). But, the optical density of the mixed culture falls to the level of

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monoculture S. epidermidis ATCC 29887 when the minimum tested concentration of
NAC (1.56 mg/ml) was added. At the same time, we found that the S. epidermidis - P.

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acnes biofilm formation has increased almost two times compared with the control at a
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concentration of 3.125 mg/ml of NAC. A similar effect was observed for monoculture
S. epidermidis ATCC 29887 at 6.25 mg/ml of NAC. The biofilm formation and growth
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of both S. epidermidis ATCC 29887 and the mixed culture were significantly slowed
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with increasing the concentration of NAC up to 12.5 mg/ml.

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Fig. 5 Growth (A) and biofilm formation (B) of mono- and mixed species culture of
S. epidermidis ATCC 29887 and P. acnes VKM Ac-1450 Rifr in the presence of NAC.
The error bars indicate the standard deviations of the means for three experiments. (2-
column fitting image).
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Unlike staphylococci, propionic acid bacteria were significantly more sensitive to
the inhibitory effect of NAC. So, even the minimal studied concentration of NAC
(1.56 mg/ml) prevents the growth and biofilm formation of P. acnes VKM Ac-1450
Rifr.
3.7 Effect of NAC on biofilm eradication
N-acetylcysteine was found to have significant inhibitory effects (p<0.05) on

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preformed mature biofilm of M. smegmatis. Exposure to NAC (12.5 mg/ml) has
reduced biomass of M. smegmatis mc2 155 biofilm in two times compared with the

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untreated biofilm (Fig. 6). Results showed that NAC also has a higher disruptive effect
on mature S. epidermidis – P. acnes mixed biofilm than biofilms formed by

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monocultures. As 12.5 mg/ml of NAC caused a reduction in the biomass of
multispecies biofilm to 61% in comparison to control while biomass of S. epidermidis

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ATCC 29887 and P. acnes VKM Ac-1450 Rifr biofilms was almost unaltered after 4h
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treatment with NAC.
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Fig. 6 Eradication of preformed mature biofilm of P. acnes VKM Ac-1450 Rifr,

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S. epidermidis ATCC 29887, mixed species, and M. smegmatis mc2 155 by treatment
of NAC (12.5 mg/ml). Mixed biofilm consist of S. epidermidis ATCC 29887 and P.
acnes VKM Ac-1450 Rifr. The error bars indicate the standard deviations of the means
for three experiments. (1-column fitting image)
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4. Discussion
In view of the absence of the toxic effect of NAC, confirmed during the long
years [12], use of this compound should not be limited to treatment of upper
respiratory tract diseases. We tested the antibacterial action of NAC against Gram-
positive skin pathogens. The MIC for majority of studied strains was 25.0 mg/ml of
NAC (Table) and the most sensitive microorganism to NAC was M. smegmatis

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mc2 155 (0.098 mg/ml). The anti-mycobacterial effect of NAC was for a long time
considered to relate with its ability NAC scavenge of ROS, which, in turn, stimulates

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the growth and development of fast-growing non-pathogenic mycobacteria [24].
However, it was recently suggested that NAC may exert a bactericidal effect on

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mycobacteria, in particular M. tuberculosis, through the generation of glutathione
(GSH) and in turn Cys-Gly [13]. Actually, GSH has been described to limit
mycobacterial growth [25].
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The greatest effect NAC against growth and biofilm formation of M. smegmatis
mc2 155 was observed on M63 medium (Fig. 4, B). So, 1.563 mg/ml of NAC caused a
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four-fold reduction in biofilm biomass. Perhaps, minimal medium composition allows

M. smegmatis mc2 155 cells go into dormant forms that is common to all
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mycobacterial cells and can be obtained in stationary-phase cultures developing under

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hypoxia and carbon, phosphorus, or nitrogen starvation stress [26].

Higher concentration of NAC (12.5 mg/ml) effectively prevents the biofilm
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development as well as destroys mature biofilm of M. smegmatis (Fig. 6),

consequently, NAC can be considered a promising agent for the control of
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mycobacterial growth.
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In the present study, the growth and biofilm formation of skin pathogens was
significantly inhibited by NAC at concentrations of 1.56–25.0 mg/ml. Notably, biofilm
formation by the E. faecalis NCIMB 13280, C. ammoniagenes IEGM 1862, three
strains of coagulase negative staphylococci (CNS 19, CNS 33, CNS 34), and S. aureus
ATCC 25923, was reduced in the presence of ≥ MIC of NAC, while NAC at a sub-
MIC in some cases even enhance bacterial biofilm formation. Thus it seems that the
effect of NAC in reducing biofilm formation by skin pathogens is mainly due to its
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inhibitory effect on bacterial cell growth. The antibacterial mechanism of NAC is not
clear, but it was suggested that NAC is likely to be achieved by preventing amino acid
(cysteine) utilization in bacteria or reaction of sulfhydryl group of NAC with bacterial
cell proteins [15]. Therefore, further studies are required to assess whether the
reduction of biofilm formation with NAC is at least partly due to changes in the
amount and composition of EPS produced by the bacteria.

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The unexpected increase of staphylococcal biofilm formation occurring
concurrently with the decrease of the plankton growth at sub-MIC of NAC (Fig. 3, 5B)

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may be associated with fact that some stress factors, as sublethal concentrations of
antibiotics, can enhance production of biofilm matrix polymers [27].

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Though P. acnes described as a commensal human skin bacterium with a low
pathogenicity, this microorganism has been considered as responsible for various types

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of infections associated with breast implants, cerebrospinal fluid shunts, ocular
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implants, or osteosynthesis and joint prosthesis [28]. But we found that the bacterial
growth and biofilm formation of P. acnes can be slowed at sub-MIC of NAC (1.56
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mg/ml) (Fig. 3). Although, no clinical isolate of P. acnes has been reported resistant to
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rifampicin so far we observed that rifampicin resistance emerged rapidly and this strain
can formed stronger biofilm then parent strain. But NAC can prevent growth of
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plantonic cells of P. acnes VKM Ac-1450 Rifr as well as their biofilm formation (Fig.
5).
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High sensitivity of P. acnes to the action of NAC keeps even in the multispecies
P.acnes – S.epidermidis community. Actually, when NAC was added to medium, the
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curve of total growth and biofilm formation of the multispecies culture mirrored that of
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S. epidermidis. Apparently, S.epidermidis ATCC 29887 dominate because of their

resistance to NAC and their high growth potential despite of fact that Rasmussen et al.
showed that the bacterial ecology of multispecies oral biofilms was nearly unchanged
with 1% NAC exposure [29]. In the light of our data about effect of NAC on bacterial
ecology within mixed P. acnes and S. epidermidis biofilms, the introduction of NAC
probably could change the ratio of microorganisms in mixed biofilm. In this way, the
long-term treatment with NAC, can perhaps lead to a change of the skin microflora
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composition, in particular a reduction of propionic acid bacteria in comparison with
staphylococci. Recently it has been shown that such imbalance in the P. acnes –
S.epidermidis ratio may cause the appearance of various skin disorders such as
dandruff [30].
In the present study, the formation of monospices as well as mixed P. acnes and
S. epidermidis biofilms was significantly inhibited by 12.5 mg/ml of NAC. But, the

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same concentration of NAC poorly destroys mature monospices biofilm of P. acnes
and S. epidermidis (Fig. 6). Thus it seems that the effect of NAC in reducing

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planktonic growth and biofilm formation is mainly due to its inhibitory effect on
bacterial cell growth. We have been previously shown that bacteria S. epidermidis

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ATCC 29887 are PIA-negative and the main component of their biofilms are proteins
[22]. So, this fact probably makes biofilm of S. epidermidis ATCC 29887 more

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resistant to NAC treatment, because NAC acts on the polysaccharide matrix of the
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biofilm, breaking the disulfide bridges that link the polysaccharide fibers [16].
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5. Conclusion.
According to our results, it may be concluded that the effect of NAC on microbial
adherence inhibition and disruption of biofilm of skin pathogen is valuable. NAC
appears to be a promising, non-antibiotic alternative to prevent biofilm-associated
infections.

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Acknowledgements
The study is supported by Program of UD RAS, project No 15-4-4-3.

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Highlights

− MICs of N-acetylcysteine ranged from 0.098 to 25.0 mg/ml.

− No correlation between adhesion and biofilm formation of CNS at 0.5xMICs

of NAC
− NAC reduce growth and biofilm formation of skin pathogens in dose-

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dependent manner
− NAC inhibit formation of monospecies and multispecies biofilm

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− M. smegmatis biofilm can be prevented and eradicated by 12.5mg/ml of
NAC

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