Ciencias de los Alimentos/Food Sciences

Cienc Tecn UTEQ (2019) 12(2) p 1-6

ISSN 1390-4051; e-ISSN 1390-4043

Interaction of Salmonella sp. and essential oils: bactericidal activity and

adaptation capacity

Interacción entre Salmonella sp. y aceites esenciales: actividad bactericida y adaptabilidad

Alcilene de Abreu Pereira¹, Jorge Pamplona Pagnossa², João Paulo Alcântara², Silas Rodrigo Isidoro², Roberta Hilsdorf Piccoli²*

Food Microbiology Laboratory, Department of Food Science, Universidade Federal de Lavras, caixa postal 3037, 37200-000,

Lavras, Minas Gerais, Brazil

¹ Instituto Federal de Minas Gerais - Campus Bambuí. Faz. Varginha - Rodovia Bambuí/Medeiros - km 05, Caixa Postal 05,

38900-000, Bambuí, MG, Brazil, alcilene.pereira@ufmg.edu.br

² Universidade Federal de Lavras, Caixa Postal 3037, 37200-000, Lavras, MG, Brazil, rhpiccoli@ufla.br

Rec.: 23.09.2019. Acept.: 05.12.2019.

Publicado el 31 de diciembre de 2019

Abstract

Resumen

acteria of the genus Salmonella, responsible for

a bacteria Salmonella, responsable de numerosos

many foodborne disease outbreaks, are capable

brotes de intoxicación alimentaria, puede formar

of forming biofilms on various surfaces in the food

biopelículas en varias superficies utilizadas en la

industry. The constant exposure of these bacteria to

industria alimentaria. La exposición constante de

sublethal concentrations of sanitizers has made them

estas bacterias a concentraciones subletales de agentes

tolerant to several of them. Seeking alternatives to

desinfectantes los hace tolerantes a muchos de estos

control of bacterial biofilms and adaptation under

agentes. Buscando alternativas para el control de

sublethal exposure, this study tested the antimicrobial

biofilms adaptadas a condiciones subletales, este

activity of Thymus vulgaris

(thyme) and Origanum

estudio evaluó la actividad antimicrobiana de los aceites

vulgare

(oregano) essential oils

(EOs) and their

esenciales Thymus vulgaris

(tomillo) y Origanum

major compounds, thymol and carvacrol, against

vulgare

(orégano) y sus componentes principales

of Salmonella enterica serovars Enteritidis and

timol y carvacrol contra Salmonella enterica serovares

Typhimurium. Carvacrol 0.25% (v/v) was the most

Enteritidis y Typhimurium. Carvacrol

0.25%

(v/v)

efficient antimicrobial agent against planktonic cells

fue más eficiente contra las células planctónicas de S.

of S. Enteritidis and biofilm were more susceptible to

Enteritidis y su biopelícula fue más sensible al 2.0% (v/v)

oregano EO at 2.0% (v/v). Differently, S. Typhimurium

de orégano EO. S. Typhimurium en forma planctónica

planktonic was inhibited at 0.25% (v/v) of thyme EO

fue inhibida por el tomillo EO a una concentración de

and biofilm was more susceptible to carvacrol (2.5%

0.25% (v/v) y su biopelícula fue más susceptible al

v/v). Adaptation of S. Enteritidis and Typhimurium was

carvacrol (2.5% v/v). S. Enteritidis y S. Typhimurium

observed on all tests (p < 0.05). This study confirms the

se adaptaron a todas las concentraciones subletales de

potential of EOs and its major compounds as alternative

los antimicrobianos probados. Este estudio confirma

sanitizers in the food industry against pathogenic

el uso potencial de EO y sus componentes principales

bacteria such as Salmonella spp. and of possible

como desinfectantes en la industria alimentaria para

adaptation due to sublethal exposure.

controlar bacterias patógenas como Salmonella spp. y su

capacidad para adaptarse a concentraciones subletales.

Keywords: biofilm, planktonic cells, carvacrol, thymol,

stress response.

Palabras clave: biopelículas, células planctónicas,

carvacrol, timol, respuesta al estrés.

Pereira et al., 2019

Introduction

2004). EOs are distinguished by high antimicrobial

activity and, in appropriate concentrations, generally

t is well known that the implementation of health

recognized as safe. Thus, it is considered exempt from

control measures, cleaning and sanitization in food

food additive tolerance requirements for its use (Smith et

industries can prevent economic loss.Aiming at the safety

al., 2005). Essential oils of Origanum vulgare (oregano)

of their products, the food industry use antimicrobial

and Thymus vulgaris (thyme) contain, among other

agents with varied modes of action, exposure time and

compounds, thymol and carvacrol, which are considered

chemical composition. However, the hygienic programs

powerful bactericides and fungicides

(Kalemba and

have been inefficient and often unable to completely

Kunicka, 2003). Induced stress conditions as exposure

remove bacterial biofilms that accumulate on surfaces

to sublethal concentrations of bactericidal compounds

and equipment of food processing environments, even

are an important evaluation to indicate adaptive capacity

considering all the misfortunes that these contaminations

of microorganisms. However, information on possible

can cause regarding microbiological aspects. Several

effects of using EOs or its compounds at sublethal

factors contribute to this situation, mainly the loss of

concentrations on microbial sensitivity to antimicrobials

susceptibility to antimicrobial agents due to the frequent

or physical processes is still scarce (Souza, 2016).

exposure of pathogenic bacteria, such as Salmonella, to

The investigation of bacteria challenged with

sublethal concentrations of sanitizers during cleaning

sublethal stresses reveal significant physiological

sessions.

changes that may enhance their ability to survive the

Bacteria of the genus Salmonella are

imposed hostile conditions. In recent literature, a plenty

foodborne pathogens of global coverage and cause

of evidences shows that exposure of bacteria to sublethal

the most outbreak-related in the world (CDC, 2018).

stresses may induce decrease of sensitivity to food

The contamination caused by these Gram-negative

antimicrobials, biocides and other food preservation

rod-shaped microorganisms can bring great losses

techniques (Erickson and Doyle, 2017). Furthermore,

to the food industry through embargoes and taxes

relevant increase of virulence and lower infectious

established by importing countries

(Shinohara et

doses in pathogenic bacteria brings even more concern

al., 2008), and Brazil is a major exporter of meat and

to food antimicrobials, such as essential oils (Gadea et

poultry (Brasil, 2015). Salmonellosis is considered a

al., 2017).

disease of major importance for human health by the

The aim of this work was to evaluate the antimicrobial

high risk of mortality and morbidity. It typically causes

effect of Thymus vulgaris

(thyme) and Origanum

gastroenteritis in humans and the infection is related to

vulgare

(oregano) EOs and its major compounds,

precarious hygiene conditions in poultry farms (Pui et

thymol and carvacrol, against Salmonella Enteritidis

al., 2011).

and Typhimurium serovars testing the adaptive response

Among all Salmonella enterica serotypes,

of their biofilms to sublethal concentrations of these

Typhimurium and Enteritidis are the two most frequently

substances and classifying their biofilm formation

observed in salmonellosis-recorded outbreaks

(Mor-

capacity.

Mur and Yuste, 2010). Salmonella enterica serotypes

can survive in a broad range of temperature (7 to 48 °C),

Material and methods

pH (4.3 to 9.3) and frequently associated to resistance

to common antibiotics and sanitizers (D’Aousts, 1997,

Essential oils and major compounds

McLaren et al., 2011). In addition, Salmonella spp. has

The essential oils of Origanum vulgare (oregano) and

great ability to form biofilms on surfaces and equipment

Thymus vulgaris (thyme) were acquired by Ferquima

of food industries (Steenackers et al., 2012, Fuente-

Indústria e Comercio Ltda (Vargem Grande Paulista, São

Núñez et al.,

2013). Biofilms are cells aggregates

Paulo, Brazil). Oregano EO composition was specified

in which increase significantly the prevalence of

by the supplier pointing carvacrol (71%), γ-terpinene

pathogenic strains in various food environments (Yaron

(4.5%), β-cariofilene (4.0%); p-cimene (3.5%), thymol

& Romling, 2014). Due to the difficulty to control the

(3.0%), while thyme EO contained thymol (47.3%),

development of biofilms formed by Salmonella spp. and

p-cimene (26.8%), γ-terpinene (6.0%), linalol (5.2%),

other microorganisms, the food industry is in need for

carvacrol

(3.1%), α-pinene

(2.2%), mircene (1.4%),

new products with active ingredients with antimicrobial

1.8-cineole (1.3%), borneol (0.9%), canfene (0.8%) and

efficiency and non-toxic to humans.

β-cariofilene (0.8%). In addition, the high-purity major

Essential oils

(EOs) and their components

compounds of thymol (99.5%) and carvacrol (98%)

are renowned to be effective against a wide range of

were purchased from Sigma-Aldrich^®.

microorganisms, including pathogenic bacteria (Burt,

Ciencia y Tecnología. 2019. 12(2):1-6

Interaction of Salmonella sp. and essential oils: bactericidal activity and

adaptation capacity

Microorganisms

0.5% Tween 80 were added in such concentrations (%)

Salmonella enterica subspecies enterica serovars

(v/v): 0.12; 0.25; 0.50; 1.00; 2.00; 2.50; 3.00; 3.50; 4.00;

Enteritidis S64 and Typhimurium S190 were donated by

4.50; 5.00 and 6.00. After 20 min, tested cultures were

the Laboratory of Enterobacteria (LABENT) at Oswaldo

washed and incubated with addition of TSB during 24h

Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil).

followed by TSA plating 37°C for 24h in order to obtain

Stock culture was stored in preservation culture medium

the MBCB. Tests were performed in triplicate and three

and reactivation occurred in Brain Heart Infusion

repetitions using negative (TSB with 0.5% Tween 80

broth (BHI) (HIMEDIA) incubation at 37°C for 24h.

and EO or major compounds) and positive (TSB with

Standard inoculum was obtained by growth curve and

0.5% Tween 80 and inoculum) control.

tests were carried out using 10⁸CFU/mL. All analysis

were performed in Laboratory of Food Microbiology of

Adaptation homologue of sessile cells to

Federal University of Lavras, Minas Gerais.

antimicrobials

Solution of TSB with 0.5% Tween 80 and

Formation and classification of biofilms

sublethal concentrations (1/4 MBCB) of thyme (0.06%)

Biofilms were formed by inoculation of 50 µL

or oregano (0.12%) EOs, carvacrol (0.12%) or thymol

aliquots of standard cultures into wells containing 150

(0.12%) were added into the wells and inoculated with

µL of TSB followed by incubation at 37°C for 48 hours.

50 µL of standard cultures. The microplates were sealed

Biofilm formation was determined by absorbance

and incubated at 37ºC for 48h. After this period, exposed

measures of crystal violet (0.1% w/v) added into each

cultures were removed, washed and tested against new

well at 600 nm in a microplate reader Anthos 2010

concentrations of EOs and major compounds: 2.00;

(Biochrom®), after wash/dry periods and addition of

2.50; 3.00; 3.50; 4.00; 4.50; 5.00 and 6.00 % (v/v). After

ethanol 95% (v/v) (Merritt et al., 2005). Classification

20 minutes, solutions were removed and washed. Then,

of biofilms followed Stepanović et al. (2000) proposal

TSB was added in order to incubate adapted biofilms

where “Dob” is optical density of biofilm and “Donc”

at 37°C for 24h, followed by TSA plating during 24h

is optical density of negative control: no biofilm former

at 37°C.

(Dob ≤ Donc), weak biofilm former (Donc < Dob ≤

2x Donc), moderate biofilm former (2x Donc < Dob

Results and discussion

≤ 4x Donc) strong biofilm former (4x Donc < Dob).

Final measures were obtained by arithmetic mean of

able

1 displays the minimal bactericidal

absorbance of eight replicates. Statistical analyses were

concentrations against planktonic

(MBC) and

performed using Kruskall-Wallis test and SPSS 19.0

sessile (MBCB) cells, and adapted biofilms (MBCB_A)

program (p < 0.05).

of EOs and major compounds. Susceptibility tests

revealed that MBC of EOs and major compounds varied

Minimal bactericidal concentration of essential oils

from 0.25 to 1.00 (% v/v) against planktonic cells of

and major compounds against planktonic and sessile

both Salmonella serovars (p < 0.05) and all MBCB were

cells

above 1.00% (v/v).

The minimum bactericidal concentration

EOs and major compounds tested against S.

against planktonic cells (MBC) and biofilms (MBCB)

Enteritidis and Typhimurium biofilms showed higher

of oregano and thyme EOs, thymol and carvacrol was

minimal bactericidal concentrations (MBCB) than those

determined using microdilution technique with

96-

obtained against planktonic cells (MBC) and significant

well polystyrene microplates according to CLSI-M100

differences between them were found (p<0.05). Several

(Clinical and Laboratory Standards Institute, 2019) with

factors are involved in this increased tolerance of cells

modifications. EOs and major compounds solutions

in biofilms to antimicrobial agents, including the matrix

were diluted in Tryptic soy broth (TSB) (HIMEDIA®),

of extrapolymeric substances (EPS) in which the cells

with addition of

0.5% Tween 80, in concentrations

are embedded limiting the diffusion of antimicrobials.

of (%): 0.03; 0.06; 0.12; 0.25; 0.50 and 1.00 (v/v).

Various substances are also found embedded in the EPS

Then, microplates with 10 µL of standard cultures and

reacting with these agents and reducing their efficiency

solutions were sealed and incubated at 37ºC for 24h,

(Bridier et al., 2011). In addition to EPS, it is known that

followed by Tryptic soy agar

(TSA) (HIMEDIA®)

when in biofilm, cells multiply more slowly, increasing

plating by microdrop technique using 10 µL of each

tolerance to antimicrobials, which is a major concern in

well and 37°C/24h incubation to obtain the MBC of

food safety standards (Srey et al,. 2013).

substances.

It was observable a significant difference

After biofilm formation, cultures were removed,

between carvacrol and thymol MBC and is well known

washed, and EOs and major compounds solutions with

in literature that inactivation of microbial

Ciencia y Tecnología. 2019. 12(2):1-6

Pereira et al., 2019

Table 1. Essential oils and major compounds minimal bactericidal concentrations (% v/v) against planktonic

and sessile cells and adapted biofilms of Salmonella serovars

S. Enteritidis

S. Typhimurium

Biocidal

% MBC

MBCB

MBC

MBCB

MBCB_A

Oregano EO

0.5 ±0.07

2.0 ±0.12

5.0 ±0.13

1.0 ± 0.13

3.0 ±0.29

6.0 ±0.25

Thyme EO

0.5 ±0.07

2.5 ±0.12

4.5 ±0.13

0.25 ±0.13

2.5 ±0.29

3.0 ±0.25

Thymol

0.5 ±0.07

3.0 ±0.12

6.0 ±0.13

0.5 ±0.13

5.0 ±0.29

6.0 ±0.25

Carvacrol

0.25 ±0.07

2.5 ±0.12

4.5 ±0.13

0.5 ±0.13

2.5 ±0.29

5.0 ±0.25

enzymes is also related to the presence of the

response to environmental stress that promote increased

hydroxyl group in monoterpenes (Bakkali et al., 2008).

and / or decreased gene expression leading to higher

The group can interact with the cell membrane causing

tolerance to this or other types of stress (e.g. thermal),

leakage of cellular components through membrane.

allowing this pathogen to survive in food processing

Thymol

(4-isopropyl-2-metylphenol) and carvacrol

environments. This adaptation can also lead to increased

(2-isopropyl-5-metylphenol) are isomers differing only

virulence and resistance to several antimicrobial

by the position of hydroxyl group. This difference in the

agents

(Spector & Kenyon, 2012). However, it is not

positions changes the reactivity of each compound since

completely understood in regard of the adaptability of

most of the reactions must occur by interaction with the

bacteria to essential oils and their compounds both in

hydroxyl group. It is possible that, in carvacrol tests, the

planktonic and sessile forms (Oloketuyi & Khan, 2017;

steric hindrance performed by methyl is much smaller

Rossi et al., 2017).

than propyl performs on thymol, due to its size and

Phenotypic changes caused in Salmonella by

number of present atoms. In methyl, there is only one

exposure to sublethal concentrations of oils and their

carbon and three hydrogen atoms to hinder interaction

compounds have been reported. The exposure to

with the hydroxyl group while in thymol, propyl offers

sub-lethal concentrations of thyme and oregano EO

three carbon and seven hydrogen atoms to that effect

and carvacrol, thymol, trans-2-hexenal and citral of

(Mastelic et al,. 2008, Hyldgaard et al., 2012, Meeran

Listeria monocytogenes, S. Enteritidis and Escherichia

et al., 2017).

coli induced a marked increase of some membrane

Both serotypes were capable to adapt to tested

associated fatty acids, particularly unsaturated fatty

antimicrobial compounds. The comparison between

acids, trans-isomers, and specific released free fatty

MBCB and MBCB_A shows significant differences

acids (Siroli et al. 2015). S. Enteritidis 86 (SE86) grown

(p<0.05) among biofilm adaptational conditions.

in sublethal concentrations of oregano EO and carvacrol

Much higher concentrations were required to inhibit S.

exhibited alteration in gene expression associated with

Enteritidis biofilm adapted in sublethal doses of EOs and

repair of cell damage caused by osmotic, oxidative, acid

major compounds solutions, suggesting an increased

stress and thermal shock (Cariri et al., 2019). However,

tolerance to antimicrobial agents when exposed to

the study was not evaluated if increased tolerance to

mild-stress conditions. Statistically significant values in

antimicrobial has occurred. In another study, Salmonella

different concentrations compared by Kruskall-Wallis

Senftenberg, isolated from an outbreak linked to the

test

(α = 0.05) revealed that thyme EO showed no

herb Ocimum basilicum L. (basil) adapted to linalool

significant difference between biofilm and adaptation to

with a minimal inhibitory concentration increasing of

sublethal concentrations to S. Typhimurium.

at least 8-fold and conferred heterologous adaptation

Table 2 displays the optical densities of biofilms

to the antibiotics trimethoprim, sulfamethoxazole,

exposed to sublethal concentrations of EOs and major

piperacillin, chloramphenicol and tetracycline (Kalily

compounds in order to classify them. According

et al., 2017)isolated from an outbreak linked to the herb

to Stepanović et al.

(2000) classification, it can be

Ocimum basilicum L. (basil. These information shows

observed that both strains, even after culturing in the

that exposure to inadequate concentrations of EOs or

presence of sublethal concentrations of antimicrobials,

major compounds can also increase bacterial tolerance

remain considered as “strongly biofilm forming”.

to other stressors in the processing environment, leading

The response to environmental stress of

to bacterial persistence in industry and food.

microorganisms is well-known by factors such as

For biofilm cells, similar results to this study

temperature, pH, osmolality, antibiotics and sanitizers.

were obtained by Zou et al. (2012). The biofilm and

In addition, Salmonella sp. activates regulators in

dispersed cells of S. Typhimurium showed higher

Ciencia y Tecnología. 2019. 12(2):1-6

Interaction of Salmonella sp. and essential oils: bactericidal activity and

adaptation capacity

Table 2. Biofilm formation capacity of the two serotypes of Salmonella grown in presence of sublethal

concentration of essential oils and major components

Sublethal

Conc.

Biofilm

Serotype

DOA

DOCN

stress

(%)

class.

Enteritidis

control

0.0

0.27+0.02

0.06+0.002

FFB

Oregano EO

0.25

0.29+0.02

0.06+0.002

FFB

Thyme EO

0.12

0.27+0.02

0.06+0.002

FFB

thymol

0.12

0.28+0.02

0.06+0.002

FFB

carvacrol

0.06

0.30+0.03

0.06+0.002

FFB

Typhimurium

control

0.0

0.29+0.02

0.06+0.002

FFB

Oregano EO

0.12

0.30+0.02

0.06+0.002

FFB

Thyme EO

0.06

0.28+0.03

0.06+0.002

FFB

thymol

0.12

0.29+0.03

0.06+0.002

FFB

carvacrol

0.12

0.34+0.02

0.06+0.002

FFB

Non biofilm forming - NF (Doa < Docn), Weakly biofilm forming - FF, (Docn < Doa ≤ 2 x Docn), moderately

biofilm forming - MF MF (2 x Docn < Doa ≤ 4 x Docn), and strongly biofilm forming- FFB (4 x Docn < Doa).

Where Doa is biofilm optical density and Docn, negative growth control optical density.

resistance to antimicrobials, allyl isothiocyanate,

2015. Projeções do Agronegócio: Brasil 2014/2015 a

thymol, eugenol and polyphenol, than the planktonic

2024/2025, Assessoria de Gestão Estratégica. Brasília,

cells after cultivation in the presence of sublethal

p. 100.

concentrations of the compounds. In this regard, the

Bridier, A., Briandet, R., Thomas, V., and Dubois-Brissonnet,

potential use of essential oils and its major compounds as

F. 2011. Resistance of bacterial biofilms to disinfectants:

alternative sanitizers raises awareness to concentration

a review. Biofouling 27(9): 1017-1032.

adjustment in order to avoid sublethal exposure leading

Burt, S. 2004. Essential oils: their antibacterial properties and

to microbial adaptation and persistence on common

potential applications in foods -a review. Int. J. Food

food industry surfaces.

Microbiol. 94(3): 223-53.

Cariri, M.L., de Melo, A.N.F., Mizzi, L., Ritter, A.C., Tondo,

Conclusions

E., de Souza, E.L., Valdramidis, V., and Magnani, M.

2019. Quantitative assessment of tolerance response to

arvacrol and thyme EO were more efficient against

stress after exposure to oregano and rosemary essential

planktonic cells of S. Enteritidis and Typhimurium,

oils, carvacrol and 1,8-cineole in Salmonella Enteritidis

respectively, while oregano EO showed better

86 and its isogenic deletion mutants ∆dps, ∆rpoS and

performance against S. Enteritidis biofilms. Adaptation

∆ompR. Food Res. Int. 122: 679-687.

was observed on all treatments and both serovars were

CDC. 2018. CDC estimates of foodborne illness in the United

classified as strong biofilm formers, proving the high

States. Centers for Disease Control and Prevention.

risk of resistance development of Salmonella sp. to

CLSI.

2019. Performance Standards for Antimicrobial

sublethal doses of EOs and major compounds.

Susceptibility Testing

(29th Ed.) CLSI supplement

M-100. Clinical and Laboratory Standards Institute.

Acknowledgement

D’Aousts J. 1997. Salmonella species. Food Microbiology

- Fundamentals and Frontiers. Microbiological

Authors thank FAPEMIG, CAPES, CNPq and

Specifications of Food Pathogens. A S M Press: 129-158,

UFLA for the financial support and scholarship.

Washinghton.

Erickson, M.C., and Doyle, M.P. 2017. The challenges of

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