Biocides, which include antiseptics and disinfectants, are widely used in hospitals and other healthcare settings to disinfect medical devices and minimize ambient bioburden. Disinfectants, in particular, serve an important role in infection control and the prevention of infectious pathogen transmission in the hospital setting. [1] Bacteria exposed to such biocides at subinhibitory concentrations changed the microbial population structure and increased tolerance to biocides, as well as resistance to the most crucial antimicrobials utilized in the elimination of resistant Gram-negative microbes. [2] Actually, P. aeruginosa strains have demonstrated cross-resistance to biocides and antibiotics with likely identical mechanisms, making bacterial removal from hospital environments a challenge. [3]The RND-type efflux pump system consists of three distinct components. These components are called proton motive force (mexB), outer membrane proteins (oprM), and periplasmic membrane fusion proteins (mexA) Certain substrates or pressures cause overexpression of the MexAB-OprM efflux pump (primarily by mutations in regulatory genes like mexR, nalC, or nalD), leading to extrusion biocides. [2] Small multidrug resistance (SMR) proteins are found in the inner layer of the cytoplasmic membrane and are classified into three categories: SUG, SMP, and PSMR. They produce resistance to biocides and a variety of antibiotics. The genes qacE and qacEΔ1 belong to the SMP subgroup, and the gene is included in integron class 1, which permits transfer and integration into the chromosome and can be found on the plasmids of many drug-resistant bacteria, causing Pseudomonas aeruginosa to be resistant to hospital disinfectants. The qacEΔ1 gene represents a mutation of the qacE gene. The SMR family consists of proton-dependent efflux pumps. [4, 5] Although the qac genes were named after one of their primary substrates quaternary ammonium compounds (QACs), their activity includes a considerably wider range. More than 30 lipophilic cationic compounds from at least 12 distinct chemical groups are identified as targets of qac-mediated resistance. [6] Organic acids have antibacterial characteristics because of their capacity to permeate the cell membrane, which changes the proton and related anion concentrations in the cytoplasm. Consequently, purine bases and crucial enzymes are extremely damaged, and bacterial viability declines. [7]

Examples of an eco-friendlier antimicrobial alternative is the organic acid acetic acid, lactic acid, citric acid, ascorbic acid, sorbic acid. Organic acids have been used for centuries to inhibit microbial growth and possess the other valuable property of being non-toxic to humans. [8]

Therefore, the aim of this study to investigate efflux pumps as mechanisms of biocides resistance in P. aeruginosa phenotypically and genotypically, and evaluation the antimicrobial activity of organic acids to use as safer and eco-friendly antiseptic and disinfectant alternative agents.

Bacterial isolates:

Twenty P. aeruginosa isolates were used in  the current study which previously isolated and diagnosed from burn infections in Department of Biology/ College of Science/ University of Mosul , Mosul city/Iraq.

Biocides:

All 12 biocides used in this study were prepared by Iraqi Ministry of Health / National Health Factory, except Surgical spirit was prepared by Areej Baghdad Factory. The biocides included: POVIDONE-IODINE 10 %, POVIDONE-SCRUB 7.5%, SURGICAL SOAP, SURFACE DISINFECTANT, SPRAY DISINFECTANT, SAFE SPRAY , CLIMTE DISINFECTANT , ENZYME PLUS , OPA PLUS,  GLUTARA 2% DISINFECTANT,  HANS AND SKIN ANTISWPTIC,  SURGICAL SPIRIT. These biocides vary in their chemical compositions.

Determination of antimicrobial activity of  biocides using agar disk diffusion method:

To conduct this test, bacteria were suspended in sterile saline solution to a final concentration of 0.5 McFarland (1.5 x 10⁸ CFU/ml) and distributed evenly across Muller-Hinton agar with sterile swabs. Meanwhile, sterile filter paper disks (Ø = 6 mm) were soaked with 10 µl of each biocide, permitted to dry for 1 minute, and then transferred to the surface of each inoculation plate. After 1 hour at room temperature, the plates were incubated at 36 °C ± 1 °C for 24 hours. The efficiency of each disinfectant was assessed by measuring the diameter of the inhibition zone around the disks. Microorganisms were considered sensitive when the diameter of the inhibition zone was greater than 8 mm. [9]

Phenotypic estimation for efflux pump in P. aeruginosa isolates by ethidium bromide cartwheel method (EtBrCw) method:

Tryptone Soya Agar (TSA) plates were newly prepared on the same day of the experiment, and they contained varied ethidium bromide concentrations (0.0 to 3 mg/L). The plates were kept hidden from light. The isolates were cultured overnight in Mueller-Hinton broth and fixed to a turbidity of 0.5 McFarland standard. To make a cartwheel pattern, TSA plates were split into eight to ten radial sections. Bacterial inocula were surface inoculated on EtBr-TSA plates and incubated at 37ºC for 16 h. TSA plates were examined under a UV transilluminator to detect the lowest concentration of EtBr that triggered the fluorescence of the bacterial mass. Plates were photographed. Isolates that produced fluorescence at concentrations greater than 2 μg/ml EtBr were considered to have positive efflux activity, while fluorescence at concentrations less than or equal to 1 μg/ml EtBr demonstrated negative efflux activity. On the other hand, the existence of fluorescence at concentrations equivalent to 2 μg/ml EtBr demonstrated intermediate efflux activity. [10]

Detection of mexB, qacE Δ1 and qacE genes by Polymerase chain reaction (PCR)

We extracted genomic DNA from our bacteria using Geneaid's PrestoTM Mini gDNA Bacteria Kit Protocol. The manufacturer recommends the following criteria: The purity and concentration of genomic DNA were tested before being stored at -20 °C for future use. [11] The PCR reaction was done in a total volume of 25 μl, involving of 12.5μl  GoTaq G2 Green Master Mix supported by Promega., 1µl of each forward and reverse primers as shown in Table (1), 3µl DNA template, and completed by 7.5μl nuclease-free water. The optimum condition used according [12, 3, 5] Then on 2% Gel agarose the PCR products and ladder of 100 bp were transferred to the well for separated by electrophoresis.

Table 1: Primer used to detect efflux pump genes

Organic acids Preparation :

Five organic acids were used in the current study , acetic acid and  lactic acid prepared using DMSO as a solvent  [13] Citric acid, Ascorbic acid, and Sorbic acid were prepared using sterilized distilled water as a solvent . [14]

Determination of antimicrobial activity of organic acids by well diffusion method: 

Bacterial suspensions have been achieved by comparing to a standard tube (McFarland number 0.5) with 1.5×10⁸ CFU/mL. Bacterial suspension was spreading over Mueller-Hinton agar plates. Cotton swabs were placed into a screw tube that includes bacterial suspension and distributed over the surface of the plates, which were then allowed to dry for approximately five to fifteen minutes at room temperature. Numerous wells (8 mm in diameter) were prepared into the medium of agar using a sterile cork borer and then organic acid in 24μl volumes containing 1.2%, 0.73%, 0.24%, 0.24%, and 0.24% were taken from acetic acid, lactic acid, citric acid, ascorbic acid, and sorbic acid, respectively were transferred into the wells, then left in incubator at 37°C for 24 hrs and the inhibition zone were measured in mm.[15, 16]

Our 11 bacterial isolates subjected to susceptibility test revealed that Pvidone-iodine10% and Climate disinfectant shown resistance at percentage (81.82%), while Povidone-scrub7.5% and spray disinfectant at a percentage (90.91%). the best biocides is Surgical soap (0%) resistance, the rest of biocides revealed (100%) resistance as shown in Table (2)

Table 2: Percentage of susceptibility pattern of P. aeruginosa isolates to the studied biocides

 (Inhibition zone by mm)

Our phenotypic estimation shown that all 20 (100%) isolates revealed efflux pump activity, 14 (70%) positive and 6 (30%) intermediate efflux activity as shown in Table (3) and Figure (1) 

Table 3: Results of Efflux pump production in P. aeruginosa isolates by EtBrCw method.

Figure 1: Phenotypic estimation of Efflux pump activity in P. aeruginosa isolates by EtBrCw method. Isolates (1, 2, 3, 6, 7, 8) which produced fluorescence at concentration greater than 2µg/ml ethidium bromide were represented positive efflux activities, while isolates (4, 5) which  fluorescence at concentration equal 2µg/ml were represented intermediate efflux activities.

Our results shown that 19 (95%) of isolates have mexB gene, while the qacE Δ1 gene was observed in all isolates (100%) and none of the isolates (0%) harboured the qacE gene as shown in Figures (2), (3), (4)

Figure 2: Percentage of the existence of efflux pumps genes in all 20 isolates.

Figure 3: Gel electrophoresis shown PCR products related to the mexB gene (244bp) .

Figure 4: Gel electrophoresis revealed PCR products related to the qacE Δ1 gene (285bp).

In our current study, two isolates of P. aeruginosa ( No. 14 & 19 ) were subjected to testing against some organic acid, to reveal the possibility of using them as therapeutic alternatives in cases of disinfection and antiseptics against drug-resistant bacteria , the results revealed that acetic acid and lactic acid have effective antimicrobial activity at a concentration 1.2% and 0.73% respectively, while citric acid, ascorbic acid and sorbic acid revealed antimicrobial activity against one isolate at concentration 0.24% as shown in Table (4) and Figure (5).

Table 4 : Susceptibility test of P. aeruginosa isolates to studied organic acids ( Inhibition zone by mm) .

S= Sensitive        R=Resistant

Figure 5: Susceptibility test of P. aeruginosa isolates to studied organic acids.

Since antibiotics and biocides have several target regions in bacteria, the most prevalent  mechanisms for cross-resistance through non-specific activities such as efflux pumps and changes in cell wall characteristics (e.g., permeability reduction due to porin down regulation). [17] Based on that, in our study selected 11 bacterial isolates which shown higher antimicrobial resistance and evaluated through in vitro study against 12 various biocides on Muller hinton agar which includes disinfectants and antiseptics as shown in Table (2). Our results shown that the bacterial isolate revealed high resistance to Povidone-Iodine 10 % and climate disinfectant at percentage 81.82%, while Povidone-Scrub 7.5% and Spray disinfectant, at percent 90.91%. The best antiseptic used which shown highest effectiveness and none of the isolates (0%) shown resistance to it was Surgical soap, the rest of biocides revealed (100%) bacterial resistance as shown in Table (2). Surgical soap which contains Chlorhexidine gluconate 4%, Chlorhexidine digluconate (CHG), a cationic broad-spectrum antiseptic, disrupts bacterial membranes. As a result, the bacterial membrane has a negative charge that is stabilized by divalent cations (Mg2+ and Ca2+), which can be disrupted by chlorhexidine via (i) binding directly to the negative charge of the lipopolysaccharide (LPS) and (ii) displaced of divalent cations (LPS-stabilizing factor) which results in intracellular bacterial component precipitation. The usage of chlorhexidine corresponds with a reduced chlorhexidine susceptibility and a cross-resistance to antibiotics, particularly colistin, in P. aeruginosa. [18] Quaternry ammonium compounds (Qacs) are the main component of most biocides used so their resistance mechanisms are efflux pump as seen here. our bacterial isolates showed high resistance to biocide compared to other studies. This may be due to misuse or overuse of biocide and also the absence of execution of the instructions of the manufacturer, which results in subihibitory concentrations being applied, which results in bacteria becoming adapted to these biocides. Also, there are differences in geographical areas and bacterial strains, which have many resistance factors.     

Our study revealed that out of Twenty isolates, 14 resistant isolates (70%) were EtBrCw-positive and 6 isolates (30%) were EtBrCw-intermediate as shown in Table (3) and Figure (1) which exhibited the method. This inducates that all isolates (100%) have efflux activity. This approach identifies overexpressed efflux systems that contribute to the MDR phenotype in bacteria. It uses EtBr as the pump substrate to verify the existence of an overexpressed efflux system in comparison to the intrinsic efflux activity of the identifying wild-type strain. [19] It is an easy, instrument-free approach that uses agar plates with increasing concentrations of ethidium bromide and is readily applicable to a standard clinical microbiology laboratory. [20] also the advantage of this method  could be uses many isolates on one agar plates. Our results are higher than the study in Nigeria, were shown only (59%)  of the isolates phenotypically revealed efflux pump activity. [21]

MexAB-OprM was the first RND pump observed to target several antibiotic classes and common wild-type bacteria, resulting in multidrug resistance. It is continuously expressed and enables the pumping of numerous antibiotics, dyes, biocides, and organic solvents out of the cell. Their antibiotics substrates include chloramphenicol, β-lactams, carbapenems, fluoroquinolones, tetracyclines, trimethoprim, and sulphonamides. [22, 23] The qac genes, particularly qacE and qacEΔ1, have been linked to resistance to biocides, particularly quaternary ammonium compounds. Biocides resistance has already been described in biocides and microbial resistance and is found in both clinical and environmental strains. [24]

Our results shown that 19 (95%) of isolates have mexB genes, while the qacE Δ1 gene was observed in all isolates (100%) and none of the isolates (0%) harboured the qacE gene as shown in Figures (2), (3), (4). Similar to study in Baghdad, Iraq was detected mexB efflux pumps gene in all isolates understudy, while  study in Egypt was detected Mex B in 18 isolates (51.4%). [12, 25]

Our results shown the qacEΔ1 gene higher than the study in Baghdad, Iraq (80%) qacEΔ1 in P.aeruginosa, in Brazil the qacEΔ1 gene was detected 88%  of the multiresistant isolates carried qacEΔ1 gene, in Turkey the qacE∆1 genes were determined in 50.7% (n=38), but the qacE gene in our result similar the same study in Baghdad, Iraq where, no any isolate carried qacE gene and disagreement with the same study in Turkey where, the qacE detected in 65.3% (n=49) and discovered the frequency of qacE and qacE∆1 genes in carbapenem resistant isolates was found to be significantly higher than in susceptible ones. [26, 27, 28] Also disagreement to study in Iran where Genomic detection of qacE and qacΔE1, showed that 111 (92.5%), and 21 (17.5%), out of 120 P. aeruginosa isolates harbor the qacE, qacΔE1. [4] These variation in the existence of the genes may be due to the differences in isolates of bacteria, types of hospitals, procedures of administration of antibiotics and application of biocides in the hospitals, and may be these genes are plasmid encoded. 

One of the worldwide health challenges is the rising prevalence of biocide resistance and the possibility of cross-resistance to certain antibiotics, which can lead to hospital-acquired infections and inefficient therapies. [29] the results of organic acids revealed that acetic acid and lactic acid have effective antimicrobial activity at a concentration 1.2% and 0.73% respectively, while citric acid, ascorbic acid and sorbic acid revealed antimicrobial activity against one isolate at concentration 0.24% as shown in Table (4) and Figure (5). So these concentration of acetic acid and lactic acid can be used as antiseptics and disinfectants and can be raised the concentration of others and testing antimicrobial activity. Energy competition, bacterial outer membrane permeabilization, increasing intracellular osmotic pressure, and suppression of biomolecule synthesis are some of the molecular mechanisms behind acid inhibition. Undissociated organic acids are lipid-soluble and can penetrate the cell through free diffusion. They dissociate to produce acid ions (ROO−) and protons (H+). The buildup of H+ in the cytosol contributes to cytoplasmic acidification, leading to rupture of cytoplasmic membrane, leakage of precursor and growth factor which result inhibiting metabolism and growth, acid ion block DNA synthesis, and denaturation of the important enzymes. [30]

Other study in India find out that 1% of acetic acid used as topical antiseptics on the burns to prevent and treat infections that have not responded to traditional therapy, including oral or injectable antibiotics and local wound care with hydrogen peroxide and betadine and the period of treatment required to eradicate MDR P. aeruginosa from the chronic burns was 4.5 days. [31] acetic acid at a concentration of 5% reveal an antibacterial and antifungal activity when tested on surfaces showed a complete reduction for P. aeruginosa, E. coli, S. aureus, E. hirae, A. brasiliensis and C. albicans. [32] Study found that acetic acid (vinegar) effectively  kills M. tuberculosis after 30 min of exposure to a 6% acetic acid solution. [33] A high concentration of AA (>0.156% v/v) could be used to sterilize biofilm-prone surgical instruments, disinfect hospitals, and treat external wounds. A low concentration of AA (0.00975-0.039% v/v) can be used as an anti-virulence agent for adjuvant treatment of colistin-resistant P. aeruginosa, pH of AA below 4.76 was effective against P. aeruginosa biofilms. [34] These differences in concentration may be due to the nature of the cell wall characteristics of bacteria and the nature of surfaces dealing with it biotics or abiotics because environmental condition often effect on the effective concentration. lactic acid (LA) is a naturally occurring weak organic acid that is considered eco-friendly and recognized as safe. LA is used as a spray to decontaminate areas. [35]

The prevalence of efflux pump system and its relation to biocides resistance, so research for novel inhibitors was recommended, and can be used organic acid as safer, eco-friendly antiseptics and disinfectants to eradicate biocides resistance P. aerugiosa, elaborate detection of the minimum inhibitory concentration and Synergistic study between these organic acid and in vivo study were recommended.

1. Vijayakumar, Rajendran, and Tim Sandle. "A review on biocide reduced susceptibility due to plasmid‐borne antiseptic‐resistant genes—special notes on pharmaceutical environmental isolates." Journal of Applied Microbiology 126.4 (2019): 1011-1022.

2. Amsalu, Anteneh, et al. "Efflux pump-driven antibiotic and biocide cross-resistance in Pseudomonas aeruginosa isolated from different ecological niches: a case study in the development of multidrug resistance in environmental hotspots." Microorganisms 8.11 (2020): 1647.

3. Namaki, Malek, et al. "Prevalence of resistance genes to biocides in antibiotic-resistant Pseudomonas aeruginosa clinical isolates." Molecular Biology Reports 49.3 (2022): 2149-2155.

4. Bakht, Mehdi, et al. "Phenotype and genetic determination of resistance to common disinfectants among biofilm-producing and non-producing Pseudomonas aeruginosa strains from clinical specimens in Iran." BMC microbiology 22.1 (2022): 124.

5. Al-Azzawi, Shahla Najim Abed, and Rana Mujahid Abdullah. "DNA sequences of qacE∆ 1 gene in Pseudomonas aeruginosa isolated from wounds and burns infections." Kirkuk Journal of Science 14.3 (2019): 10-20.

6. Mahzounieh, Mohammadreza, et al. "Detection of antiseptic-resistance genes in Pseudomonas and Acinetobacter spp. isolated from burn patients." Jundishapur journal of natural pharmaceutical products 9.2 (2014).

7. Gómez-García, Manuel, et al. "Antimicrobial activity of a selection of organic acids, their salts and essential oils against swine enteropathogenic bacteria." Porcine health management 5 (2019): 1-8.

8. Zhitnitsky, Daniel, Jessica Rose, and Oded Lewinson. "The highly synergistic, broad spectrum, antibacterial activity of organic acids and transition metals." Scientific Reports 7.1 (2017): 44554.

9. Montagna, Maria Teresa, et al. "Study on the in vitro activity of five disinfectants against nosocomial bacteria." International journal of environmental research and public health 16.11 (2019): 1895.

10. Baiomy, Amr A., Ghada H. Shaker, and Hisham A. Abbas. "Sensitizing multi drug resistant Staphylococcus aureus isolated from surgical site infections to antimicrobials by efflux pump inhibitors." African Health Sciences 20.4 (2020): 1632.

11. Salih, Mohammad M., and Rawi Amera M. Al. "Morphological and molecular identification of a new local strain of Eikenella corrodens isolated from chronic periodontitis." Medicinski časopis 57.3 (2023): 91-97.

12. Yaseen, Najlaa Nabhan, and Dalia Azher Ahmed. "Detection of mexB multidrug efflux gene in some local isolates of Pseudomonas aeruginosa." Iraqi Journal of Science (2023): 111-118.

13. Al-Muslih, R. M. ; Al-Kaisey, M.Tand Al-Obaidi, A. K. ‘’Pathogenic Bacterial Isolated From Fresh veal”. Iraqi J. Agric., (Special Issue) (2009).14 (4):164-174

14. Al-Rousan, Walid M., et al. "Use of acetic and citric acids to inhibit Escherichia coli O157: H7, Salmonella Typhimurium and Staphylococcus aureus in tabbouleh salad." Food microbiology 73 (2018): 61-66.

15. Al-Noamy, Najlaa AF. "Detection of the Inhibitory Effect of the Leaves, Seed and Fruits of Cydonia oblonga on some Gram Positive and Negative Bacteria." Rafidain Journal of Science 29.1 (2020): 10-19.

16. Al-Ahbabi, H. H., et al. "Antimicrobial activity of Aloe vera extract on cases of keratoconjunctivitis in sheep (in vivo and in vitro study) and compared with penicillin-streptomycin." (2016): 227-245.

17. Adkin, Pat, et al. "Priming with biocides: A pathway to antibiotic resistance?." Journal of Applied Microbiology 133.2 (2022): 830-841.

18. Singkham-In, Uthaibhorn, et al. "Chlorhexidine promotes Psl expression in Pseudomonas aeruginosa that enhances cell aggregation with preserved pathogenicity demonstrates an adaptation against antiseptic." International Journal of Molecular Sciences 23.15 (2022): 8308.

19. Martins, Marta, et al. "Identification of efflux pump-mediated multidrug-resistant bacteria by the ethidium bromide-agar cartwheel method." In vivo 25.2 (2011): 171-178.

20. Silpa, K. N., et al. "Study of antimicrobial resistance profile and efflux mediated drug resistance in clinical isolates of Pseudomonas aeruginosa detected by ethidium bromide-agar Cartwheel method." International Journal of Research in Medical Sciences 10.12 (2022): 2837.

21. Ugwuanyi, Florence Chijindu, et al. "Evaluation of efflux pump activity and biofilm formation in multidrug resistant clinical isolates of Pseudomonas aeruginosa isolated from a Federal Medical Center in Nigeria." Annals of Clinical Microbiology and Antimicrobials 20 (2021): 1-7.

22. Okafor, J. U., & Nwodo, U. U. (2023). Detection of Multidrug-Resistant RND Efflux Pumps and Regulatory Proteins in Antibiotic-Resistant P. aeruginosa Recovered from Hospital Wastewater Effluent in the Eastern Cape Province of South Africa. Applied Sciences, 13(20), 11241.

23. Mohi-Aldeen, Zahraa Khairi-Aldeen, Muhsin Ayoub Essa, and Mahmod Zeki Al-Hasso. "Detection of Efflux Pumps Genes in Pseudomonasaeruginosa Isolates Isolated from Open Fracture Infections." Kirkuk Journal of Science 13.4 (2018): 155-169.

24. Verdial, Cláudia, et al. "Mechanisms of antibiotic and biocide resistance that contribute to Pseudomonas aeruginosa persistence in the hospital environment." Biomedicines 11.4 (2023): 1221.

25. Abdallah, Alshimaa L., et al. "Expression of Mex AB-Opr M efflux pump system and meropenem resistance in Pseudomonas aeruginosa isolated from surgical intensive care unit." Microbes and Infectious Diseases 2.4 (2021): 781-789.

26. Hassan, Noor S., Mohamed F. Al–marjani, and Nadema H. Hussain. "Detection of Antiseptic Resistant Genes in Colistin-Resistant Pseudomonas aeruginosa and MDR Klebsiella penumoniae." Indian Journal of Forensic Medicine & Toxicology 15.1 (2021).

27. Romão, Célia, et al. "Presence of qacEΔ1 gene and susceptibility to a hospital biocide in clinical isolates of Pseudomonas aeruginosa resistant to antibiotics." Current Microbiology 63 (2011): 16-21.

28. Gülbudak, Harun, et al. "Investigation of qacE, qacEΔ1 and cepA biocide resistance genes in carbapenem resistant Pseudomonas aeruginosa isolates." (2023): 277-284.

29. Anari, Raana Kazemzadeh, et al. "Evaluation of antibacterial activity of five biocides and the synergistic effect of biocide/EDTA combinations on biofilm-producing and non-producing Stenotrophomonas maltophilia strains isolated from clinical specimens in Iran." BMC microbiology 22.1 (2022): 257.

30. Ji, Qing-Yang, et al. "The Effect of Different Organic Acids and Their Combination on the Cell Barrier and Biofilm of Escherichia coli." Foods 12.16 (2023): 3011.

31. Madhusudhan, V. L. "Efficacy of 1% acetic acid in the treatment of chronic wounds infected with Pseudomonas aeruginosa: prospective randomised controlled clinical trial." International wound journal 13.6 (2016): 1129-1136.

32. Zinn, Marc-Kevin, and Dirk Bockmühl. "Did granny know best? Evaluating the antibacterial, antifungal and antiviral efficacy of acetic acid for home care procedures." BMC microbiology 20 (2020): 1-9.

33. De Pascalis, Roberto, et al. "Models derived from in vitro analyses of spleen, liver, and lung leukocyte functions predict vaccine efficacy against the Francisella tularensis Live Vaccine Strain (LVS)." MBio 5.2 (2014): 10-1128.

34. Feng, Luozhu, et al. "Evaluation of the antibacterial, antibiofilm, and anti-virulence effects of acetic acid and the related mechanisms on colistin-resistant Pseudomonas aeruginosa." BMC microbiology 22.1 (2022): 306.

35. Malheiro, Joana F., et al. "Biocide potentiation using cinnamic phytochemicals and derivatives." Molecules 24.21 (2019): 3918.