Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

In vitro efficacy of bismuth thiols against biofilms formed by bacteria isolated from human chronic wounds

Aims: The purpose of this study was to evaluate the antimicrobial efficacy of thirteen bismuth thiol preparations for bactericidal activity against established biofilms formed by two bacteria isolated from human chronic wounds. Methods: Single species biofilms of a Pseudomonas aeruginosa or a methicillin‐resistant Staphylococcus aureus were grown in either colony biofilm or drip‐flow reactors systems. Biofilms were challenged with bismuth thiols, antibiotics or silver sulfadiazine, and log reductions were determined by plating for colony formation. Conclusions: Antibiotics were ineffective or inconsistent against biofilms of both bacterial species tested. None of the antibiotics tested were able to achieve >2 log reductions in both biofilm models. The 13 different bismuth thiols tested in this investigation achieved widely varying degrees of killing, even against the same micro‐organism in the same biofilm model. For each micro‐organism, the best bismuth thiol easily outperformed the best conventional antibiotic. Against P. aeruginosa biofilms, bismuth‐2,3‐dimercaptopropanol (BisBAL) at 40–80 μg ml^?1 achieved >7·7 mean log reduction for the two biofilm models. Against MRSA biofilms, bismuth‐1,3‐propanedithiol/bismuth‐2‐mercaptopyridine N‐oxide (BisBDT/PYR) achieved a 4·9 log reduction. Significance and Impact of the Study: Bismuth thiols are effective antimicrobial agents against biofilms formed by wound bacteria and merit further development as topical antiseptics for the suppression of biofilms in chronic wounds.     

Campanula portenschlagiana Roem. et Schult.: Chemical and Antimicrobial Activities

The phytochemical profile and the antimicrobial effects of the volatile oil and the aqueous extract of Campanula portenschlagiana, a wild growing plant endemic to Croatia, were described. In the volatile oil, 53 compounds were identified by GC‐FID and GC/MS analyses. Diterpene alcohols constituted the major compound class with labda‐13(16),14‐dien‐8‐ol as the main compound. The aqueous extract was characterized by the total phenolic content. The antimicrobial potential of the volatile oil and the aqueous extract was evaluated against a diverse range of microorganisms comprising food‐spoilage and food‐borne pathogens. The volatile oil exhibited interesting and promising antimicrobial effects against the tested species, which were generally more pronounced against Gram‐negative bacteria. In addition, the inhibitory effect of this volatile oil was also evaluated against eleven extended‐spectrum β‐lactamase (ESBL)‐producing isolates. The results suggest that the C. portenschlagiana volatile oil might be used as antimicrobial agent against ESBL‐producing isolates and Gram‐negative bacteria.     

Chemical Variability,Antioxidant and Antifungal Activities of Essential Oils and Hydrosol Extract of Calendula arvensis L. from Western Algeria

The chemical composition of the essential oils and hydrosol extract from aerial parts of Calendula arvensis L. was investigated using GC‐FID and GC/MS. Intra‐species variations of the chemical compositions of essential oils from 18 Algerian sample locations were investigated using statistical analysis. Chemical analysis allowed the identification of 53 compounds amounting to 92.3 – 98.5% with yields varied of 0.09 – 0.36% and the main compounds were zingiberenol 1 (8.7 – 29.8%), eremoligenol (4.2 – 12.5%), β‐curcumene (2.1 – 12.5%), zingiberenol 2 (4.6 – 19.8%) and (E,Z)‐farnesol (3.5 – 23.4%). The study of the chemical variability of essential oils allowed the discrimination of two main clusters confirming that there is a relation between the essential oil compositions and the harvest locations. Different concentrations of essential oil and hydrosol extract were prepared and their antioxidant activity were assessed using three methods (2,2‐diphenyl‐1‐picrylhydrazyl, Ferric‐Reducing Antioxidant Power Assay and β‐carotene). The results showed that hydrosol extract presented an interesting antioxidant activity. The in vitro antifungal activity of hydrosol extract produced the best antifungal inhibition against Penicillium expansum and Aspergillus niger, while, essential oil was inhibitory at relatively higher concentrations. Results showed that the treatments of pear fruits with essential oil and hydrosol extract presented a very interesting protective activity on disease severity of pears caused by P. expansum.     

Antimicrobial susceptibility and sessile behaviour of bacteria isolated from a minimally processed vegetables plant

Abstract

In this study, 20 heterotrophic bacteria from a minimally processed vegetables (MPV) plant were tested for their susceptibilities to five antibiotics (tetracycline, erythromycin, ampicillin, levofloxacin and ciprofloxacin), their (co)aggregation abilities and their survival under gastric simulated conditions. Peracetic acid (PA) and sodium hypochlorite (SH), both at 50?ppm, were evaluated for their abilities to control biofilms of these bacteria. In general, the Gram-negative bacteria were found to be more resistant to the selected antibiotics. Two isolates, Rhanella aquatilis and Stenotrophomonas maltophilia, demonstrated multidrug resistance. Only Rhodococcus erythropolis presented aggregation potential, while no bacterium survived under the gastric conditions. The biofilm experiments showed PA as less efficient than SH in killing biofilms and neither of the disinfectants was able to fully eliminate the biofilms. Significant regrowth was observed for most of the biofilms. The results indicate that alternative and/or complementary disinfection strategies are required to guarantee food safety.     

Bacterial disinfectant resistance—a challenge for the food industry

The focus on hygiene in the food industry has resulted in an increasing use of chemical disinfection and it has been speculated that this will impose a selective pressure and contribute to the emergence of disinfectant-resistant microorganisms. The frequency of strains with a low-level resistance to quaternary ammonium compounds (QAC) is relatively high for Listeria monocytogenes (10%), Staphylococcus spp. (13%) and Pseudomonas spp. (30%) and lower for lactic acid bacteria (1.5%) and coliforms (1%) isolated from food and food processing industry. In general, bacteria isolated after disinfection are more resistant and represent a potential food safety or food spoilage problem. Adaptation to disinfectants may be accompanied by cross-resistance to related disinfectants. We have recently found a genetic linkage between resistance to QAC and antibiotics in food associated staphylococci, and there is a growing concern about cross-resistance between antibiotics and disinfectants.Disinfectant resistance can in most cases be prevented by effective cleaning and disinfection procedures. More effort should be made to avoid build-up of resistance in food production environments.     

Dittrichia graveolens (L.) Greuter Essential Oil: Chemical Composition,Multivariate Analysis,and Antimicrobial Activity

The chemical composition and in vitro antimicrobial activities of Dittrichia graveolens (L.) Greuter essential oil was studied. Moreover, using agglomerative hierarchical cluster (AHC) and principal component analyses (PCA), the interrelationships of the D. graveolens essential‐oil profiles characterized so far (including the sample from this study) were investigated. To evaluate the chemical composition of the essential oil, GC‐FID and GC/MS analyses were performed. Altogether, 54 compounds were identified, accounting for 92.9% of the total oil composition. The D. graveolens oil belongs to the monoterpenoid chemotype, with monoterpenoids comprising 87.4% of the totally identified compounds. The major components were borneol (43.6%) and bornyl acetate (38.3%). Multivariate analysis showed that the compounds borneol and bornyl acetate exerted the greatest influence on the spatial differences in the composition of the reported oils. The antimicrobial activity against five bacterial and one fungal strain was determined using a disk‐diffusion assay. The studied essential oil was active only against Gram‐positive bacteria.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p?>?0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p?<?0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p?<?0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1?–?0.5%) and exposure period were noted (p?<?0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p?>?0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Activity of disinfectants against multispecies biofilms formed by Staphylococcus aureus,Candida albicans and Pseudomonas aeruginosa

Microbial biofilms are a serious threat to human health. Recent studies have indicated that many clinically relevant biofilms are polymicrobial. In the present study, multispecies biofilms were grown in a reproducible manner in a 96-well microtiter plate. The efficacy of nine commercially available disinfectants against Staphylococcus aureus, Candida albicans, and Pseudomonas aeruginosa in multispecies biofilms was determined and compared. The results showed that the direction and the magnitude of the effect in a multispecies biofilm depend on the strain and the disinfectant used and challenge the common belief that organisms in multispecies biofilms are always less susceptible than in monospecies biofilms.     

Comparative susceptibility of planktonic and 3‐day‐old Salmonella Typhimurium biofilms to disinfectants

Aims: To compare the susceptibility of a 3‐day‐old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella. Methods and Results: The susceptibility of planktonic cells to disinfectants was tested by a modified version of the Council of Europe suspension test EN 1276. Salmonella biofilms were formed using the Calgary Biofilm Device. Results show that 3‐day‐old Salmonella biofilms are less susceptible to the disinfectants benzalkonium chloride, chlorhexidine gluconate, citric acid, quaternary ammonium compounds, sodium hypochlorite (SH) and ethanol, compared to planktonic Salmonella. Surprisingly, the results also demonstrate that low concentrations of SH were more effective against a 3‐day‐old biofilm compared to high concentrations of SH. Conclusions: While all the disinfectants evaluated were able to reduce biofilm‐associated cells at concentrations and contact times sufficient to eliminate planktonic cells, there were still sufficient viable cells remaining in the biofilm to cause further contamination and potential infection. Significance and Impact of the Study: Protocols for the use of chemical disinfectants need to include biofilm susceptibility testing. There is a requirement for an effective and standardized tool for determining the susceptibility of biofilms to disinfectants.     

The ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency

The penetration ability of 12 antimicrobial agents, including antibiotics and biocides, was determined against biofilms of B. cereus and P. fluorescens using a colony biofilm assay. The surfactants benzalkonium chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB), and the antibiotics ciprofloxacin and streptomycin were of interest due to their distinct activities. Erythromycin and CTAB were retarded by the presence of biofilms, whereas ciprofloxacin and BAC were not. The removal and killing efficacies of these four agents was additionally evaluated against biofilms formed in microtiter plates. The most efficient biocide was CTAB for both bacterial biofilms. Ciprofloxacin was the best antibiotic although none of the selected antimicrobial agents led to total biofilm removal and/or killing. Comparative analysis of the results obtained with colony biofilms and microtiter plate biofilms show that although extracellular polymeric substances and the biofilm structure are considered a determining factor in biofilm resistance, the ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency. Also, the results reinforce the role of an appropriate antimicrobial selection as a key step in the design of disinfection processes for biofilm control.     

Chemical Composition of Hydrodistilled Essential Oil of Artemisia incana (L.) Druce and Antimicrobial Activity against Foodborne Microorganisms

The oil obtained by hydrodistillation from the aerial parts of Artemisia incana (L.) Druce from Turkey was analyzed by GC and GC/MS. Sixty‐three compounds were characterized, representing 97.2% of the total components detected, and camphor (19.0%), borneol (18.9%), 1,8‐cineole (14.5%), bornyl acetate (7.8%), camphene (4.9%), and α‐thujone (4.8%) were identified as predominant components. The essential oil was also tested for its antimicrobial activity against 44 different foodborne microorganisms, including 26 bacteria, 15 fungi, and 3 yeast species. The essential oil of A. incana exhibited considerable inhibitory effects against all bacteria, fungi, and yeast species tested. However, the oil showed lower inhibitory activity against the tested bacteria than the reference antibiotics.     

Unsaturated Fatty Acid,cis-2-Decenoic Acid,in Combination with Disinfectants or Antibiotics Removes Pre-Established Biofilms Formed by Food-Related Bacteria

Biofilm formation by food-related bacteria and food-related pathogenesis are significant problems in the food industry. Even though much disinfection and mechanical procedure exist for removal of biofilms, they may fail to eliminate pre-established biofilms. cis-2 decenoic acid (CDA), an unsaturated fatty acid messenger produced by Pseudomonas aeruginosa, is reportedly capable of inducing the dispersion of established biofilms by multiple types of microorganisms. However, whether CDA has potential to boost the actions of certain antimicrobials is unknown. Here, the activity of CDA as an inducer of pre-established biofilms dispersal, formed by four main food pathogens; Staphylococcus aureus, Bacillus cereus, Salmonella enterica and E. coli, was measured using both semi-batch and continuous cultures bioassays. To assess the ability of CDA combined biocides treatments to remove pre-established biofilms formed on stainless steel discs, CFU counts were performed for both treated and untreated cultures. Eradication of the biofilms by CDA combined antibiotics was evaluated using crystal violet staining. The effect of CDA combined treatments (antibiotics and disinfectants) on biofilm surface area and bacteria viability was evaluated using fluorescence microscopy, digital image analysis and LIVE/DEAD staining. MICs were also determined to assess the probable inhibitory effects of CDA combined treatments on the growth of tested microorganisms'' planktonic cells. Treatment of pre-established biofilms with only 310 nM CDA resulted in at least two-fold increase in the number of planktonic cells in all cultures. While antibiotics or disinfectants alone exerted a trivial effect on CFU counts and percentage of surface area covered by the biofilms, combinational treatments with both 310 nM CDA and antibiotics or disinfectants led to approximate 80% reduction in biofilm biomass. These data suggests that combined treatments with CDA would pave the way toward developing new strategies to control biofilms with widespread applications in industry as well as medicine.     

Chemical Characteristics,Antimicrobial, and Cytotoxic Activities of the Essential Oil of Egyptian Cinnamomum glanduliferum Bark

The essential oil isolated from the bark of Cinnamomum glanduliferum (Wall ) Meissn grown in Egypt was screened for its composition as well as its biological activity for the first time. The chemical composition was analyzed by GC and GC/MS. The antimicrobial activity of the oil was assessed using agar‐well diffusion method toward representatives for each of Gram‐positive bacteria, Gram‐negative bacteria, and fungi. The cytotoxic activity was checked using three human cancer cell lines. Twenty seven compounds were identified, representing 99.07% of the total detected components. The major constituents were eucalyptol (65.87%), terpinen‐4‐ol (7.57%), α‐terpineol (7.39%). The essential oil possessed strong antimicrobial activities against Escherichia coli, with an activity index of one and minimum inhibitory concentration (MIC) equaling to 0.49 μg/ml. The essential oil possessed good antimicrobial activities against methicillin‐resistant Staphylococcus aureus, Geotrichum candidum, Pseudomonas aeruginosa, Bacillus subtilis, Helicobacter pylori, Aspergillus fumigatus (MIC: 7.81, 1.95, 7.81, 0.98, 31.25, and 32.5 μg/ml, respectively). A considerable activity was reported against S. aureus and Mycobacterium tuberculosis (MIC; 32.5 and 31.25 μg/ml, respectively). The extracted oil was cytotoxic to colon (HCT‐116), liver (HepG2), and breast (MCF‐7) carcinoma cell lines with IC₅₀ of 9.1, 42.4, and 57.3 μg/ml, respectively. These results revealed that Egyptian Cinnamomum glanduliferum bark oil exerts antimicrobial and cytotoxic activities mainly due to eucalyptol and other major compounds.     

Chemical Composition and Antimicrobial Activity of the Essential Oils from New Caledonian Citrus macroptera and Citrus hystrix

The essential oils from the leaves of Citrus macroptera and C. hystrix, collected in New Caledonia, have been analyzed by gas chromatography/mass spectrometry (GC/MS) and evaluated for their antimicrobial activity. A total of 35 and 38 constituents were identified, representing 99.1 and 89.0% of the essential oils, respectively. Both essential oils were rich in monoterpenes (96.1 and 87.0%, resp.), with β‐pinene as major component (33.3 and 10.9%, resp.), and poor in limonene (2.4 and 4.7%, resp.). Other main components of C. macroptera oil were α‐pinene (25.3%), p‐cimene (17.6%), (E)‐β‐ocimene (6.7%), and sabinene (4.8%). The essential oil of C. hystrix was characterized by high contents of terpinen‐4‐ol (13.0%), α‐terpineol (7.6%), 1,8‐cineole (6.4%), and citronellol (6.0%). The antimicrobial activity was evaluated against five bacteria and five fungi strains. Both oils were inactive against bacteria. However, the C. macroptera leaf oil exhibited a pronounced activity against Trichophyton mentagrophytes var. interdigitale, with a minimal‐inhibitory concentration (MIC) of 12.5 μg/ml.     

Biofilm formation and proteolytic activities of Pseudoalteromonas bacteria that were isolated from fish farm sediments

In order to save natural resources and supply good fishes, it is important to improve fish‐farming techniques. The survival rate of fish fry appears to become higher when powders of foraminifer limestone are submerged at the bottom of fish‐farming fields, where bacterial biofilms often grow. The observations suggest that forming biofilms can benefit to keep health status of breeding fishes. We employed culture‐based methods for the identification and characterization of biofilm‐forming bacteria and assessed the application of their properties for fish farming. Fifteen bacterial strains were isolated from the biofilm samples collected from fish farm sediments. The 16S rRNA gene sequences indicated that these bacteria belonged to the genera, Pseudoalteromonas (seven strains), Vibrio (seven strains) and Halomonas (one strain). It was found that Pseudoalteromonas strains generally formed robust biofilms in a laboratory condition and produced extracellular proteases in a biofilm‐dependent manner. The results suggest that Pseudoalteromonas bacteria, living in the biofilm community, contribute in part to remove excess proteineous matters from the sediment sludge of fish farms.     

Chemical and Biological Evaluation of Hypericum maculatum Crantz Essential Oil

The chemical composition and the antimicrobial activity of the essential oil isolated from the aerial parts of Hypericum maculatum Crantz were determined. In total, 109 compounds were identified, with germacrene D (21.5%), nonane (6.5%), (E)‐β‐farnesene (5.3%), δ‐cadinene (4.5%), and ledol (4.4%) as the main constituents. The chemical compositions of this oil and of four previously studied H. maculatum oils were compared using multivariate statistical analyses, viz., agglomerative hierarchical cluster and principal component analyses. Based on the results, the interrelationship among the hitherto studied H. maculatum oil samples, including the oil characterized here, was discussed. The study of the antimicrobial potential of the oil against five bacterial and two fungal strains showed that the oil had mainly moderate antimicrobial effects.     

Composition and Antimicrobial Activity of the Essential Oils of Laserpitium latifolium L. and L. ochridanum Micevski (Apiaceae)

The chemical composition and antimicrobial activity of essential oils of Laserpitium latifolium and L. ochridanum were investigated. The essential oils were isolated by steam distillation and characterized by GC‐FID and GC/MS analyses. All essential oils were distinguished by high contents of monoterpenes, and α‐pinene was the most abundant compound in the essential oils of L. latifolium underground parts and fruits (contents of 44.4 and 44.0%, resp.). The fruit essential oil was also rich in sabinene (26.8%). Regarding the L. ochridanum essential oils, the main constituents were limonene in the fruit oil (57.7%) and sabinene in the herb oil (25.9%). The antimicrobial activity of these essential oils as well as that of L. ochridanum underground parts, whose composition was reported previously, was tested by the broth‐microdilution method against four Gram‐positive and three Gram‐negative bacteria and two Candida albicans strains. Except the L. latifolium underground‐parts essential oil, the other investigated oils showed a high antimicrobial potential against Staphylococcus aureus, S. epidermidis, Micrococcus luteus, or Candida albicans (minimal inhibitory concentrations of 13.0–73.0 μg/ml), comparable to or even higher than that of thymol, which was used as reference compound.     

Comparison of the Composition and Antimicrobial Activities of the Essential Oils of Green Branches and Leaves of Egyptian Navel Orange (Citrus sinensis (L.) Osbeck var. malesy)

The essential oils isolated from the leaves and green branches of the Egyptian navel orange trees were analyzed by GC and GC/MS. A total of 33 and 24 compounds were identified from the oils of the leaves and branches accounting for 96.0% and 97.9%, respectively, of the total detected constituents. The major ones were sabinene (36.5; 33.0%), terpinen‐4‐ol (8.2; 6.2%), δ‐3‐carene (7.0; 9.4%), limonene (6.8; 18.7%), trans‐ocimene (6.7; 6.1%), and β‐myrcene (4.5; 4.4%). The antimicrobial activities of both oils were evaluated using the agar‐well diffusion method toward three representatives for each of Gram‐positive bacteria, Gram‐negative bacteria, and fungi. The oil of leaves was more effective as antimicrobial agent than that of the branches. Streptococcus pyogenes, Staphylococcus aureus, Salmonella typhimurium, and Aspergillus fumigatus were the most sensitive bacteria and fungi by the leaves oil.     

Chemical Composition and Antipathogenic Activity of Artemisia annua Essential Oil from Romania

The essential oil extracted by hydrodistillation from Romanian Artemisia annua aerial parts was characterized by GC/MS analysis, which allowed the identification of 94.64% of the total oil composition. The main components were camphor (17.74%), α‐pinene (9.66%), germacrene D (7.55%), 1,8‐cineole (7.24%), trans‐β‐caryophyllene (7.02%), and artemisia ketone (6.26%). The antimicrobial activity of this essential oil was evaluated by determining the following parameters: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), minimal fungicidal concentration (MFC), and minimal biofilm eradication concentration (MBEC). Moreover, the soluble virulence factors were quantified with different biochemical substrates incorporated in the culture media. The reference and resistant, clinical strains proved to be susceptible to the A. annua oil, with MICs ranging from 0.51 to 16.33 mg/ml. The tested essential oil also showed good antibiofilm activity, inhibiting both the initial stage of the microbial cell adhesion to the inert substratum and the preformed mature biofilm. When used at subinhibitory concentrations, the essential oil proved to inhibit the phenotypic expression of five soluble virulence factors (hemolysins, gelatinase, DNase, lipases, and lecithinases). Briefly, the present results showed that the A. annua essential oil contained antimicrobial compounds with selective activity on Gram‐positive and Gram‐negative bacterial strains as well as on yeast strains and which also interfere with the expression of cell‐associated and soluble virulence factors.