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Comparative study of growth temperature impact on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells to benzalkonium chloride

Comparative study of growth temperature impact on the susceptibility of biofilm-detached and... The present study investigated and compared the effect of growth temperature on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells, to benzalkonium chloride (BAC). This study also highlights the impact of BAC on the bacterial physiology and the role of membrane fluidity regulation as a bacterial resistance mechanism. The minimum inhibitory concentration of BAC was characterized with micro-dilution growth inhibition assay. The BAC treatment was performed on S. aureus cultured at 20 °C and 37 °C, for 24 h. The morphology of S. aureus cells was examined using scanning electron microscopy. The loss of bacterial membrane integrity after BAC treatment was studied by monitoring the intracellular potassium ion leakage using the atomic absorption spectroscopy. The bacterial membrane total fatty acid composition, controlling the membrane fluidity, was analyzed by GC/MS. The results showed that the resistance of S. aureus cells to BAC increased with the increase of growth temperature. The planktonic cells were more susceptible to BAC than biofilm-detached ones. The rise of growth temperature resulted in an increase of S. aureus membrane rigidity. Furthermore, a higher membrane fluidity was observed in planktonic cells when compared to that in the biofilm-detached ones. The resistance of S. aureus seems to depend on the growth temperature. Compared to planktonic cells, biofilm-detached cells showed a greater resistance to BAC. The BAC targets and disturbs the bacterial membrane. Membrane fluidity modulation is likely a one of resistance mechanisms for S. aureus to BAC at the cellular scale. Therefore, disinfection procedures, in food sector, should be adapted for bacteria detached from biofilm. . . . . . Keywords Staphylococcus aureus Biofilm-detached cells Planktonic cells Growth temperature Membrane fluidity Susceptibility to BAC Introduction disinfectants are constantly applied to maintain a high level of surface hygiene in food fields (Khelissa et al. 2017a). Such The emergence of Staphylococcus aureus in food sector rep- procedure is thought to be effective to decrease the microbio- resents a significant risk for public health. In fact, this bacte- logical risk in the highly affected areas. Bacteria are often rium is commonly involved in foodborne diseases (FBDs) attached to surfaces and form a complex structure, called bio- (Denayer et al. 2017). In order to fight against FBDs, film (Tuson and Weibel 2013). The biofilm-associated cells are physiologically different from the planktonic ones (Chua et al. 2014). Furthermore, biofilm-structured cells have a bet- * Nour-Eddine Chihib ter resistance to disinfectants than their planktonic counter- nour-eddine.chihib@univ-lille.fr parts (Bridier et al. 2011). In the food sector, bacteria are usually exposed to different environmental constraints such CNRS, INRA, UMR 8207-UMET-PIHM, Université de Lille, 369 as temperature changes, shear forces, and pH (Simões et al. rue Jules Guesde, 59651 Villeneuve d’Ascq, France 2010; Berne et al. 2018). The exposure of biofilms to high CNRS, ENSCL, UMR 8207-UMET-PSI, Université de Lille, shear forces may result in cell dispersal (Berne et al. 2018). Avenue Dimitri Mendeleïev, 59652 Villeneuve d’Ascq, France Thus, the biofilm represents a bacterial reservoir which, once Université Lyon 1, ISARA Lyon, Laboratoire BioDyMIA, Equipe detached, serves as a continuous source of contamination Mixte d’Accueil, no. 3733, IUT Lyon 1, Technopole Alimentec, rue resulting in severe FBDs. Henri de Boissieu, F-01000 Bourg en Bresse, France 292 Ann Microbiol (2019) 69:291–298 The biofilm resistance mechanisms to antimicrobial Stainless steel slide preparation agents may be explained in several ways (Bridier et al. 2011). The biofilm resistance mechanisms should be ob- The stainless steel (SS) (304 L, Equinox, France) slides were served at the macroscopic and the microscopic levels. At first immersed in acetone (Fluka, Sigma-Aldrich, France) for the macroscopic scale, it seems to be related to the pro- 1 h, then rinsed under distilled water. The slides were soaked duction of an extracellular matrix, composed of extracel- in DDM ECO detergent (1%) for 10 min at 20 °C (ANIOS, lular polymeric substances (EPS), that hinder the disinfec- France) and rinsed under distilled water then under ultrapure tant diffusion (Bridier et al. 2011). However, several stud- water (Milli-Q Academic, Millipore, France). The SS slides ies highlighted the fact that the biofilm matrix cannot were dried and autoclaved (20 min, 120 °C). always explain the biofilm resistance to disinfectants (Abdallah et al. 2014). Therefore, the biofilm resistance at the microscopic or cellular scale, which is thought to be Development of biofilm linked to the modification of bacterial physiology, should be explored. In addition, bacterial cells may adapt to un- The SS slides (90 × 90 mm) were covered with 12 mL of the favorable growth conditions by modifying their mem- corresponding cell suspension (20 °C or 37 °C) adjusted to brane lipid composition (Chihib et al. 2005). The bacterial 10 CFU/mL and incubated at 20 °C for 1 h to allow the membrane, composed of phospholipids and proteins, con- bacterial adhesion. Afterwards, slides were rinsed twice, by stitutes the first line of bacterial defense against antimi- immersion in PPB, to remove loosely attached cells. Then, crobial. The fatty acid composition controls the fluidity of each slide was covered with 12 mL of TSB and incubated bacterial membrane and may hinder the penetration of statically, at 20 °C or 37 °C for 24 h. Following the 24-h antimicrobial into cells (Dubois-Brissonnet et al. 2016; incubation, slides covered with biofilm were rinsed with Malanovic and Lohner 2016). PPB. The strongly attached bacteria were recovered into In this regard, the purpose of this work was to study the 10 mL of PPB by surface scraping, harvested by centrifuga- effect of growth temperature (20 °C and 37 °C) on the resis- tion (5000g, 5 min, 20 °C), and then washed once with 20 mL tance of S. aureus cells, issued from planktonic culture or of PPB. The bacterial suspension concentrations were adjust- detached from biofilm formed on stainless steel, to BAC. ed in PPB to 10 CFU/mL as above cited. This study also aimed to study the membrane fluidity, of planktonic and biofilm-detached S. aureus, in order to charac- terize its involvement as a resistance mechanism to BAC treat- BAC minimum inhibitory concentration ment at the cellular level. determination The minimum inhibitory concentration (MIC) of BAC was determined by micro-dilution growth inhibition as- Materials and methods says, using a Bioscreen C (Labsystems, Helsinki, Finland). Staphylococcus aureus cells were cultured, as Bacterial culture conditions and suspension previously, in Mueller-Hinton Broth (MHB) (Bio-Rad, preparation France). One hundred microliters of S. aureus suspension (10 CFU/mL) were added to the plate wells containing Staphylococcus aureus CIP4.83strainwas stored(− decreasing BAC concentrations ranging from 25 to 0 mg/ 80 °C) in tryptic soy broth (TSB) (Biokar Diagnostics, L (in MHB). Two BAC-free controls were included, one France) with 40% (v/v) of glycerol. Precultures were pre- with only MHB (sterility control) and the other with MHB paredbyadding100 μL from stock tube to 5 mL of + bacteria (growth control). The micro-dilution plates TSBand incubatedat20°C(for48h)or37°C(for were incubated in the Bioscreen C at 37 °C under contin- 24 h). The cultures were started by inoculating 10 CFU/ uous shaking. The OD wasmeasuredevery2hfor 600 nm mL from the preculture in 50 mL of TSB and incubation 48 h. The MIC was the lowest concentration of the BAC under continuous shaking (160 rpm) at 20 °C or 37 °C. that prevented growth, as measured by optical density. Cultures were stopped at the late exponential phase and Log OD values were plotted versus time (h). The 600 nm cells were pelleted (5000g, 5 min, 20 °C). Bacteria were lag time, of each growth curve, was estimated by extrap- washed twice with potassium phosphate buffer (PPB; olating the linear portion of Log OD versus time 600 nm 100 mM, pH 7). Finally, the bacterial suspensions were plot back to the initial OD . The growth rate (μ) 600 nm adjusted to 10 CFU/mL in PPB, by fixing the optical was calculated during the exponential growth phase using density at 600 nm to 0.110 ± 0.005, using a Jenway the formula: dN/dt = kN,where N is the Log OD of 600 nm 6320D UV/visible light spectrophotometer. cells, t the time, and k is thegrowth rateconstant. Ann Microbiol (2019) 69:291–298 293 Disinfection of S. aureus cells (Shimadzu, Japan) with a capillary column (Zebron ZB- FFAP, Phenomenex, Australia) and connected to mass For the disinfection assay, 1 mL of bacterial suspension, ad- spectrometer (Thermo-Finnigan Trace DSQ, Thermo justed to 10 CFU/mL, was introduced to 1 mL of 6 mg/L Fisher Scientific, USA). BAC solution (prepared in PPB). After 5-min contact time at 20 °C, 1 mL of this mixture was transferred into 9 mL of Scanning electron microscope observation neutralizing solution (Abdallah et al. 2014) to stop the anti- bacterial action. Thereafter, tenfold serial dilutions were done The morphology of S. aureus cells upon BAC treatment was in PPB. Samples of 100 μL were spread onto tryptic soy agar assessed by scanning electron microscope (SEM). One milli- broth plates (TSA; Biokar Diagnostics, France) and incubated liter volume of the BAC- or PPB-treated then neutralized at 37 °C for 24 h. After the incubation time, the number of planktonic or biofilm-detached cell suspensions was filtered viable and culturable cells was counted on the plates and the through a 0.2-μm-pore-size polycarbonate membrane filter results were expressed in Log CFU/mL. For the control as- (Schleicher & Schuell, Dassel, Germany) then fixed for 4 h says, the disinfectant solution was replaced by PPB. with 2% glutaraldehyde, in cacodylate buffer 0.1 M pH 7, at 20 °C. Fixed samples were then dehydrated in an ascending BAC-induced cell membrane permeabilization ethanol series (50, 70, 95, and 2 × 100% (v/v) ethanol), for 15 min at each concentration. Samples were critical point Biofilm-detached and planktonic S. aureus cells grown at dried and coated with thin carbon film before examination in 20 and 37 °C were concentrated to 10 CFU/mL. Five the SEM. Microscopy was performed with a Hitachi S4700 milliliters of the concentrated bacterial suspensions was microscope at 3 kV. introduced into silicone cap glass reaction vessels (100-mL wide-necked flasks) containing 45 mL of BAC Statistical analysis solution prepared in HEPES buffer (final concentration of 3 mg/L in 50 mL final volume) or HEPES buffer (nega- The results are presented as mean values and their standard tive control). The K concentration at time zero was mea- error of the mean. Data analysis was performed using Sigma sured in a tenfold dilution of the bacterial suspension fil- Plot 11.0 (Systat Software Inc.), using one-way ANOVA ™ ™ trate (0.2 μm, Sartorius Minisart NML Syringe Filters, (Tukey’s method). Results were considered significant at a P France) before contact with BAC solution. After the in- value of <0.05. troduction of bacterial suspension to the reaction vessel containing the BAC solution, samples (4 mL) were filter sterilized at 5, 10, 15, 20, 30, 60, and 90 min. Each sam- Results ple was removed using a sterile plastic syringe attached to a sterile needle to enable easy access to the reaction mix- Determination of the BAC minimum inhibitory ture suspension through the silicon cap. The K concen- concentration tration in filtrate samples was determined using a Varian SpectrAA 55/B atomic absorption spectrometer in flame The results of Fig. 1 showed that at a concentration of 0.2 mg/ emission mode (wavelength 766.5 nm; slit 0.7 nm high; L, cells have the same growth behavior of the control (cells air-acetylene flame). without BAC). At a concentration of 0.4, 0.8, and 1.5 mg/L of BAC, the growth profile was different from the control (P< Cellular fatty acid extraction and analysis 0.05). The results showed that the BAC minimum inhibitory concentration was of 3 mg/L. Figure 1 showed also that the Biofilm-detached and planktonic S. aureus cells were har- growth rate (μ)and lagtime(λ) were dependent on the BAC vested as cited above, either by scrapping cells embedded concentration. In fact, our results showed that the μ value of S. −1 in biofilm from the rinsed coupons or by centrifuging aureus cultures was of 0.056 h when the BAC concentra- planktonic culture, then resuspended in 10 mL of PPB. tions were ranging from 0 to 1.5 mg/L (Fig. 1). Furthermore, Cells were sonicated (37 kHz, 5 min) and vortexed for the lag time consistently extended with increased BAC con- 30 s. Cells were pelleted by centrifugation (10,000g, centrations (Fig. 1). When the BAC concentrations increased 10 min, 4 °C), and pellets, containing 10 CFU/mL, were from 0 to 1.5 mg/L, the λ increased from 8 to 20 h. washed twice with cold distilled water. Cells were sub- jected to the saponification and methylation (Chihib Susceptibility of S. aureus cells to BAC treatment et al. 2005). Fatty acid methyl ester extraction was real- ized as described previously by Chihib et al. (2005). Data The susceptibility of biofilm-detached and planktonic S. analysis was performed with GC-2014 gas chromatograph aureus cells, grown at 20 °C and 37 °C, to BAC treatment 294 Ann Microbiol (2019) 69:291–298 Fig. 1 Growth curves of Time (h) Staphylococcus aureus grown at 0 5 10 15 20 25 30 35 40 45 37 °C for 48 h, measured at the optic density (OD) of 600 nm -0.1 under different benzalkonium -0.2 chloride concentrations. The concentration at which there was -0.3 a linear growth inhibition was -0.4 considered the MIC. The OD at 600 nm in Mueller-Hinton Broth -0.5 without bacterial inoculum was -0.6 measured to ensure the sterility of the growth medium (Only MHB) -0.7 -0.8 -0.9 -1.0 -1.1 -1.2 -1.3 25 mg/L 12.5 mg/L 6.25 mg/L 3 mg/L 1.5 mg/L 0.8 mg/L 0.4 mg/L 0.2 mg/L was studied. The results showed that the PPB treatment, used Effect of BAC on S. aureus cell membrane as the negative control, had no significant effect on the initial permeability population of S. aureus, whatever the studied conditions (P< 0.05)(Fig. 2). The average of viable and culturable counts of Measurements of K efflux from the bacterial cells were real- cells treated with PPB was of ca 7.3 Log CFU/mL (Fig. 2). ized to assess the cell resistance to BAC treatment. This was The treatment of biofilm-detached cells grown at 20 °C carried out by monitoring the extracellular K concentration in and37°C, for5 minata BACconcentration of 3 mg/L, biofilm-detached and planktonic S. aureus cells grown at resulted in a significant reduction of the initial viable and 20 °C and 37 °C for 24 h (Fig. 3). Our results also showed culturable count of 3.2 and 1.8 Log CFU/mL, respectively that the HEPES buffer addition (control) had no effect on K (P<0.05)(Fig. 2). However, the treatment of planktonic efflux which remained stable whatever the studied condition. cells grown at 20 °C and 37 °C, for 5 min at a BAC The addition of BAC at a final concentration of 3 mg/L result- concentration of 3 mg/L, significantly reduced the initial ed in an immediate increase of extracellular K . Figure 3 viable and culturable count by 4.3 and 3.1 Log CFU/mL, showed that K efflux was higher in planktonic than in respectively (P < 0.05)(Fig. 2). Furthermore, the results biofilm-detached cell suspensions whatever the also showed that the remained viable and culturable count growth temperature condition (P < 0.05). Five minutes after of biofilm-detached cells after BAC treatment was of ca. the BAC addition, the extracellular K concentration in- 1.3 Log CFU/mL higher than that of planktonic cells creased to 4.8 and 1.7 mg/L in the planktonic cell suspensions whatever the cell growth temperature (P < 0.05)(Fig. 2). grown at 20 °C and 37 °C, respectively (P < 0.05)(Fig. 3). Fig. 2 Susceptibility of biofilm- detached and planktonic Staphylococcus aureus cells, grown at 20 °C and 37 °C for 24 h, to benzalkonium chloride treatment (BAC). The bacterial count is presented in Log CFU/ mL after 100 mM potassium phosphate buffer (control) or BAC treatment (3 mg/L for 5min) Log OD 600 nm Ann Microbiol (2019) 69:291–298 295 Fig. 3 Kinetics of intracellular potassium leakage from biofilm- detached and planktonic Staphylococcus aureus cells, grown at 20 °C and 37 °C for 24 h. The K concentration is presented in milligrams per liter. The treatment with HEPES buffer represents the negative control Under the same conditions, the extracellular K increased to a compared with untreated cells (Fig. 4). The treated S. aureus concentration of 1.9 mg/L and 0.5 mg/L in the biofilm-detached biofilm-detached cells, grown at 37 °C, became generally cell suspensions grown at 20 °C and 37 °C, respectively (Fig. 3). distorted in shape and had few holes in their cell walls (Fig. 4). After 90 min of BAC addition into bacterial suspensions grown at 20 °C, the extracellular K concentration reached Effect of growth temperature on S. aureus membrane concentrations of 10 and 15 mg/L in suspension of biofilm- fatty acid profiles detached and planktonic cells (P < 0.05)(Fig. 3). However, when cells were issued from cultures grown at 37 °C, the K Membrane fatty acid (FA) profile of biofilm-detached and extracellular concentration in planktonic and biofilm-detached planktonic S. aureus cells, grown for 24 h at 20 °C and suspensions increased to a concentration of 5 and 2.5 mg/L, 37 °C, was analyzed (Fig. 5). This investigation was per- respectively (P < 0.05)(Fig. 3). formed to study the effect of growth temperature on the mem- brane fatty acid composition which controls the membrane fluidity. Figure 5 showed that the amounts of anteiso C15 Impact of BAC treatment on S. aureus morphology (aC15) were maintained at a stable level whatever the growth changes temperature (P > 0.05). The results also showed that the aC15 amount of biofilm-detached cell membranes was significantly To investigate structural modifications of S. aureus biofilm- lower than that of planktonic cells (P < 0.05). Moreover, the detached and planktonic cells after the exposition to BAC total long-chain FA amounts, the aC19, C18, and C20, of treatment, bacterial samples were analyzed by SEM. The un- biofilm-detached cells were 1.3-fold higher than their plank- treated biofilm-detached and planktonic S. aureus cells culti- tonic counterparts whatever the growth temperature (P< vated at 20 °C and 37 °C looked round and exhibited an 0.05)(Fig. 5). The increase of growth temperature from 20 undamaged normal smooth lining (Fig. 4). However, when to 37 °C promoted an increase in long-chain FA amounts biofilm-detached and planktonic S. aureus cells were exposed including aC19, C18, and C20 (P < 0.05)(Fig. 5). for 5 min to a BAC concentration of 3 mg/L, significant mor- phological changes were observed (Fig. 4). Staphylococcus aureus planktonic cells grown at 20 °C showed holes in their cell walls (Fig. 4). Staphylococcus aureus biofilm-detached Discussion cells, cultivated at 20 °C, showed multiple dents on their sur- faces (Fig. 4). However, the morphological changes of S. Biofilm-detached cells constitute a major source of bacterial aureus biofilm-detached and planktonic cells, grown at dissemination and contamination of food contact surfaces 37 °C, seemedtobelesspronouncedthanthoseof their (Khelissa et al. 2017b). Thus, it is of importance to conduct 20 °C counterparts (Fig. 4). Hence, the BAC-treated plank- research on biofilm-detached cells to further assess their asso- tonic cells, grown at 37 °C, were less round and their mem- ciated microbiological risk and to optimize appropriate disin- brane seemed to be rougher, wrinkled, and deformed fection procedures. Staphylococcus aureus is a pathogenic 296 Ann Microbiol (2019) 69:291–298 Fig. 4 Scanning electron 20°C 37°C micrographs of biofilm-detached and planktonic Staphylococcus aureus cells grown at 20 °C and 37 °C for 24 h, after treatment with benzalkonium chloride at MIC. The control represents cells treated with potassium phosphate buffer bacterium, associated with serious FBDs, and able to adhere work is to investigate the resistance at the cellular level when and form biofilms on food contact surfaces (Kadariya et al. bacteria are detached from biofilm. 2014;Abdallah et al. 2015;Khelissa et al. 2017a). Our find- Our results underlined that the rise of growth temperature ings showed that biofilm-detached cell phenotype is highly from 20 to 37 °C increased the resistance of both biofilm- different from that of the planktonic one. Cells grown under detached and planktonic S. aureus cells to BAC treatment. biofilm state are known to be more resistant to antimicrobial Moreover, the resistance of biofilm-detached cells to BAC agents than those grown under floating state (Davies 2003; was significantly higher than that of planktonic cells what- Batoni et al. 2016). This resistance is often associated with ever the studied growth temperature. However, Rollet et al. the extracellular matrix, a compact structure, which may pre- (2009) reported that the sessile, biofilm-detached, and vent disinfectants from penetrating and reaching the bacterial planktonic Pseudomonas aeruginosa showed the same an- cells (Abdallah et al. 2014, 2015). The goal in the present tibiotic susceptibility profile. Our results highlight the fact Planktonic S. aureus Biofilm-detached S. aureus Control BAC-treated Control BAC-treated Ann Microbiol (2019) 69:291–298 297 cell membranes, promotes the release of intracellular material and thereby significantly changes cell homeostasis. Regarding our data, and in order to explain the observed results, the modification of the cellular membrane fatty acid composition, which controls the membrane fluidity, was in- vestigated. In the present study, S. aureus biofilm-detached cells displayed a significantly higher SFA amount compared to planktonic cells, and this is due to the high increase in the amount of long-chain SFA, including aC19, C18, and C20. Furthermore, the amounts of aC19, C18, and C20 of biofilm- detached and planktonic cells incubated at 37 °C were signif- icantly higher than those incubated at 20 °C. Zhang and Rock (2008) have underlined that the straight-chain saturated fatty acids are linear and are also known to pack together to make a rigid membrane bilayer with a high phase transition. Nevertheless, the amounts of aC15 remained at a stable level in both in S. aureus studied cell populations. At the same time, aC15 amounts were lower in biofilm-detached cells than in planktonic ones whatever the studied growth temperature. It has been reported that aC15 has a low melting point which makes it a major determinant of membrane fluidity for many Gram-positive bacteria (Kaneda 1991). In addition, the fatty acid melting point decreases as their chain length shortens (Annous et al. 1997). The phase transition temperatures of the phosphatidylcholine containing aC19:0 (36.7 °C) and C18 (26 °C) are significantly higher than those of phosphati- Fig. 5 Membrane fatty acid composition of biofilm-detached and plank- tonic Staphylococcus aureus cells, grown at 20 °C and 37 °C for 24 h. a: dylcholine containing aC15 (− 13.9 °C) (Schindler 1980; anteiso Suutari and Laakso 1994). Thus, the membrane fatty acid profiles of the studied S. aureus cells would have probably resulted in a lower fluidity of the biofilm-detached cell mem- branes when compared to those of planktonic cells. This could that biofilm-detached cells in food processing industry rep- resent a serious public health problem. In fact, after being explain the greater resistance of the biofilm-detached cells to released from biofilm, these cells represent a real threat as BAC treatment. Furthermore, the transition to a fatty acid they acquired a high resistance profile and require an effec- profile with stable amounts of aC15, the increase of aC19:0, tive antimicrobial treatment. It has been reported that qua- C18, and C20 amounts in biofilm-detached and planktonic ternary ammonium compounds have the bacterial mem- cells incubated at 37 °C, suggests that the bacterial membranes brane as a main target (Gilbert and Moore 2005;Abdallah may be less fluid at high growth temperatures. Wang et al. et al. 2014). In this context, our results showed that when (2016) recently showed that the increase of growth tempera- biofilm-detached and planktonic S. aureus cells were ex- ture decreased the fluidity of S. aureus membrane in response posed to a BAC concentration of 3 mg/L, an immediate to electroporation. Thus, the increase of growth temperature + + K leakage was measured. The K efflux rate decreased probably decreased the permeability of S. aureus membranes with the increase of the growth temperature from 20 to to BAC. Taken together, our findings may explain the increase 37 °C. These results also showed that at a given growth of planktonic and biofilm-detached cells resistance to BAC temperature, the K leakage was higher in planktonic than treatment with the increase of growth temperature and as well in biofilm-detached cells. Thus, the BAC bactericidal activ- as the greater resistance of biofilm-detached cells to BAC ity depends both on the physiological state and on the treatment compared to their planktonic counterparts. Overall, growth temperature. These findings were comforted by the the results related to the membrane fluidity corroborate the morphological modifications of bacterial cells when ex- membrane integrity monitored by K efflux findings. posedtoBAC andanalyzedby SEM.Cells treatedwitha In conclusion, our work showed that the resistance of S. BAC concentration of 3 mg/L were less bulky, and their aureus to BAC is dependent on the growth temperature. In membrane seemed to be rougher, wrinkled, and deformed addition, the bacterial physiological state, whether biofilm- compared with untreated cells. This could be a result of the detached or planktonic, is a determinant parameter related to high cell wall–BAC interaction that, in addition to disrupting bacterial susceptibility to disinfectant. Our approach aimed to 298 Ann Microbiol (2019) 69:291–298 Bridier A, Briandet R, Thomas V, Dubois-Brissonnet F (2011) Resistance identify the aspects of bacterial physiology that are affected by of bacterial biofilms to disinfectants: a review. Biofouling 27:1017– BAC activity, beginning with an initial focus on antibacterial 1032. https://doi.org/10.1080/08927014.2011.626899 activity followed by an assessment of cell membrane integrity Chihib N-E, Tierny Y, Mary P, Hornez JP (2005) Adaptational changes in and changes in membrane fluidity. Staphylococcus aureus is cellular fatty acid branching and unsaturation of Aeromonas species as a response to growth temperature and salinity. Int J Food extremely susceptible to BAC which had high antimicrobial Microbiol 102:113–119. https://doi.org/10.1016/j.ijfoodmicro. effect leading to membrane collapse and irreversible loss of 2004.12.005 membrane integrity with consequent leakage of intracellular Chua SL, Liu Y, Yam JKH et al (2014) Dispersed cells represent a distinct K . The resistance behavior correlated with the membrane stage in the transition from bacterial biofilm to planktonic lifestyles. Nat Commun 5:4462 fluidity. Therefore, it is suggested that modification of mem- Davies D (2003) Understanding biofilm resistance to antibacterial agents. brane fatty acid composition seems to be a possible resistance Nat Rev Drug Discov 2:114–122. https://doi.org/10.1038/nrd1008 mechanism, at the cellular scale, for S. aureus cells. Denayer S, Delbrassinne L, Nia Y, Botteldoorn N (2017) Food-borne outbreak investigation and molecular typing: high diversity of Acknowledgements The authors thank the Haut de France region and Staphylococcus aureus strains and importance of toxin detection. FEDER (Fonds européen de développement régional) for their financial Toxins 9:407. https://doi.org/10.3390/toxins9120407 support. Dubois-Brissonnet F, Trotier E, Briandet R (2016) The biofilm lifestyle involves an increase in bacterial membrane saturated fatty acids. Front Microbiol 7:1673. https://doi.org/10.3389/fmicb.2016.01673 Funding This work was carried out within the framework of ALIBIOTECH (Agroalimentaire et Biotechnologie, Nord Pas-de-Calais Gilbert P, Moore LE (2005) Cationic antiseptics: diversity of action under region) program. a common epithet. J Appl Microbiol 99:703–715. https://doi.org/10. 1111/j.1365-2672.2005.02664.x Kadariya J, Smith TC, Thapaliya D (2014) Staphylococcus aureus and Compliance with ethical standards staphylococcal food-borne disease: an ongoing challenge in public health. Biomed Res Int 2014:9 Conflict of interest The authors declare that they have no conflict of Kaneda T (1991) Iso- and anteiso-fatty acids in bacteria: biosynthesis, interest. function, and taxonomic significance. Microbiol Rev 55:288–302 Khelissa SO, Abdallah M, Jama C et al (2017a) Bacterial contamination Research involving human participants and/or animals This article and biofilm formation on abiotic surfaces and strategies to overcome does not contain any studies with human participants or animals per- their persistence. J Mater Environ Sci 8:3326–3346 formed by any of the authors. Khelissa SO, Jama C, Abdallah M et al (2017b) Effect of incubation duration, growth temperature, and abiotic surface type on cell sur- face properties, adhesion and pathogenicity of biofilm-detached Informed consent N/A Staphylococcus aureus cells. AMB Express 7:191. https://doi.org/ 10.1186/s13568-017-0492-0 Publisher’sNote Springer Nature remains neutral with regard to jurisdic- Malanovic N, Lohner K (2016) Antimicrobial peptides targeting gram- tional claims in published maps and institutional affiliations. positive bacteria. Pharmaceuticals 9:59. https://doi.org/10.3390/ ph9030059 Rollet C, Gal L, Guzzo J (2009) Biofilm-detached cells, a transition from References a sessile to a planktonic phenotype: a comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa. FEMS Microbiol Lett 290:135–142. https://doi.org/10.1111/j. Abdallah M, Chataigne G, Ferreira-Theret P et al (2014) Effect of growth 1574-6968.2008.01415.x temperature, surface type and incubation time on the resistance of Schindler H (1980) Introduction to biological membranes. Trends Staphylococcus aureus biofilms to disinfectants. Appl Microbiol Neurosci 3:XXII. https://doi.org/10.1016/S0166-2236(80)80081-6 Biotechnol 98:2597–2607. https://doi.org/10.1007/s00253-013- Simões M, Simões LC, Vieira MJ (2010) A review of current and emer- 5479-4 gent biofilm control strategies. LWT Food Sci Technol 43:573–583. Abdallah M, Benoliel C, Ferreira-Theret P et al (2015) Effect of culture https://doi.org/10.1016/j.lwt.2009.12.008 conditions on the resistance of Pseudomonas aeruginosa biofilms to Suutari M, Laakso S (1994) Microbial fatty acids and thermal adaptation. disinfecting agents. Biofouling 31:49–59. https://doi.org/10.1080/ Crit Rev Microbiol 20:285–328. https://doi.org/10.3109/ 08927014.2014.993390 Annous BA, Becker LA, Bayles DO et al (1997) Critical role of anteiso- C15:0 fatty acid in the growth of Listeria monocytogenes at low Tuson HH, Weibel DB (2013) Bacteria-surface interactions. Soft Matter 9:4368–4380. https://doi.org/10.1039/C3SM27705D temperatures. Appl Environ Microbiol 63:3887–3894 Batoni G, Maisetta G, Esin S (2016) Antimicrobial peptides and their Wang L-H, Wang M-S, Zeng X-A, Liu Z-W (2016) Temperature- interaction with biofilms of medically relevant bacteria. mediated variations in cellular membrane fatty acid composition of Antimicrob Pept Cell Membr Microb Surf Interact 1858:1044– Staphylococcus aureus in resistance to pulsed electric fields. 1060. https://doi.org/10.1016/j.bbamem.2015.10.013 Biochim Biophys Acta BBA Biomembr 1858:1791–1800. https:// Berne C, Ellison CK, Ducret A, Brun YV (2018) Bacterial adhesion at the doi.org/10.1016/j.bbamem.2016.05.003 single-cell level. Nat Rev Microbiol. https://doi.org/10.1038/ Zhang Y-M, Rock CO (2008) Membrane lipid homeostasis in bacteria. s41579-018-0057-5 Nat Rev Microbiol 6:222–233. https://doi.org/10.1038/nrmicro1839 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Comparative study of growth temperature impact on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells to benzalkonium chloride

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Publisher
Springer Journals
Copyright
Copyright © 2018 by Università degli studi di Milano
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-018-1419-y
Publisher site
See Article on Publisher Site

Abstract

The present study investigated and compared the effect of growth temperature on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells, to benzalkonium chloride (BAC). This study also highlights the impact of BAC on the bacterial physiology and the role of membrane fluidity regulation as a bacterial resistance mechanism. The minimum inhibitory concentration of BAC was characterized with micro-dilution growth inhibition assay. The BAC treatment was performed on S. aureus cultured at 20 °C and 37 °C, for 24 h. The morphology of S. aureus cells was examined using scanning electron microscopy. The loss of bacterial membrane integrity after BAC treatment was studied by monitoring the intracellular potassium ion leakage using the atomic absorption spectroscopy. The bacterial membrane total fatty acid composition, controlling the membrane fluidity, was analyzed by GC/MS. The results showed that the resistance of S. aureus cells to BAC increased with the increase of growth temperature. The planktonic cells were more susceptible to BAC than biofilm-detached ones. The rise of growth temperature resulted in an increase of S. aureus membrane rigidity. Furthermore, a higher membrane fluidity was observed in planktonic cells when compared to that in the biofilm-detached ones. The resistance of S. aureus seems to depend on the growth temperature. Compared to planktonic cells, biofilm-detached cells showed a greater resistance to BAC. The BAC targets and disturbs the bacterial membrane. Membrane fluidity modulation is likely a one of resistance mechanisms for S. aureus to BAC at the cellular scale. Therefore, disinfection procedures, in food sector, should be adapted for bacteria detached from biofilm. . . . . . Keywords Staphylococcus aureus Biofilm-detached cells Planktonic cells Growth temperature Membrane fluidity Susceptibility to BAC Introduction disinfectants are constantly applied to maintain a high level of surface hygiene in food fields (Khelissa et al. 2017a). Such The emergence of Staphylococcus aureus in food sector rep- procedure is thought to be effective to decrease the microbio- resents a significant risk for public health. In fact, this bacte- logical risk in the highly affected areas. Bacteria are often rium is commonly involved in foodborne diseases (FBDs) attached to surfaces and form a complex structure, called bio- (Denayer et al. 2017). In order to fight against FBDs, film (Tuson and Weibel 2013). The biofilm-associated cells are physiologically different from the planktonic ones (Chua et al. 2014). Furthermore, biofilm-structured cells have a bet- * Nour-Eddine Chihib ter resistance to disinfectants than their planktonic counter- nour-eddine.chihib@univ-lille.fr parts (Bridier et al. 2011). In the food sector, bacteria are usually exposed to different environmental constraints such CNRS, INRA, UMR 8207-UMET-PIHM, Université de Lille, 369 as temperature changes, shear forces, and pH (Simões et al. rue Jules Guesde, 59651 Villeneuve d’Ascq, France 2010; Berne et al. 2018). The exposure of biofilms to high CNRS, ENSCL, UMR 8207-UMET-PSI, Université de Lille, shear forces may result in cell dispersal (Berne et al. 2018). Avenue Dimitri Mendeleïev, 59652 Villeneuve d’Ascq, France Thus, the biofilm represents a bacterial reservoir which, once Université Lyon 1, ISARA Lyon, Laboratoire BioDyMIA, Equipe detached, serves as a continuous source of contamination Mixte d’Accueil, no. 3733, IUT Lyon 1, Technopole Alimentec, rue resulting in severe FBDs. Henri de Boissieu, F-01000 Bourg en Bresse, France 292 Ann Microbiol (2019) 69:291–298 The biofilm resistance mechanisms to antimicrobial Stainless steel slide preparation agents may be explained in several ways (Bridier et al. 2011). The biofilm resistance mechanisms should be ob- The stainless steel (SS) (304 L, Equinox, France) slides were served at the macroscopic and the microscopic levels. At first immersed in acetone (Fluka, Sigma-Aldrich, France) for the macroscopic scale, it seems to be related to the pro- 1 h, then rinsed under distilled water. The slides were soaked duction of an extracellular matrix, composed of extracel- in DDM ECO detergent (1%) for 10 min at 20 °C (ANIOS, lular polymeric substances (EPS), that hinder the disinfec- France) and rinsed under distilled water then under ultrapure tant diffusion (Bridier et al. 2011). However, several stud- water (Milli-Q Academic, Millipore, France). The SS slides ies highlighted the fact that the biofilm matrix cannot were dried and autoclaved (20 min, 120 °C). always explain the biofilm resistance to disinfectants (Abdallah et al. 2014). Therefore, the biofilm resistance at the microscopic or cellular scale, which is thought to be Development of biofilm linked to the modification of bacterial physiology, should be explored. In addition, bacterial cells may adapt to un- The SS slides (90 × 90 mm) were covered with 12 mL of the favorable growth conditions by modifying their mem- corresponding cell suspension (20 °C or 37 °C) adjusted to brane lipid composition (Chihib et al. 2005). The bacterial 10 CFU/mL and incubated at 20 °C for 1 h to allow the membrane, composed of phospholipids and proteins, con- bacterial adhesion. Afterwards, slides were rinsed twice, by stitutes the first line of bacterial defense against antimi- immersion in PPB, to remove loosely attached cells. Then, crobial. The fatty acid composition controls the fluidity of each slide was covered with 12 mL of TSB and incubated bacterial membrane and may hinder the penetration of statically, at 20 °C or 37 °C for 24 h. Following the 24-h antimicrobial into cells (Dubois-Brissonnet et al. 2016; incubation, slides covered with biofilm were rinsed with Malanovic and Lohner 2016). PPB. The strongly attached bacteria were recovered into In this regard, the purpose of this work was to study the 10 mL of PPB by surface scraping, harvested by centrifuga- effect of growth temperature (20 °C and 37 °C) on the resis- tion (5000g, 5 min, 20 °C), and then washed once with 20 mL tance of S. aureus cells, issued from planktonic culture or of PPB. The bacterial suspension concentrations were adjust- detached from biofilm formed on stainless steel, to BAC. ed in PPB to 10 CFU/mL as above cited. This study also aimed to study the membrane fluidity, of planktonic and biofilm-detached S. aureus, in order to charac- terize its involvement as a resistance mechanism to BAC treat- BAC minimum inhibitory concentration ment at the cellular level. determination The minimum inhibitory concentration (MIC) of BAC was determined by micro-dilution growth inhibition as- Materials and methods says, using a Bioscreen C (Labsystems, Helsinki, Finland). Staphylococcus aureus cells were cultured, as Bacterial culture conditions and suspension previously, in Mueller-Hinton Broth (MHB) (Bio-Rad, preparation France). One hundred microliters of S. aureus suspension (10 CFU/mL) were added to the plate wells containing Staphylococcus aureus CIP4.83strainwas stored(− decreasing BAC concentrations ranging from 25 to 0 mg/ 80 °C) in tryptic soy broth (TSB) (Biokar Diagnostics, L (in MHB). Two BAC-free controls were included, one France) with 40% (v/v) of glycerol. Precultures were pre- with only MHB (sterility control) and the other with MHB paredbyadding100 μL from stock tube to 5 mL of + bacteria (growth control). The micro-dilution plates TSBand incubatedat20°C(for48h)or37°C(for were incubated in the Bioscreen C at 37 °C under contin- 24 h). The cultures were started by inoculating 10 CFU/ uous shaking. The OD wasmeasuredevery2hfor 600 nm mL from the preculture in 50 mL of TSB and incubation 48 h. The MIC was the lowest concentration of the BAC under continuous shaking (160 rpm) at 20 °C or 37 °C. that prevented growth, as measured by optical density. Cultures were stopped at the late exponential phase and Log OD values were plotted versus time (h). The 600 nm cells were pelleted (5000g, 5 min, 20 °C). Bacteria were lag time, of each growth curve, was estimated by extrap- washed twice with potassium phosphate buffer (PPB; olating the linear portion of Log OD versus time 600 nm 100 mM, pH 7). Finally, the bacterial suspensions were plot back to the initial OD . The growth rate (μ) 600 nm adjusted to 10 CFU/mL in PPB, by fixing the optical was calculated during the exponential growth phase using density at 600 nm to 0.110 ± 0.005, using a Jenway the formula: dN/dt = kN,where N is the Log OD of 600 nm 6320D UV/visible light spectrophotometer. cells, t the time, and k is thegrowth rateconstant. Ann Microbiol (2019) 69:291–298 293 Disinfection of S. aureus cells (Shimadzu, Japan) with a capillary column (Zebron ZB- FFAP, Phenomenex, Australia) and connected to mass For the disinfection assay, 1 mL of bacterial suspension, ad- spectrometer (Thermo-Finnigan Trace DSQ, Thermo justed to 10 CFU/mL, was introduced to 1 mL of 6 mg/L Fisher Scientific, USA). BAC solution (prepared in PPB). After 5-min contact time at 20 °C, 1 mL of this mixture was transferred into 9 mL of Scanning electron microscope observation neutralizing solution (Abdallah et al. 2014) to stop the anti- bacterial action. Thereafter, tenfold serial dilutions were done The morphology of S. aureus cells upon BAC treatment was in PPB. Samples of 100 μL were spread onto tryptic soy agar assessed by scanning electron microscope (SEM). One milli- broth plates (TSA; Biokar Diagnostics, France) and incubated liter volume of the BAC- or PPB-treated then neutralized at 37 °C for 24 h. After the incubation time, the number of planktonic or biofilm-detached cell suspensions was filtered viable and culturable cells was counted on the plates and the through a 0.2-μm-pore-size polycarbonate membrane filter results were expressed in Log CFU/mL. For the control as- (Schleicher & Schuell, Dassel, Germany) then fixed for 4 h says, the disinfectant solution was replaced by PPB. with 2% glutaraldehyde, in cacodylate buffer 0.1 M pH 7, at 20 °C. Fixed samples were then dehydrated in an ascending BAC-induced cell membrane permeabilization ethanol series (50, 70, 95, and 2 × 100% (v/v) ethanol), for 15 min at each concentration. Samples were critical point Biofilm-detached and planktonic S. aureus cells grown at dried and coated with thin carbon film before examination in 20 and 37 °C were concentrated to 10 CFU/mL. Five the SEM. Microscopy was performed with a Hitachi S4700 milliliters of the concentrated bacterial suspensions was microscope at 3 kV. introduced into silicone cap glass reaction vessels (100-mL wide-necked flasks) containing 45 mL of BAC Statistical analysis solution prepared in HEPES buffer (final concentration of 3 mg/L in 50 mL final volume) or HEPES buffer (nega- The results are presented as mean values and their standard tive control). The K concentration at time zero was mea- error of the mean. Data analysis was performed using Sigma sured in a tenfold dilution of the bacterial suspension fil- Plot 11.0 (Systat Software Inc.), using one-way ANOVA ™ ™ trate (0.2 μm, Sartorius Minisart NML Syringe Filters, (Tukey’s method). Results were considered significant at a P France) before contact with BAC solution. After the in- value of <0.05. troduction of bacterial suspension to the reaction vessel containing the BAC solution, samples (4 mL) were filter sterilized at 5, 10, 15, 20, 30, 60, and 90 min. Each sam- Results ple was removed using a sterile plastic syringe attached to a sterile needle to enable easy access to the reaction mix- Determination of the BAC minimum inhibitory ture suspension through the silicon cap. The K concen- concentration tration in filtrate samples was determined using a Varian SpectrAA 55/B atomic absorption spectrometer in flame The results of Fig. 1 showed that at a concentration of 0.2 mg/ emission mode (wavelength 766.5 nm; slit 0.7 nm high; L, cells have the same growth behavior of the control (cells air-acetylene flame). without BAC). At a concentration of 0.4, 0.8, and 1.5 mg/L of BAC, the growth profile was different from the control (P< Cellular fatty acid extraction and analysis 0.05). The results showed that the BAC minimum inhibitory concentration was of 3 mg/L. Figure 1 showed also that the Biofilm-detached and planktonic S. aureus cells were har- growth rate (μ)and lagtime(λ) were dependent on the BAC vested as cited above, either by scrapping cells embedded concentration. In fact, our results showed that the μ value of S. −1 in biofilm from the rinsed coupons or by centrifuging aureus cultures was of 0.056 h when the BAC concentra- planktonic culture, then resuspended in 10 mL of PPB. tions were ranging from 0 to 1.5 mg/L (Fig. 1). Furthermore, Cells were sonicated (37 kHz, 5 min) and vortexed for the lag time consistently extended with increased BAC con- 30 s. Cells were pelleted by centrifugation (10,000g, centrations (Fig. 1). When the BAC concentrations increased 10 min, 4 °C), and pellets, containing 10 CFU/mL, were from 0 to 1.5 mg/L, the λ increased from 8 to 20 h. washed twice with cold distilled water. Cells were sub- jected to the saponification and methylation (Chihib Susceptibility of S. aureus cells to BAC treatment et al. 2005). Fatty acid methyl ester extraction was real- ized as described previously by Chihib et al. (2005). Data The susceptibility of biofilm-detached and planktonic S. analysis was performed with GC-2014 gas chromatograph aureus cells, grown at 20 °C and 37 °C, to BAC treatment 294 Ann Microbiol (2019) 69:291–298 Fig. 1 Growth curves of Time (h) Staphylococcus aureus grown at 0 5 10 15 20 25 30 35 40 45 37 °C for 48 h, measured at the optic density (OD) of 600 nm -0.1 under different benzalkonium -0.2 chloride concentrations. The concentration at which there was -0.3 a linear growth inhibition was -0.4 considered the MIC. The OD at 600 nm in Mueller-Hinton Broth -0.5 without bacterial inoculum was -0.6 measured to ensure the sterility of the growth medium (Only MHB) -0.7 -0.8 -0.9 -1.0 -1.1 -1.2 -1.3 25 mg/L 12.5 mg/L 6.25 mg/L 3 mg/L 1.5 mg/L 0.8 mg/L 0.4 mg/L 0.2 mg/L was studied. The results showed that the PPB treatment, used Effect of BAC on S. aureus cell membrane as the negative control, had no significant effect on the initial permeability population of S. aureus, whatever the studied conditions (P< 0.05)(Fig. 2). The average of viable and culturable counts of Measurements of K efflux from the bacterial cells were real- cells treated with PPB was of ca 7.3 Log CFU/mL (Fig. 2). ized to assess the cell resistance to BAC treatment. This was The treatment of biofilm-detached cells grown at 20 °C carried out by monitoring the extracellular K concentration in and37°C, for5 minata BACconcentration of 3 mg/L, biofilm-detached and planktonic S. aureus cells grown at resulted in a significant reduction of the initial viable and 20 °C and 37 °C for 24 h (Fig. 3). Our results also showed culturable count of 3.2 and 1.8 Log CFU/mL, respectively that the HEPES buffer addition (control) had no effect on K (P<0.05)(Fig. 2). However, the treatment of planktonic efflux which remained stable whatever the studied condition. cells grown at 20 °C and 37 °C, for 5 min at a BAC The addition of BAC at a final concentration of 3 mg/L result- concentration of 3 mg/L, significantly reduced the initial ed in an immediate increase of extracellular K . Figure 3 viable and culturable count by 4.3 and 3.1 Log CFU/mL, showed that K efflux was higher in planktonic than in respectively (P < 0.05)(Fig. 2). Furthermore, the results biofilm-detached cell suspensions whatever the also showed that the remained viable and culturable count growth temperature condition (P < 0.05). Five minutes after of biofilm-detached cells after BAC treatment was of ca. the BAC addition, the extracellular K concentration in- 1.3 Log CFU/mL higher than that of planktonic cells creased to 4.8 and 1.7 mg/L in the planktonic cell suspensions whatever the cell growth temperature (P < 0.05)(Fig. 2). grown at 20 °C and 37 °C, respectively (P < 0.05)(Fig. 3). Fig. 2 Susceptibility of biofilm- detached and planktonic Staphylococcus aureus cells, grown at 20 °C and 37 °C for 24 h, to benzalkonium chloride treatment (BAC). The bacterial count is presented in Log CFU/ mL after 100 mM potassium phosphate buffer (control) or BAC treatment (3 mg/L for 5min) Log OD 600 nm Ann Microbiol (2019) 69:291–298 295 Fig. 3 Kinetics of intracellular potassium leakage from biofilm- detached and planktonic Staphylococcus aureus cells, grown at 20 °C and 37 °C for 24 h. The K concentration is presented in milligrams per liter. The treatment with HEPES buffer represents the negative control Under the same conditions, the extracellular K increased to a compared with untreated cells (Fig. 4). The treated S. aureus concentration of 1.9 mg/L and 0.5 mg/L in the biofilm-detached biofilm-detached cells, grown at 37 °C, became generally cell suspensions grown at 20 °C and 37 °C, respectively (Fig. 3). distorted in shape and had few holes in their cell walls (Fig. 4). After 90 min of BAC addition into bacterial suspensions grown at 20 °C, the extracellular K concentration reached Effect of growth temperature on S. aureus membrane concentrations of 10 and 15 mg/L in suspension of biofilm- fatty acid profiles detached and planktonic cells (P < 0.05)(Fig. 3). However, when cells were issued from cultures grown at 37 °C, the K Membrane fatty acid (FA) profile of biofilm-detached and extracellular concentration in planktonic and biofilm-detached planktonic S. aureus cells, grown for 24 h at 20 °C and suspensions increased to a concentration of 5 and 2.5 mg/L, 37 °C, was analyzed (Fig. 5). This investigation was per- respectively (P < 0.05)(Fig. 3). formed to study the effect of growth temperature on the mem- brane fatty acid composition which controls the membrane fluidity. Figure 5 showed that the amounts of anteiso C15 Impact of BAC treatment on S. aureus morphology (aC15) were maintained at a stable level whatever the growth changes temperature (P > 0.05). The results also showed that the aC15 amount of biofilm-detached cell membranes was significantly To investigate structural modifications of S. aureus biofilm- lower than that of planktonic cells (P < 0.05). Moreover, the detached and planktonic cells after the exposition to BAC total long-chain FA amounts, the aC19, C18, and C20, of treatment, bacterial samples were analyzed by SEM. The un- biofilm-detached cells were 1.3-fold higher than their plank- treated biofilm-detached and planktonic S. aureus cells culti- tonic counterparts whatever the growth temperature (P< vated at 20 °C and 37 °C looked round and exhibited an 0.05)(Fig. 5). The increase of growth temperature from 20 undamaged normal smooth lining (Fig. 4). However, when to 37 °C promoted an increase in long-chain FA amounts biofilm-detached and planktonic S. aureus cells were exposed including aC19, C18, and C20 (P < 0.05)(Fig. 5). for 5 min to a BAC concentration of 3 mg/L, significant mor- phological changes were observed (Fig. 4). Staphylococcus aureus planktonic cells grown at 20 °C showed holes in their cell walls (Fig. 4). Staphylococcus aureus biofilm-detached Discussion cells, cultivated at 20 °C, showed multiple dents on their sur- faces (Fig. 4). However, the morphological changes of S. Biofilm-detached cells constitute a major source of bacterial aureus biofilm-detached and planktonic cells, grown at dissemination and contamination of food contact surfaces 37 °C, seemedtobelesspronouncedthanthoseof their (Khelissa et al. 2017b). Thus, it is of importance to conduct 20 °C counterparts (Fig. 4). Hence, the BAC-treated plank- research on biofilm-detached cells to further assess their asso- tonic cells, grown at 37 °C, were less round and their mem- ciated microbiological risk and to optimize appropriate disin- brane seemed to be rougher, wrinkled, and deformed fection procedures. Staphylococcus aureus is a pathogenic 296 Ann Microbiol (2019) 69:291–298 Fig. 4 Scanning electron 20°C 37°C micrographs of biofilm-detached and planktonic Staphylococcus aureus cells grown at 20 °C and 37 °C for 24 h, after treatment with benzalkonium chloride at MIC. The control represents cells treated with potassium phosphate buffer bacterium, associated with serious FBDs, and able to adhere work is to investigate the resistance at the cellular level when and form biofilms on food contact surfaces (Kadariya et al. bacteria are detached from biofilm. 2014;Abdallah et al. 2015;Khelissa et al. 2017a). Our find- Our results underlined that the rise of growth temperature ings showed that biofilm-detached cell phenotype is highly from 20 to 37 °C increased the resistance of both biofilm- different from that of the planktonic one. Cells grown under detached and planktonic S. aureus cells to BAC treatment. biofilm state are known to be more resistant to antimicrobial Moreover, the resistance of biofilm-detached cells to BAC agents than those grown under floating state (Davies 2003; was significantly higher than that of planktonic cells what- Batoni et al. 2016). This resistance is often associated with ever the studied growth temperature. However, Rollet et al. the extracellular matrix, a compact structure, which may pre- (2009) reported that the sessile, biofilm-detached, and vent disinfectants from penetrating and reaching the bacterial planktonic Pseudomonas aeruginosa showed the same an- cells (Abdallah et al. 2014, 2015). The goal in the present tibiotic susceptibility profile. Our results highlight the fact Planktonic S. aureus Biofilm-detached S. aureus Control BAC-treated Control BAC-treated Ann Microbiol (2019) 69:291–298 297 cell membranes, promotes the release of intracellular material and thereby significantly changes cell homeostasis. Regarding our data, and in order to explain the observed results, the modification of the cellular membrane fatty acid composition, which controls the membrane fluidity, was in- vestigated. In the present study, S. aureus biofilm-detached cells displayed a significantly higher SFA amount compared to planktonic cells, and this is due to the high increase in the amount of long-chain SFA, including aC19, C18, and C20. Furthermore, the amounts of aC19, C18, and C20 of biofilm- detached and planktonic cells incubated at 37 °C were signif- icantly higher than those incubated at 20 °C. Zhang and Rock (2008) have underlined that the straight-chain saturated fatty acids are linear and are also known to pack together to make a rigid membrane bilayer with a high phase transition. Nevertheless, the amounts of aC15 remained at a stable level in both in S. aureus studied cell populations. At the same time, aC15 amounts were lower in biofilm-detached cells than in planktonic ones whatever the studied growth temperature. It has been reported that aC15 has a low melting point which makes it a major determinant of membrane fluidity for many Gram-positive bacteria (Kaneda 1991). In addition, the fatty acid melting point decreases as their chain length shortens (Annous et al. 1997). The phase transition temperatures of the phosphatidylcholine containing aC19:0 (36.7 °C) and C18 (26 °C) are significantly higher than those of phosphati- Fig. 5 Membrane fatty acid composition of biofilm-detached and plank- tonic Staphylococcus aureus cells, grown at 20 °C and 37 °C for 24 h. a: dylcholine containing aC15 (− 13.9 °C) (Schindler 1980; anteiso Suutari and Laakso 1994). Thus, the membrane fatty acid profiles of the studied S. aureus cells would have probably resulted in a lower fluidity of the biofilm-detached cell mem- branes when compared to those of planktonic cells. This could that biofilm-detached cells in food processing industry rep- resent a serious public health problem. In fact, after being explain the greater resistance of the biofilm-detached cells to released from biofilm, these cells represent a real threat as BAC treatment. Furthermore, the transition to a fatty acid they acquired a high resistance profile and require an effec- profile with stable amounts of aC15, the increase of aC19:0, tive antimicrobial treatment. It has been reported that qua- C18, and C20 amounts in biofilm-detached and planktonic ternary ammonium compounds have the bacterial mem- cells incubated at 37 °C, suggests that the bacterial membranes brane as a main target (Gilbert and Moore 2005;Abdallah may be less fluid at high growth temperatures. Wang et al. et al. 2014). In this context, our results showed that when (2016) recently showed that the increase of growth tempera- biofilm-detached and planktonic S. aureus cells were ex- ture decreased the fluidity of S. aureus membrane in response posed to a BAC concentration of 3 mg/L, an immediate to electroporation. Thus, the increase of growth temperature + + K leakage was measured. The K efflux rate decreased probably decreased the permeability of S. aureus membranes with the increase of the growth temperature from 20 to to BAC. Taken together, our findings may explain the increase 37 °C. These results also showed that at a given growth of planktonic and biofilm-detached cells resistance to BAC temperature, the K leakage was higher in planktonic than treatment with the increase of growth temperature and as well in biofilm-detached cells. Thus, the BAC bactericidal activ- as the greater resistance of biofilm-detached cells to BAC ity depends both on the physiological state and on the treatment compared to their planktonic counterparts. Overall, growth temperature. These findings were comforted by the the results related to the membrane fluidity corroborate the morphological modifications of bacterial cells when ex- membrane integrity monitored by K efflux findings. posedtoBAC andanalyzedby SEM.Cells treatedwitha In conclusion, our work showed that the resistance of S. BAC concentration of 3 mg/L were less bulky, and their aureus to BAC is dependent on the growth temperature. In membrane seemed to be rougher, wrinkled, and deformed addition, the bacterial physiological state, whether biofilm- compared with untreated cells. This could be a result of the detached or planktonic, is a determinant parameter related to high cell wall–BAC interaction that, in addition to disrupting bacterial susceptibility to disinfectant. Our approach aimed to 298 Ann Microbiol (2019) 69:291–298 Bridier A, Briandet R, Thomas V, Dubois-Brissonnet F (2011) Resistance identify the aspects of bacterial physiology that are affected by of bacterial biofilms to disinfectants: a review. Biofouling 27:1017– BAC activity, beginning with an initial focus on antibacterial 1032. https://doi.org/10.1080/08927014.2011.626899 activity followed by an assessment of cell membrane integrity Chihib N-E, Tierny Y, Mary P, Hornez JP (2005) Adaptational changes in and changes in membrane fluidity. Staphylococcus aureus is cellular fatty acid branching and unsaturation of Aeromonas species as a response to growth temperature and salinity. Int J Food extremely susceptible to BAC which had high antimicrobial Microbiol 102:113–119. https://doi.org/10.1016/j.ijfoodmicro. effect leading to membrane collapse and irreversible loss of 2004.12.005 membrane integrity with consequent leakage of intracellular Chua SL, Liu Y, Yam JKH et al (2014) Dispersed cells represent a distinct K . The resistance behavior correlated with the membrane stage in the transition from bacterial biofilm to planktonic lifestyles. Nat Commun 5:4462 fluidity. Therefore, it is suggested that modification of mem- Davies D (2003) Understanding biofilm resistance to antibacterial agents. brane fatty acid composition seems to be a possible resistance Nat Rev Drug Discov 2:114–122. https://doi.org/10.1038/nrd1008 mechanism, at the cellular scale, for S. aureus cells. Denayer S, Delbrassinne L, Nia Y, Botteldoorn N (2017) Food-borne outbreak investigation and molecular typing: high diversity of Acknowledgements The authors thank the Haut de France region and Staphylococcus aureus strains and importance of toxin detection. FEDER (Fonds européen de développement régional) for their financial Toxins 9:407. https://doi.org/10.3390/toxins9120407 support. Dubois-Brissonnet F, Trotier E, Briandet R (2016) The biofilm lifestyle involves an increase in bacterial membrane saturated fatty acids. Front Microbiol 7:1673. https://doi.org/10.3389/fmicb.2016.01673 Funding This work was carried out within the framework of ALIBIOTECH (Agroalimentaire et Biotechnologie, Nord Pas-de-Calais Gilbert P, Moore LE (2005) Cationic antiseptics: diversity of action under region) program. a common epithet. J Appl Microbiol 99:703–715. https://doi.org/10. 1111/j.1365-2672.2005.02664.x Kadariya J, Smith TC, Thapaliya D (2014) Staphylococcus aureus and Compliance with ethical standards staphylococcal food-borne disease: an ongoing challenge in public health. Biomed Res Int 2014:9 Conflict of interest The authors declare that they have no conflict of Kaneda T (1991) Iso- and anteiso-fatty acids in bacteria: biosynthesis, interest. function, and taxonomic significance. Microbiol Rev 55:288–302 Khelissa SO, Abdallah M, Jama C et al (2017a) Bacterial contamination Research involving human participants and/or animals This article and biofilm formation on abiotic surfaces and strategies to overcome does not contain any studies with human participants or animals per- their persistence. J Mater Environ Sci 8:3326–3346 formed by any of the authors. Khelissa SO, Jama C, Abdallah M et al (2017b) Effect of incubation duration, growth temperature, and abiotic surface type on cell sur- face properties, adhesion and pathogenicity of biofilm-detached Informed consent N/A Staphylococcus aureus cells. AMB Express 7:191. https://doi.org/ 10.1186/s13568-017-0492-0 Publisher’sNote Springer Nature remains neutral with regard to jurisdic- Malanovic N, Lohner K (2016) Antimicrobial peptides targeting gram- tional claims in published maps and institutional affiliations. positive bacteria. Pharmaceuticals 9:59. https://doi.org/10.3390/ ph9030059 Rollet C, Gal L, Guzzo J (2009) Biofilm-detached cells, a transition from References a sessile to a planktonic phenotype: a comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa. FEMS Microbiol Lett 290:135–142. https://doi.org/10.1111/j. Abdallah M, Chataigne G, Ferreira-Theret P et al (2014) Effect of growth 1574-6968.2008.01415.x temperature, surface type and incubation time on the resistance of Schindler H (1980) Introduction to biological membranes. Trends Staphylococcus aureus biofilms to disinfectants. Appl Microbiol Neurosci 3:XXII. https://doi.org/10.1016/S0166-2236(80)80081-6 Biotechnol 98:2597–2607. https://doi.org/10.1007/s00253-013- Simões M, Simões LC, Vieira MJ (2010) A review of current and emer- 5479-4 gent biofilm control strategies. LWT Food Sci Technol 43:573–583. Abdallah M, Benoliel C, Ferreira-Theret P et al (2015) Effect of culture https://doi.org/10.1016/j.lwt.2009.12.008 conditions on the resistance of Pseudomonas aeruginosa biofilms to Suutari M, Laakso S (1994) Microbial fatty acids and thermal adaptation. disinfecting agents. Biofouling 31:49–59. https://doi.org/10.1080/ Crit Rev Microbiol 20:285–328. https://doi.org/10.3109/ 08927014.2014.993390 Annous BA, Becker LA, Bayles DO et al (1997) Critical role of anteiso- C15:0 fatty acid in the growth of Listeria monocytogenes at low Tuson HH, Weibel DB (2013) Bacteria-surface interactions. Soft Matter 9:4368–4380. https://doi.org/10.1039/C3SM27705D temperatures. Appl Environ Microbiol 63:3887–3894 Batoni G, Maisetta G, Esin S (2016) Antimicrobial peptides and their Wang L-H, Wang M-S, Zeng X-A, Liu Z-W (2016) Temperature- interaction with biofilms of medically relevant bacteria. mediated variations in cellular membrane fatty acid composition of Antimicrob Pept Cell Membr Microb Surf Interact 1858:1044– Staphylococcus aureus in resistance to pulsed electric fields. 1060. https://doi.org/10.1016/j.bbamem.2015.10.013 Biochim Biophys Acta BBA Biomembr 1858:1791–1800. https:// Berne C, Ellison CK, Ducret A, Brun YV (2018) Bacterial adhesion at the doi.org/10.1016/j.bbamem.2016.05.003 single-cell level. Nat Rev Microbiol. https://doi.org/10.1038/ Zhang Y-M, Rock CO (2008) Membrane lipid homeostasis in bacteria. s41579-018-0057-5 Nat Rev Microbiol 6:222–233. https://doi.org/10.1038/nrmicro1839

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Annals of Microbiology – Springer Journals

Published: Dec 13, 2018

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