Photoinactivation of Methicillin-Resistant S. Aureus Biofilm using a

New Chlorin as Photosensitizer

L. S. Amaral

, I. A. P. Linares

and J. R. Perussi

1,2 c

Programa de Pós-Graduação Interunidades em Bioengenharia EESC/FMRP/IQSC, Universidade de São Paulo,

Av. Trabalhador Sãocarlense, 400, 13566-590, São Carlos - SP, Brazil

Instituto de Química de São Carlos, Universidade de São Paulo,

Av. Trabalhador Sãocarlense, 400, 13566-590, São Carlos - SP, Brazil

Keywords: Bacterial Biofilm, Membrane, Resistant Bacteria, Photoinactivation, Photosensitizer, Chlorin.

Abstract: Due to the increase in bacterial resistance to antibiotics, the development of new drugs and technologies for

the eradication of microorganisms is a priority. Photodynamic Therapy (PDT) depends on the interaction

between a light-sensitive compound (photosensitizer), light, and molecular oxygen. The reaction generates

reactive oxygen species (ROS), which induce cell death by oxidative stress. Antimicrobial Photodynamic

Therapy (A-PDT) may be a promising alternative for microbial infections since its action occurs by multiple

targets, which hinders the development of resistance. The main goal of this study was the evaluation of the

potential of a newly synthesized chlorin derivative sterically prevented from self-aggregation as a

photosensitizer to photoinactivation Methicillin-Resistant S. aureus (MRSA) biofilm and to investigate the

membrane integrity after the treatment. The results showed a high potential of this chlorin for

photoinactivation of MRSA biofilms reducing the survival index more than 5 log CFU mL

-1

leading to the

unstructured membrane and consequent cell death by photooxidation of membrane components after A-PDT.

1 INTRODUCTION

Due to the increasing worldwide resistance of

bacteria to antibiotics, the development of new drugs

and technologies for the eradication of

microorganisms is a priority (Sobotta et al., 2019).

The biggest problem with using antibiotics is that

bacteria have different resistance mechanisms, which

can result in the formation of biofilms that are even

more refractory to treatments. Antimicrobial

Photodynamic Therapy (A-PDT) may be a promising

alternative for microbial infections since its action

occurs by multiple targets, which hinders the

development of resistance (Stanislaw et al., 2018).

PDT involves the combination of a photosensitizer

(PS), molecular oxygen, and visible light of adequate

wavelength to produce reactive oxygen species

(ROS), causing the cell to die through the oxidation

of its constituent biological molecules (Fig. 1).

PS is a substance that induces light sensitivity to

chemical, physical, or both processes usually

insensitive to light. Most photosensitizers have a

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heterocyclic ring similar to chlorophyll and the

hemoglobin heme group. Photons are absorbed in the

band of the electromagnetic absorption spectrum

characteristic of photosensitizers and can transfer this

energy to other molecules, especially to molecular

oxygen, which will result in the release of short-lived

energy species, leading to damage to the biological

system involved (Hamblin et al., 2008).

Chlorins are molecules of high abundance and

importance in nature, present in most plants that make

photosynthesis (De Oliveira et al., 2014). CHL-Ph-A

is a new chlorin derivative sterically prevented from

aggregation due to the structural shape in “L”. Diels-

Alder reaction was used to synthesize CHL-Ph-A

from protoporphyrin IX (Linares et al., 2017).

The objective of this study was to photoinactivate

Methicillin-Resistant S. aureus (MRSA) biofilm

using a new chlorin (CHL-Ph-A) with the aid of Full

Factorial Design 2

and microscopy techniques to

evaluate the integrity of the bacterial membrane.

Amaral, L., Linares, I. and Perussi, J.

Photoinactivation of Methicillin-Resistant S. Aureus Bioﬁlm using a New Chlorin as Photosensitizer.

DOI: 10.5220/0008938100920096

In Proceedings of the 8th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2020), pages 92-96

ISBN: 978-989-758-401-5; ISSN: 2184-4364

Figure 1: Mechanisms of oxygen reactive species production:

when the photosensitizer (PS) is incubated with the bacteria,

it absorbs energy from light and goes to the excited singlet

state (S

 S

or S

decaying to S

by Internal Conversion −

IC). The relaxation can occur by fluorescence (F) or by

intersystem crossing (ISC). In the triplet state, the PS

molecule may lose energy by phosphorescence (P) or energy

transfer to molecular oxygen, generating singlet oxygen (

)

by type II mechanism. When charge transfer occurs

generating free radicals (type I), death of the bacteria may

happen due to the oxidation of their cellular components.

2 METHODS

2.1 Bacterial Strain and Biofilm

Culture Conditions

Methicillin-Resistant Staphylococcus aureus –

MRSA (ATCC® 33591 ™) was grown of the

planktonic form in Brain Heart Infusion (BHI)

medium at 37° C for 18 h with orbital agitation at 250

rpm. After planktonic cultivation, the optical density

of each bacterium was standardized to OD 600 in

phosphate-buffered saline (PBS), using a HITACHI

U-2800 spectrophotometer. For biofilm formation,

150 μL of the suspension was deposited on the 96-

well plate and supplemented with 150 μL of the BHI

medium. The plates were kept in an incubator for 48

h at 37° C.

2.2 Photosensitizer and Light Source

CHL-Ph-A (Fig. 2) was synthesized in our research

group, and the procedure and full characterization are

described in Linares et al., 2018.

The light source used for photoinactivation of

MRSA was an illumination platform called Biotable

(Fig. 3) developed by the LAT at the Instituto de

Física de São Carlos, Brazil, composed by 40 red

LEDs (660 ± 10 nm).

Figure 2: Molecular structure of chlorin CHL-Ph-A.

Figure 3: Red biotable 660 ± 10 nm used in irradiation

procedures.

2.3 Photodynamic Inactivation of

Bacteria in Biofilm Form

The MRSA biofilm photoinactivation was performed

with the aid of Full Factorial Design 2

( FFD 2

= 8

experiments), using three parameters with two levels

and a central point allowing to perform a smaller

number of experiments, less time consuming and

lower expenses to obtain the results. The three

parameters used were CHL-Ph-A concentration (C

5; 7.5 and 10 µmol L

-1

) incubation time (IT: 20; 30

and 40 min), and light dose (LD: 15; 22 and 30 J cm

) at 660nm combined among them in a multivariate

form, submitting the results to Two-way ANOVA.

2.4 Determination of the Bacterial

Viability

MRSA biofilm was slowly homogenized, removing a

100 µL aliquot from each well of the 96-well plate

(before and after A-PDT) and diluting from 10

-1

to 10

. Four aliquots of 15 µL were taken from each

dilution and deposited on BHI agar plates and

incubated at 37° C for 18 h. Each BHI agar plate was

divided into four quadrants, each assigned to a

dilution. Quantification was performed by counting

the colonies at the dilution in which they had 5 to 50

colony forming units (CFU). The number of survivors

Photoinactivation of Methicillin-Resistant S. Aureus Bioﬁlm using a New Chlorin as Photosensitizer

present in the sample was determined by the average

number of colonies, multiplied by the dilution, and

the number of CFU per milliliter of the solution was

obtained.

2.5 Membrane Integrity Analysis by

Fluorescence Microscopy

MRSA Biofilm before and after photoinactivation

was submitted to membrane integrity analysis.

Biofilm was cultivated in microscopy slides and

submitted to a mixture of SYTO

9 and Propidium

Iodide (PI) (mixture of the LIVE/DEAD™ kit,

Invitrogen Molecular Probes

) being finally analyzed

by a Fluorescence Microscope (Olympus BX41) with

100X objective, 500 nm dichromatic filter, excitation

at 460-490 nm and emission at 520 nm.

2.6 Morphostructural Analysis of

Photoxidized MRSA

For the morpho-structural analysis by Scanning

Electron Microscopy (SEM), the biofilm was

cultivated under polystyrene slides 1x1 cm arranged

in the bottom of 12 well plates. Then 1 mL of BHI

broth was added, keeping them in the oven for 48 h at

37° C. Alternatively, in place of the BHI, 1 mL of the

PS solution was placed in a previously defined

concentration and irradiated. Then the biofilms were

washed with PBS and fixed with 1 ml 2.5%

glutaraldehyde for 1 hour. Then, the sample was

dehydrated with ethyl alcohol in different

concentrations: 10, 25, 50, 75, 90, and 100% for 20

min each. After drying, the slides were metalized and

visualized in the LEO scanning electron microscope,

model 440, with a magnitude of 60.00 KX.

3 RESULTS

3.1 Biofilm Photoinactivation

The inactivation obtained using CHL-Ph-A by the

multivariate form presented in Table 1. The best

photoinactivation obtained was 53 % for the more

significant variation in the survival index (Δ log

) of

5.13 corresponding to nine assays. This decrease in

the survival index can be considered good (or

enough) when dealing with biofilm, which is very

difficult to inactivate. So, the best parameters were PS

concentration of 5 µmol L

-1

, IT of 40 min, and LD 30

J cm

-2

reaching maximum photoinactivation of 4,52 ±

0,02 log CFU.

In a biofilm, bacteria have the same genetics as in

planktonic culture, but their biochemical activities differ by

40%, presenting a greater difficulty to be eliminated due to

acquired resistance (Wiesch et al., 2011). Given this

difficulty, antimicrobial photodynamic therapy can be

employed as an option for indiscriminate use of antibiotics,

thus reducing the problem related to bacterial resistance.

The methodology does not entail resistance to bacteria due

to the vast number of possible targets that ROS can act in

preventing any bacterial adaptation/mutation. However,

according to the American Society of Microbiology, the

reduction must be more significant than required (> 3 log

CFU mL

-1

) for a new approach to be called antimicrobial

(ASM, 2015). Fortunately, photodynamic therapy using

CHL-Ph-A fulfills this requirement.

Table 1: Bacterial viability of MRSA biofilm after a-PDT

with CHL-Ph-A. The results of the experiments are

arranged according to the experimental matrix FFD 2

where IT: incubation time (min), LD: light dose (J cm

-2

and C: chlorin concentration (µmol L

-1

). Nine different

experiments were performed as described according to the

values of the parameters used. After the procedures, the

results of each assay (Colony Forming Unit – CFU) are

described and presented as average ± standard deviation

(SD) with n = 4 replicas.

Assay

(min)

(J cm

-2

)

(µmol L

-1

)

CFU Average ± SD

CHL-Ph-A

- 0 0 0 9,65 ± 0,03

1 20 15 5 7,78 ± 0,11

2 20 15 10 7,36 ± 0,14

3 20 30 5 7,46 ± 0,06

4 20 30 10 7,40 ± 0,04

5 40 15 5 6,11 ± 0,16

6 40 15 10 5,90 ± 0,12

7 40 30 5 4,52 ± 0,02

8 40 30 10 6,48 ± 0,03

9 30 22 7.5 6,48 ± 0,05

3.2 Membrane Rupture after

Chlorin-PDT

The integrity of the bacteria membrane present in the

biofilm was determined by Fluorescence Microscopy

using the Live/Dead kit, which contains two markers,

the fluorescent green SYTO

9 (S) and the fluorescent

red propidium iodide (PI). The probe S penetrates

both into intact cells or not because of its low

molecular weight; however, the PI only penetrates

cells with the damaged cytoplasmic membrane

because of its high molecular weight resulting in the

reduction of S intensity when both dyes coexist in the

cell. Figure 4 shows the results.

The CHL-Ph-A

chlorin associated with photodynamic therapy

enabled the cytoplasmic membrane disruption of

MRSA in the biofilm, revealing the red color (Fig.4

PHOTOPTICS 2020 - 8th International Conference on Photonics, Optics and Laser Technology

B). On the other hand, the presence of membrane

integrity is represented in green (Fig.4 A).

Figure 4: Fluorescence microscopy of MRSA biofilm: (A)

Control and (B) after photoinactivation using CHL-Ph-A.

3.3 Analysis of MRSA Photoxidized

after Chlorin-PDT

Scanning Electron Microscopy (SEM) was used to

characterize the biofilm structure, bacterial

morphology, as well as to evidence the photodynamic

process. For this, S. aureus biofilm was submitted to

SEM analysis in the best experimental conditions for

CHL-Ph-A photooxidation using FFD 2

. The

parameters used were: C = 5 µmol L

-1

; IT = 40 min,

and LD = 30 J cm

-2

Through Figure 5, it is possible to observe by

Scanning Electron Microscopy the bacterial

arrangements of Staphylococci colonies as well as the

bacterium-bacterium interactions in biofilm

presenting links between them through fimbria

(highlighted in yellow). After A-PDT (Fig. 5B), a

damaged structure was observed (highlighted in red),

denoting photooxidation of cell membrane

components in which cocci do not have a definite

shape but a turgid structure as a deformation.

Figure 5: Scanning Electron Microscopy of MRSA

Biofilm: (A) Control and (B) After photoinactivation using

CHL-Ph-A.

4 CONCLUSIONS

The results suggest that A-PDT of MRSA biofilm

using CHL-Ph-A has excellent potential to combat

MRSA biofilm since reductions up to 5.13 Δlog

were reached. The efficiency of photooxidation and

the potential to eradicate biofilm was observed and

proven by SEM and Fluorescence Microscopy. The

results suggest that antimicrobial photodynamic

therapy using this new chlorin may be a good

alternative for the treatment of antibiotic-resistant

bacterial infections.

Overall, A-PDT is expected to be implemented to

ensure a broad bacterial inactivation. It is also

expected that the use of multivariate models, such as

complete factorial design 2

be usefull in future

experiments aiming to reduce costs and experimental

time in order to search for better responses that

enable even greater photoinactivation.

Photoinactivation of Methicillin-Resistant S. Aureus Bioﬁlm using a New Chlorin as Photosensitizer

ACKNOWLEDGMENTS

This study was financed in part by the Coordenação

de Aperfeiçoamento de Pessoal de Nível Superior -

Brasil (CAPES) - Finance Code 001; Conselho

Nacional de Desenvolvimento Científico e

Tecnológico (CNPq) and Fundação de Amparo à

Pesquisa do Estado de São Paulo/Centro de Pesquisas

de Ótica e Fotônica (FAPESP/CEPOF) 2013/07276-

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PHOTOPTICS 2020 - 8th International Conference on Photonics, Optics and Laser Technology