Edited by: Miklos Fuzi, Semmelweis University, Hungary
Reviewed by: George Seghal Kiran, Pondicherry University, India; Eduardo J. Gudiña, University of Minho, Portugal; Joseph Selvin, Pondicherry University, India
This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
One current strategy to deal with the serious issue of antibiotic resistance is to use biosurfactants, weak antimicrobials in their own right, with antibiotics in order to extend the efficacy of antibiotics. Although an adjuvant effect has been observed, the underlying mechanisms are poorly understood. To investigate the nature of the antibiotic and biosurfactant interaction, we undertook a scanning electron microscopy (SEM) and atomic force microscopy (AFM) microscopic study of the effects of the tetracycline antibiotic, combined with sophorolipid and rhamnolipid biosurfactants, on Methicillin-resistant
Antimicrobial resistance (AMR) is a serious challenge (
The joint use of biosurfactants and antibiotics is a recent development and published data on this synergetic effect is scarce. In one study (
In this report, we used scanning electron microscopy (SEM) and atomic force microscopy (AFM) to investigate the joint action of biosurfactants and antibiotics for both fixed and live cells. To the best of our knowledge, it is the first time that these two microscopic techniques have been used together to study such a system. The aim is to measure morphological changes of the bacterial surfaces as well as changes in local surface properties (stiffness, adhesion, measured by AFM), to help understand the effect of these antimicrobials on bacteria. The system of choice was methicillin-resistant
The bacterial film were prepared on glass slides 15 × 20 mm2, cleaned with 70% ethanol and autoclaved. To optimize the adsorption of the bacteria on these substrates, the glass was first treated with polyethyleneimine (PEI); a polycationic polymer which has already proved successful in giving high coverage of
The
The PEI coated glass slides (as described above), positioned in sterilized petri dishes, were fully immersed in ∼10 ml of bacterial suspension at OD = 0.1 and left to incubate for 24 h at 37°C. Following incubation, the slides were removed, dried under nitrogen and stored in an airtight container at room temperature until needed. Throughout the manuscript, these bacterial samples which have not been treated with either biosurfactant or antbiotic are referred as control samples.
The biosurfactants were extract of RL composed of Mono and di-rhamnolipids (Jeneil Biosurfactants JO LLC, product. num. JBR 425) and extract of SL (Ecover, product num. SL18-SF-D/E01-C-5.) containing a mixture of acidic and lactonic SL, obtained through personal arrangements with Jeneil Biosurfactants and Ecover company.
According to the product datasheet, the RL was produced from sterilized and centrifuged fermentation broth that has had all protein removed and partially decolorized. Two major rhamnolipids, RLL (R1 = C26 H48 O9) and RRLL (R2 = C32 H58 O13) are present. For the SL biosurfactant the manufacturer information stipulates that those correspond to a mixture of acidic and lactonic sophorolipids. Hence, we consider those biosurfactants to be clarified crude extracts and provide further compositional analysis in section “Antimicrobial Treatments.”
Liquid chromatography electrospray ionization mass spectrometry (LC /ESI /MS) is a highly selective and sensitive analysis technique (
Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) is a soft ionization mass spectrometry technique that allows the identification of intact compounds (
It has the advantage of high sensitivity, speed of analysis, wide applicability with a good tolerance toward contaminants (
The biosurfactant’s surface activity and ability to form micelle were studied using a pendant drop technique (CAM 200 from KSV Instruments Ltd.). The size and shape of the drop depends on the competition between gravitational forces and interfacial tension, hence the surface tension of the liquid can be obtained by this technique. The system was calibrated with a glass sphere with diameter 3.90 ± 0.05 mm, giving a surface tension for DI water of 72 ± 1 mN/m. The biosurfactants were tested in solution of increasing concentration to avoid cross-contamination. The critical micelle concentration (CMC) is that at which the surface tension becomes independent of concentration. The measurements were done for a range of drop volumes, with at least five drop measurements per drop volume. The saving of the optical micrographs of the pendant drops and the subsequent estimation of the surface tension by the instrument software were carried out only a few seconds after each dispensation to avoid evaporation effects. The software also permitted to calculate the drop volume more accurately, as this variable is required in the calculation of the surface tension. Generally, it is found that one needs to use experimental conditions giving a Worthington number W
Where V
The measurements of MIC for the two biosurfactants was carried out using a protocol adapted from a published microdilution assay (
As antimicrobials, it was found that MIC of the RL and SL biosurfactants were, respectively, 1.25 and 2% (see “Results” below). These fairly large MIC values are indicative of these biosurfactants being weak antimicrobials in their own right, as expected.
As the focus of this microscopic study is to examine how RL and SL can assist tetracycline, rather than the other way around, the biosurfactant concentrations were kept five times below their respective MIC value; 20% of the MIC, i.e., at 0.25% for RL and 0.4% for SL. Hence, in terms of their antimicrobial effect, those biosurfactant concentrations are low. In addition, sophorolipid is recognized as having low toxicity (
A number of bacterial films were prepared to investigate the effect of biosurfactants and antibiotics on the bacteria. The samples are described in
Sample labeling and concentrations.
Sample name | Concentrations of biosurfactant and antibiotic in final solutions |
Control | Not treated |
Sub | 0.4 μg/ml tetracycline (~80% of MIC |
Supra | 0.6 μg/ml tetracycline (~120% of MIC |
RL | 0.25% Rhamnolipid (~20% of MIC |
SL | 0.4% Sophorolipid (~20% of MIC |
Sub-RL | 0.4 μg/ml tetracycline and 0.25% Rhamnolipid |
Sub-SL | 0.4 μg/ml tetracycline and 0.4% Sophorolipid |
Supra-RL | 0.6 μg/ml tetracycline and 0.25% Rhamnolipid |
Supra-SL | 0.6 μg/ml tetracycline and 0.4% Sophorolipid |
One should note that the seemingly low tetracycline concentrations used in this study (0.4 μg/ml and 0.6 μg/ml) are in line with the MIC for tetracycline (i.e., respectively, 80 and 120% of the MIC), hence, in terms of its potential effect on
The following procedure was adopted. The
HMDS drying is a standard protocol for preparing biological samples for SEM observation (
All samples examined by AFM were allowed to warm up to room temperature before measurements were made. A Bruker Dimension 3100 SPM system was used. Tapping mode atomic force microscopy (TAFM) imaging of the deposited
CAFM images were acquired at room temperature (∼20°C), at 1 Hz scanning rate with a 0.3 V deflection set point. The force curves were acquired with a relative deflection trigger of 0.2–0.3 V at a 1 Hz frequency, equivalent to a Z displacement rate and loading rates of 1 μm/s and 30 nN/s, respectively. To ensure that force curves were carried out on target (i.e., on adsorbed bacteria), the following protocol was followed. For dense
The deformation of the bacterial surface was also investigated, using the approach segments of the force curves obtained in PBS and transforming the cantilever deflection δ into the surface deformation
where
The samples were prepared in triplicates and the statistical analysis was performed using Microsoft Excel. Data in bar charts are presented as mean and standard error for at least 10 measurements. A Student
The compositional analysis resulting from the HPLC spectra is shown in
The pendant drop measurements showed that the rhamnolipid reduced the surface tension of DI water to 38 ± 3 mN/m at a CMC of 180 ± 5 ppm and that the sophorolipid reduced the surface tension of the solution to 46 ± 3 mN/m at a CMC of 177 ± 5 ppm. Previous studies found CMC values in line with the present measurements of ∼100 ppm for sophorolipid (
The results for the MIC measurements for the two biosurfactants are shown in
We found that our simple preparation technique for adsorbed bacteria on PEI-treated glass allowed for a clear differentiation of the combined effects of biosurfactants and antibiotics on a multi-resistant bacterial pathogen, as observed by SEM and AFM microscopy.
Low kV FESEM micrographs of adsorbed
Low kV FESEM micrographs of adsorbed
FESEM micrographs of six
Bacterial diameters for
Bacteria treated with rhamnolipids were covered by rough deposits (see
Once the antibiotic and biosurfactants were used together, the trends were very different. The rhamnolipid in the sub-RL and supra-RL samples did not change the morphology of the adsorbed bacterial cells, they retain a shape and diameter similar to that of the control
The bacterial samples in their hydrated state were examined by AFM microscopy. In
In
Four micrometer AFM height
AFM microscopy on the bacterial film treated with tetracycline and/or biosurfactants proved difficult, essentially because, as observed in FESEM microscopy, the bacterial coverage was poor.
Fourty five micrometer TAFM height images in air of
Thirty micrometer AFM images of
Fifteen micrometer AFM images of
Thirty micrometer CAFM height images of
Thirty micrometer CAFM height images of
Representative AFM force curves are shown in
Representative AFM force curves for adsorbed
Bar chart of average AFM pull-off forces for the adsorbed
The deformation analysis of the bacterial surfaces is shown in
Elastic deformation of adsorbed
A few studies have shown that there is merit in combining biosurfactants and antibiotics to address the issue of antibiotic resistance (
The main results exposed in the previous section have been summarized in
Summary of results; effects of treatments with respect to the control sample of
The discussion below is organized in two sections; one focused on the control bacteria, another examining the effect of the antimicrobial treatments, with references to previously published results when relevant.
The AFM force curves showed that the control
Firstly, despite the uniform coverage of adsorbed untreated
Looking at the bacteria treated with supra-MIC concentration of tetracycline, its hydrophilic character increased with respect to that of the control bacteria. This could be due to adsorption of tetracycline onto the bacterial surface (i.e., both hydrophilic). In addition, the positively charged dimethyl-amino group of zwitterionic tetracycline can bond with the anionic teichoic acid chains extending though the peptidoglycan film (
When the bacteria are exposed solely to the biosurfactants, we observe the following. The rhamnolipid treatment gives the largest increases in adhesive force, particularly of its hydrophilic component. As rhamnolipids are amphiphilic, singly-adsorbed rhamnolipid molecules would present their hydrophilic head to the hydrophilic
Examiming now the combined treatments, a striking result from the SEM micrographs is the observation of enlarged and, sometimes, damaged and punctured bacteria for the sub-SL treatment. Comparing these results to the published literature on this particular system (i.e., tetracycline and
Looking back to the comparison with the control sample, and examining all combined treatments,
Treating
The datasets generated for this study are available on request to the corresponding author.
PL, PN, JD, and IB conceived and designed the investigation. AJ prepared the samples. BO’H and JM carried out the treatments for SEM observation. PL and AS performed the SEM and AFM experiments. PL drafted the manuscript with helpful suggestions and contributions from all. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
HPLC analysis spectrum of rhamnolipid and table of results with approximate probable molecular weights.
MALDI-TOF analysis spectrum of sophorolipid and table of results with approximate probable molecular weights.
Results for microdilution assay: The bacterial number are in CFU/ml.
30 μm TAFM height images of glass surface