Biofilm Formation and Inhibitory Effect of Essential Oils in Multidrug-Resistant Pseudomonas aeruginosa

Research Article

J Bacteriol Mycol. 2021; 8(7): 1192.

Biofilm Formation and Inhibitory Effect of Essential Oils in Multidrug-Resistant Pseudomonas aeruginosa

Benie CKD1,2*, Attien Paul Y3, Toe E5, Tra Bi YC4, Atobla K1 and Dadié A2

1University of Félix Houphouët Boigny, Abidjan, Côte d’Ivoire, Laboratory of Biotechnology, Agriculture and valorization of Biological Resources, Department of Biosciences (LBAVR), Abidjan, Côte d’Ivoire

2Department of Bacteriology and Virology, Institut Pasteur of Côte d’Ivoire (IPCI), Abidjan, Côte d’Ivoire

3University of Jean Lorougnon GUEDE, Department of Biochemistry-Microbiology, Agrovalorisation Laboratory, UFR Agroforestry, Daloa, Côte d’Ivoire

4University of Nangui-Abrogoua, Institute for Research on New Energies (IREN), Biomass-Energy Laboratory, Abidjan, Côte d’Ivoire

5University of Peleforo Gon Coulibaly, UFR of Biological Sciences, Department of Biochemistry-Genetic, Korhogo, Côte d’Ivoire

*Corresponding author: Comoé Koffi Donatien Benie, Department of Bacteriology and Virology, Institut Pasteur of Côte d’Ivoire (IPCI), 01 BP 490 Abidjan 01, Côte d’Ivoire

Received: November 23, 2021; Accepted: December 16, 2021; Published: December 23, 2021


Biofilm formation is a major concern in medicine, as well as in the food industry. Some infections related to bacterial biofilms such as Pseudomonas aeruginosa are a real public health challenge. This study aimed to show the activity of essential oils on P. aeruginosa biofilm formation. Pseudomonas aeruginosa isolates consisting of animal (100), environmental (20) and clinical strains (42) were identified by PCR and sequenced. Biofilm formation was assessed by the microplate method. Antibiotic susceptibility testing was performed by using Kirby–Bauer disc-diffusion method. The average biofilm formation percentages vary from 1.2 to 2.1 in 24h and from 2.3 to 3.2 in 48h. The median biofilm formation value was higher in environmental strains (1.4 ± 0.2) than in clinical (1.2 ± 0.4) and animal (1.1 ± 0.4). In decreasing order of importance, the essential oils of Mentha piperita (90 ± 5.12% at 100%), Eucalyptus globulus (34 ± 0.08% at 100%) and Lavandula angustifolia (12 ± 0.71% to 100%) showed distinct inhibitory effects on biofilm formation (p <0.05). The rate of resistance of P. aeruginosa to the antibiotics imipenem, ceftazidime, cefepime, fosfomycin and colistin varied from 12.7% to 48.4% in the biofilm status while that of plankton cells ranged from 2.3% to 15.0%. Moreover, resistance to ticarcillin, ticarcillin clavulanic acid, piperacillin and ciprofloxacin ranged from 56.4% to 83.1% in biofilm and from 29.4% to 51.4% in planktonic cells. In general, biofilm is more resistant to different antibiotics than free cells. The tested essential oils could be an effective natural control against microbial biofilm formation.

Keywords: P. aeruginosa; Biofilm; Essential oils; Antibiotics resistance


Biofilms are an aggregate of microorganisms frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS) [1]. These microorganisms adhere to each other and/ or to a surface, protecting them from environmental stresses. Biofilms are the source of persistent infections caused by many pathogenic microbes [2]. Moreover, bacterial biofilms can survive antibiotics due to impaired antibiotic diffusion, antibiotic efflux, expression of biofilm-specific genetic mechanisms, selection of resistant mutants or nutrient and oxygen limitation [3-5]. Over the last few decades, the study of microbial biofilms has been gaining interest among the scientific community.

Various studies indicate that the lifestyle of biofilms, their structure and their composition lead to an increase in resistance to antimicrobial agents [6]. Thus, biofilm bacteria resist the host’s immune response and may be 10 to 1000 times more resistant to antimicrobial agents than planktonic bacterial cells [7]. The biofilm formation can have a negative impact in different sectors within society; namely in agriculture, food industries, veterinary and human health, as it could lead to substantial economic losses [2].

In the food industry, biofilms are the source of many problems, in terms of hygiene and deterioration of the organoleptic qualities of food products [8,9]. In dairy industries, for example, bacterial species can remain on certain parts of the equipment, which promotes their development in the form of biofilms and thus contaminate the finished product [10,11]. Likewise, the presence of biofilm on surfaces found on the farm, at the slaughterhouse in water pipes or at the processing plant will affect the effectiveness of the disinfection protocol [9].

In medicine, biofilms are of particular importance because they are implicated in a wide range of infections in humans. In addition, nearly 80% of human bacterial infections are biofilm-associated. Infections resulting from biofilms pose real public health problems [8,9].

Among the germs involved in the formation of biofilms, Pseudomonas aeruginosa is increasingly mentioned for each health insecurity factor, whether in terms of food poisoning or nosocomial infections [6,12]. Pseudomonas aeruginosa is an opportunistic pathogen frequently implicated in biofilm-related infections [1].

This microorganism has several virulence mechanisms that promote its pathogenesis, in particular the production of biofilm [13]. Although P. aeruginosa has been widely used as a study model in biofilm, to date there are no guidelines for the treatment of biofilm infections caused by P. aeruginosa [14].

Therefore, P. aeruginosa biofilm infections present a pharmacological and medical challenge. Anti-infective agents that selectively interrupt virulence pathways to prevent or cure infection are less likely to promote the emergence of resistance [13]. Among these anti-infective agents, certain natural essential oils show promising therapeutic properties to fight against emerging resistance [15].

This study aimed to determine the inhibition of biofilm formation by essential oils in P. aeruginosa isolates obtained from various origins.

Materials and Methods

Isolation and identification of P. aeruginosa

A total of 162 strains of P. aeruginosa isolated from animal infections (100), clinical (42) and environmental (20) settings were studied. The isolates were identified by classical microbiology and biochemical characters using API 20NE (bioMérieux, Marcy l’Etoile, France). The molecular identification of P. aeruginosa strains using Polymerase Chain Reaction (PCR) was conducted using the 16S gene. The reference strain P. aeruginosa PA14 was used as quality control.

Extraction and purification of the genomic DNA of P. aeruginosa

Pseudomonas aeruginosa strains were harvested from an overnight broth culture. Genomic DNA was extracted and purified according to the method described by Amutha and Kokila [16]. After extraction, DNA was diluted and stored at -20°C to serve as a DNA template for polymerase chain reactions (PCR).

Amplification of the 16S rDNA Gene for P. aeruginosa detection

The 50μL reaction mixture consisted of 38μL of sterile Milli-Q water (milli-QTM, Millipore Corporation, Foster City, CA, USA) ; 5μL of 10 X concentration loading buffer; 1μL of Mgcl2, 25mM (Promega Corporation, Madison, WI, USA) ; 1μL of d’NTPs, 10mM; 1μL of each primer 27F and 1492R, 10mM (TranS, AP111 5U, Macau City, China); 0.5μL of BSA, 20mg/mL and 0.5μL of Easy Tag® DNA polymerase with a final concentration of 1.5U (TranS, AP111 5U, Macau City, China) and 2μL of the DNA matrix.

Sterile Milli-Q water and the reference strain P. aeruginosa PA14 were used as negative control and positive control, respectively, for each PCR reaction run.

Amplification of the 16S rDNA gene was performed according to the method described by Amutha and Kokila [16] using primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (3'-TACGGYTACCTTGTTACGACTT-5'). The amplification program included an initial denaturation of 5min at 94°C followed by 35 cycles of denaturation (94°C for 30s), annealing (55°C for 40s) and extension (72°C for 30s), with a single final extension of 10min at 72°C. The samples were stored at 4°C until the Thermocycler was stopped.

Sequencing of P. aeruginosa strains

After purification of the PCR products using a commercial kit (EZ-10Spin Column PCR Products Purification Kit, Foster City, CA, USA), these products were sequenced with primers 27F and 1492R in an automated DNA sequencer 310 (Applied Biosystem, Foster City, CA, USA). The sequences obtained were analyzed in the NCBI database using BLAST for strains confirmation, as described in our previous publications [17].

Biofilm formation by the microplate method

Biofilm formation: Biofilm-forming ability of P. aeruginosa was measured in 96-well polystyrene microplates by using the method described by [18,19] with some adaptations. Different suspensions with a final volume of 1.2mL consisting of LB medium diluted 1:10 and Casamino acid with a final concentration of 0.5% are produced in different Eppendorf tubes for each strain of P. aeruginosa. The overnight cultures were adjusted to a 0.5 McFarland (108CFU/ mL). Each strain was tested in 5 replicates after inoculation of a standardized culture in Tryptic Soy Broth (TSB) added with 0.2% of glucose. Negative control wells contained noninoculated Tryptic Soy Broth (TSB) with glucose. Samples (200μL) were dispensed into wells, and the microplates were covered and incubated aerobically without agitation at 37°C for 24 and 48 h.

Biofilm assay: In detail, after 24h and 48h, the bacterial suspension was aspirated, and each well was washed three times with phosphate-buffered saline (PBS) (Sigma). The plates were dried at room temperature and stained with 200μL 1% (v/v) of crystal violet solution used for Gram staining (Merck Millipore) for 30min. The excess crystal violet is removed by 3 to 10 successive manual washings of the plates with milliQ water and dried at room temperature. After that, the biofilm was fixed with 300μL of ethanol (95%) for 15min, and was later removed. The dye bound to the adherent cells was resolubilized with 160μL of 33% (v/v) glacial acetic acid (Sigma) per well. The dye concentration or absorbance (OD) is measured with a spectrophotometer at 595nm wavelength with the CytationTM 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Innovation, CA, USA), linked to the analysis software Data Gen 5 v. 2.04 TM (BioTek Instruments, Innovation, Foster City, CA, USA).

Finally, the strains were grouped into: OD595 <0.1, nonproducers (NP) ; OD595 = 0.1-1.0, weak producers (WP); OD595 = 1.1-3.0, moderate producers (MP); and OD595 >3.0, strong producers (SP).

Determination of the antibiofilm effect of essential oils

The antibiofilm activity of three essential oils (Lavandula angustifolia (true lavender), Mentha piperita (peppermint) and Eucalyptus globulus (eucalyptus)) was evaluated. This antibiofilm activity was compared with that of furanone (reference antibiofilm molecule) according to different doses of essential oils and the biofilm formation time. Thus, furanone and essential oils were introduced at a dose of 100%, 50% and 25% after formation of the biofilm in 24h.

The percentage inhibition of biofilm formation (PI) is determined according to the formula below [20]. On the one hand, it was a question of comparing the optical densities (OD) of the white wells without bacteria (B) with the OD of the control wells without antibiofilm molecule (C). On the other hand, to compare the optical densities (OD) of the same white wells without bacteria (B) with the ODs of the wells containing the antibiofilm molecules (S), we used the following equation.

Antimicrobial susceptibility of planktonic cell and biofilm

Antibiotic susceptibility testing was performed by using the Kirby–Bauer disc-diffusion method according to the CA-SFM/ EUCAST recommendations [21]. The antibiotics tested and their sensi-disk concentrations are mentioned in Table 1. The standard reference strain of P. aeruginosa ATCC 27,853 was used as a quality control. The method remains the same for the determination of biofilms resistance, but the inoculum of approximately 106CFU/mL is obtained from the 24h biofilm taken from a well of microplates.