Microbial drug resistance is considered a global public health problem. The spreading of multidrug-resistant (MDR) bacteria threatens the healthcare system. The increasing cost of treatment, prolonged hospitalization, and failure to prevent serious infections are affected by the overuse of antibiotics [1
]. Because of the lack of effective antimicrobial agents, the medical procedures for immune-compromised patients such as those needing organ transplantation, cancer chemotherapy, diabetes management, and major surgery become seriously high risk [3
]. For these reasons, in 2014, the World Health Organization emphasized the urgent need to develop new alternative antimicrobial agents for a post-antibiotic era [4
]. Among the MDR pathogenic bacteria, Acinetobacter baumannii
and Pseudomonas aeruginosa
are among the common causes of morbidity and mortality in hospitals [5
These non-fermenting Gram-negative pathogens are quickly resistant to almost all antibiotic classes such as β-lactam, aminoglycosides, and quinolones agents because of their improved intrinsic resistant mechanisms [8
]. Recent publications have reported the prevalence of extensively drug resistance Acinetobacter
spp. in nosocomial infections all around the world [11
]. There are many reports of nosocomial infections associated with the MDR Acinetobacter
spp. from Iran [16
]. Regarding the current world-wide reports, the World Health Organization (WHO) categorized these mentioned bacteria in a critical group of MDR bacteria which need new antibiotic agents urgently [20
]. The ability of Acinetobacter
spp. to attach and form biofilms on both biotic and abiotic surfaces is a critical role to their pathogenesis in hospitalized patients. Their ability to form MDR microbial biofilms causes a varied range of infections such as skin, wound, and urinary tract infections, to septicemia in immune-suppressed or immune-compromised patients [22
]. Because of the physiological properties of the microbial biofilms, bacterial masses are more resistant to various conventional antibiotic agents. Their increased resistance to antimicrobial agents (up to 1000-fold compared to planktonic cells), making the treatment of biofilm-associated infection extremely challenging. Recently, biofilm-associated infections have emerged as a major problem in clinical settings. It seems that the current clinical antibiotics do not have the potential to combat and remove microbial biofilms [25
]. Therefore, the discovery of potentially powerful new compounds with novel mechanisms of action to eradicate biofilm-forming cells needs to be developed.
One of the alternative agents to conventional antibiotics is antimicrobial peptides (AMPs), which have been under focus regarding their potential activities against microorganisms. The AMPs are defined as small molecules (10–50 amino acids) with a positive net charge (+2–+9) that exhibit amphipathic properties. They have critical roles in modulating the innate immune system of the host’s defenses [27
AMPs are generally bactericidal and exhibit broad-spectrum antimicrobial effects against Gram-positive and Gram-negative bacteria via the electrostatic interaction between the negatively charged microbial cell surfaces (lipopolysaccharide and lipoteichoic acid of Gram-negative and Gram-positive bacteria, respectively) and the positively charged peptides [30
]. They inhibit bacterial growth via membrane disruption or pore formation or efflux the entire cell’s content. Some AMPs have intracellular targets. They pass through the cell membrane and bind to their targets. During this process, critical biological processes including cell wall formation or DNA, RNA, and protein synthesis, are inhibited and lead to cell death [32
]. In addition, it has been shown that many AMPs can prevent the biofilm formation or remove the attached bacterial biofilms due to different specific mechanisms, which include the inhibition of bacterial cell attachment to the surfaces, inducing the motility gene expression, the down-regulation of extracellular matrix synthesis, inhibition quorum sensing, and rapid bacterial killing ability [34
Nevertheless, there are some limitations for the clinical applications of AMPs, such as their potential toxicity to human cells, the susceptibility to proteases, and the high cost of industrial peptide (>20 residues in length) synthesis [32
]. In order to obtain a large quantity of AMPs for further analysis, the recombinant production strategies are utilized. The bacterial expression system is a great candidate for this purpose, owing to its rapid growth, cost effectiveness, and the different accessibilities of commercial vectors. The cloning of AMP genes in a suitable vector towards fusion proteins has been developed so as to cover the toxicity of the expressed cationic peptides for the host microorganism and to protect the AMPs from the proteases [38
Cathelicidins are a major group of cationic antimicrobial peptides and have been detected in the immune system of several vertebrates. The cathelicidin’s structure consists of two different regions: a cathelin-like domain at the N-terminus that displayed a high similarity, at the intra-species and a heterologous domain at the C-terminus that represented the antimicrobial activities [40
Recently, a new type of cathelicidin was determined in the venom glands of the Bungarus fasciatus
snake (Cath-BF), which exhibited potential antimicrobial activity against the MDR pathogenic bacteria, with minimal hemolytic and cytotoxic effects on human cells [41
]. It is well defined that by increasing the net positive charge of cationic peptides, the interaction of the AMPs with the negatively bacterial cell membrane is developed [43
]. As reported previously, through the substitution of the positively charged amino acid (lysine) in the peptide’s sequence, the peptide (Cath-A: KRFKKFFRKLKKSVKKRKKEFKKKPRVIKVSIPF) displayed a high efficacy against the bacteria, with decreased hemolytic and cytotoxic exhibitions on the eukaryotic cells [45
The possible application of developed AMPs as anti-biofilm agents on biomaterial surfaces is useful in the hospital setting. They can exert for combating MDR and/or biofilm forming bacterial infections in the healthcare system [46
In the present study, Cath-A was used for anti-biofilm tests. In order to establish cost-effective production with the potential activity of the peptide, the recombinant Cath-A (rec-Cath-A) gene sequence was expressed by the E. coli
utilization system. The developed expression systems in E. coli
have provided an opportunity to produce large quantities of various AMPs [47
]. However, codon optimization problems, a lethal toxicity of the expressed peptides to the E. coli
host, instability of AMPs against bacterial proteases, the correct fold of expressed products, are challenges to achieve a biologically active form of AMPs. For this reason, several fusion partners have been introduced to facilitate the expression and purification of AMPs [38
According to the nature of the expressed peptide, the plasmid of pET-32a with a T7
promoter and TrxA as a fusion partner was applied in order to reduce the toxicity of the AMPs to the host cell and to express the foreign protein in a soluble form [51
]. This procedure was followed by an enzymatic cleavage (enterokinase) which resulted in the production of an intact Cath-A sequence. The antimicrobial activity of the recombinant peptide was examined against two strains of A. baumannii
and P. aeruginosa.
2. Materials and Methods
The peptide with a purity ≥90% was synthesized by GL Biochem Ltd. (Shanghai, China). The antibiotic powders (ampicillin, piperacillin, levofloxcacin, imipenem, ceftazidime, tetracycline), enterokinase, isopropyl β-d-1-thiogalactopyranoside (IPTG) and chemical reagents were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). The antibiotic disks prepared from MAST House (Merseyside, UK). The colistin disk was prepared from Rosco (Taastrup, Denmark). The Luria–Bertani (LB) broth and Mueller–Hinton (MH) broth (Pronadisa, Madrid, Spain), and the Tryptic Soy Broth (TSB), LB and MH agar (Merck KGaA, Darmstadt, Germany) were prepared. The polymerase chain reaction (PCR) reagents and plasmid extraction kit were purchased from Bioneer (Daejeon, South Korea). All of the chemicals that were used were of the analytical grade. The expression vector pET-32a (+) was purchased from Novagen (Billerica, MA, USA). E. coli strain DH5α was used for sub-cloning and plasmid amplification. The E. coli Bl21 (DE3) was a host for the expression transformed plasmids. The clinical bacterial isolates were prepared by the Milad Hospital, Tehran, Iran. The ATCC standard controls were kindly donated by the referral laboratory of Iran’s Ministry of Health and Medical Education. The HisTrap FF 5 mL column (GE Healthcare Europe GmbH, Freiburg, Germany) was used as an affinity chromatography for the purification of the histidine-tagged recombinant protein.
2.2. Clinical Bacterial Strains
The MDR P. aeruginosa
and A. baumannii
isolates were collected for the current study during three months. All of the bacteria were isolated from the clinical instruments (catheters, ventilators, etc.) of the patients who were submitted to the intensive care unit (ICU) of the Milad Hospital, Tehran, Iran. The bacterial strains were confirmed by standard microbiological tests. The antibiotic susceptibility tests against the conventional antibiotics were performed by the disk diffusion method based on the Clinical and Laboratory Standards Institute (CLSI) [52
]. The strains were assessed against the following antibiotic disks: piperacillin (100 μg), ceftazidime (30 μg), cefepime (30 μg), imipenem (10 μg), gentamicin (10 μg), amikacin (30 μg), tetracycline (30 μg), and ciprofloxacin (5 μg). All of the P. aeruginosa
isolates were tested against colistin (10 μg). The minimum inhibitory concentration (MIC) of the isolates was also tested using the broth microdilution method against ampicillin, tetracycline, levofloxacin, ceftazidime, cefepime, piperacillin, and imipenem based on the CLSI recommendation [52
]. The E. coli ATCC 25922
and P. aeruginosa
ATCC 27853 were used as controls for the antibiotic tests.
2.3. Antimicrobial Activity of the Peptide
The antimicrobial activity of the synthetic peptide was tested against the clinical and standard isolates by the modified broth microdilution method as described previously [53
]. Briefly, the bacterial strains were cultured in a TSB medium at 37 °C overnight. The fresh bacteria were diluted in MH broth. Serial doubling dilutions of the peptide, with a range of 1–512 μg/mL, were prepared in 0.01% acetic acid and 0.2% bovine serum albumin (BSA). A total of 50 μL of the peptide dilution was added to a 96-well plate, which was followed by 50 μL of bacterial suspensions added to each well in order to give a final 2 × 105
colony-forming unit (CFU)/mL inoculum. The plates were incubated for 24 h at 37 °C. The MH broth with untreated bacterial suspension was used as the positive control. The negative controls were in un-inoculated media with normal saline. The MIC of the peptide was defined as the minimum concentration which inhibited the bacterial growth visually. All of the experiments were performed in triplicates.
2.4. Investigation of Antimicrobial Effects on an Established Biofilm
Two clinical strains P. aeruginosa
no. 1 and A. boumannii
no. 1 (according to Table 1
) were selected to assess the biofilm inhibitory effect of the peptide in a 96-well flat bottom tissue culture plate (TCP), with a few modifications [54
]. Briefly, the bacteria were cultured overnight in an MH broth and then diluted in the same medium, which was supplemented with 0.2% glucose. Then, 100 μL of 2 × 105
CFU/mL of bacterial suspensions were added to the individual wells of polystyrene TCP and were incubated at 37 °C for 24 h in order to allow for the biofilm formation. The media containing normal saline were defined as the negative control. During the following day, the plates were washed three times with phosphate-buffered saline (PBS) 1× to remove the non-attached cells. The established biofilms were treated with 10 μL of 10× concentrations of antimicrobial agents (Cath-A and levofloxacin) to reach a final concentration of 4–256 μg/mL and 90 μL of fresh MH broth. The bacterial suspension without any treatment agents was performed as the positive control. The plates were incubated for 24 h at 37 °C. Following the incubation time, the plates were washed with PBS so as to remove the planktonic cells and left to dry at room temperature. For the fixation of the biofilms, 100 µL of 100% methanol was added for 10 min, then, by removing the methanol, the wells were stained with 100 μL of 0.1% crystal violet (cv) for 15 min. The TCPs were washed with sterile distilled water and were left to dry. Finally, 100 μL of glacial acetic acid (30% v
) was added to dissolve the cv. The absorbance of the stained biofilm was measured at an optical density (OD) of 595 nm by a microplate reader (BioTek Instruments, PowerWave XS, Winooski, VT USA). The experiments were conducted in triplicates.
2.5. Recombinant Vector Construct
The entire gene sequence, including the rec- Cath-A with the additional enterokinase cleavage site was designed, as seen in Figure 1
. The whole construct was synthesized by Bioneer Company (Daejeon, South Korea). According to the E. coli
expression system, the codon optimization was done and the gene was inserted into pET-32a as an expression vector. The recombinant plasmid (pET-32(a)—rec Cath-A) and non-recombinant (original vector of pET-32(a)) were transformed into competent E. coli
DH5α cells. The positive transformed cells were confirmed by the colony PCR. The PCR tests were performed using T7
universal primers. The PCR products were purified with a PCR product purification kit and were sequenced to exhibit the fidelity of the transformed plasmids by an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The plasmids of the positive competent cells were extracted using a Bioneer plasmid mini extraction kit and were transformed into competent E. coli Bl21 (DE3)
2.6. Expression of the Fusion Protein
To optimize the highest protein expression, different media cultures, IPTG concentration, incubating times, and temperatures after the IPTG induction, were tested.
A fresh colony of transformed E. coli BL21 (DE3) with expression vector was inoculated in 50 mL of the 2XYT medium containing 100 mg/L of ampicillin and were cultured in a shaking incubator at 37 °C for 12 h. Then, 10 mL of the culture was transferred to 500 mL of a fresh Terrific broth (TB) medium containing 100 mg/L ampicillin and 1% glucose. The bacteria were cultured at 37 °C with 200 rpm shaking until the optical density (OD 600) reached 0.6. The protein expression was induced by IPTG to the final concentration of 1 mM. The transformed cells were cultured for a further 6 h of cultivation at 25 °C. Subsequently, the cells were harvested by centrifugation at 5000 rpm, at a temperature of 4 °C for 10 min. The bacterial pellets were re-suspended in the binding buffer (20 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 7.5) and were lysed on ice by sonication for 10 cycles (20 s working and 40 s rest). The supernatant of the lysate was collected by centrifugation at 14,000 rpm for 20 min at 4 °C. The extracted proteins were detected by a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and were stained with coomassie brilliant blue R-250.
2.7. Recombinant Proteins Isolation and Purification
The proteins were applied to His Trap FF columns in order to detect the His-tag proteins. The column was washed with the binding buffer several times. The extracted proteins were loaded on a column and were eluted by a gradient of the imidazole concentration (0–100%) from an elution buffer (20 mM NaH2PO4, 500 mM NaCl, 500 mM imidazole; pH 7.5). The peak fractions were collected and concentrated overnight at 4 °C by polyethylene glycol (PEG) with a 20,000 molecular weight. The concentrated proteins were analyzed on SDS-PAGE.
The fraction, which contained the recombinant peptide (rec-Cath-A) were dialyzed against a 500-mL volume of the enterokinase buffer (10 mM Tris HCI, pH 8.0, with 10 mM CaCl2) overnight at 4 °C.
2.8. Proteolytic Cleavage of Fusion Protein
The rec- Cath-A fusion protein was incubated with enterokinase at a ratio of 5 U:1 µg of the fusion protein in 10 mM of Tris HCI, pH 8.0, and 10 mM CaCl2. The reaction mixture was incubated at 37 °C overnight. After the cleavage process, the reaction was subjected to amicon ultra-centrifugal filters (Merk, Darmstad, Germany) with 30 kDa as well as a 3 kDa molecular weight cut-off in order to remove the enterokinase and non-specific proteins. The peptide solution was dialyzed (14 kDa cut-off) in 5 mL of PBS the 1X buffer in order to achieve a released Cath-A peptide and was analyzed by SDS-PAGE.
2.9. Analysis of Protein Concentration
The standard Bradford protein assay was displayed for the quantitative determination of the purified protein concentration [56
2.10. Antimicrobial Activity Assay of Expressed Peptide
The antimicrobial activity of the purified peptide was determined using the agar disk diffusion method, based on the standard assay which was recommended by the CLSI against standards and clinical MDR bacteria [52
Briefly, the 0.5 Mcfarland of the fresh colonies of bacteria were sub-cultured on MH agar and blank disks with different concentrations of synthetic peptide were placed on the plate. Approximately 20 μL of the dialyzed peptide was inoculated on the blank disk. The plates were incubated at 37 °C overnight. The inhibition zones were measured and compared to the synthetic peptide. All of the experiments were performed in triplicates.
2.11. Statistical Analysis
The experiments were performed in triplicates. Data were analyzed by one-way analysis of variance (ANOVA), and standard deviations of the mean (mean ± SD) were presented for each test. p values < 0.05 and 0.01 were indicated as significant.
An imminent need for new antibiotics dictates the necessity for studies aimed at designing clinically useful AMPs. The present study was carried out on such a basis and it provides a 34 amino-acid long AMP sequence named Cath-A. In previous reports, this peptide was proven to be effective in ex vivo studies using different pathogen microorganisms containing standard and wild-type strains, especially the methicillin-resistant Staphylococcus aureus
(MRSA). Additionally, the cell culture and hemolysis studies proved that the peptide was safe in its effective concentrations [45
Bacterial cells form biofilm matrixes under several environmental conditions including nutritional signs and starvation, attachment to the host tissues or non-living surfaces, exposure to sublethal concentrations of antibiotics, and environmental stresses. The biofilm structures are resistance to the stress, especially the conventional antimicrobial agents and the host defense mechanisms. It is estimated that microbes growing in the biofilm structures are more resistant to antibiotics compared to the planktonic cells. So the treatment of biofilm-related infections is a significant problem in healthcare systems. To clear the biofilm forming bacteria, the various antibiotics classes (e.g., ß-lactams, aminoglycosides or fluoroquinolones) are generally used. Hence, new developed antibacterial agents with alternative strategies are required for the treatment and eradication of established biofilms in clinical settings [57
]. Applying AMPs as an anti-biofilm strategy has been considered, which may represent promising approaches to control biofilms. AMPs have been considered as alternative therapeutic candidates for conventional antibiotics. Their important roles in the modulation of innate host immune defenses, broad-spectrum antimicrobial activity against microorganisms, efficacy on neutralizing lipopolysaccharide endotoxin, rapid mechanism of actions on MDR bacteria, and low incidence in selecting resistance to AMPs are the benefits of these molecules [32
Therefore, in this report, the antimicrobial potential of Cath-A was examined against the A. baumannii
and P. aeruginosa
biofilm isolates. Among the opportunistic bacteria, MDR P. aeruginosa
and A. baumannii
are considered medically important pathogens. These pathogens are commonly associated with nosocomial and hospital-acquired infections, especially in the ICU and burn sites. [23
]. The strategy of applying AMPs as an anti-biofilm has been considered, which may represent promising approaches to control biofilms [46
]. Other researchers have also reported an anti-biofilm efficacy of the AMPs against A. baumanii
and P. aeruginosa
]. Most of the medically relevant biofilms are resistant to commercial antimicrobial agents because of their structural and functional properties. In the present study, the isolates exhibited MDR properties in the phenotypic antimicrobial assays, as reported in previous scientific reports [65
]. The anti-biofilm activities of Cath-A were substantial, particularly for the MDR A. baumanii
isolates. In the case of the P. aeruginosa
strains, Cath-A had anti-biofilm effects comparable to commercially available antibiotics. However, some isolates among P. aeroginusa
exhibited a resistance to Cath-A (MIC ≥ 265 µg/mL) which may be related to the bacterial resistance mechanisms such as proteolytic degradation, the modification of cell wall components, or the presence of alginate in the biofilm structure [68
]. Additionally, as there are few candidate molecules available against biofilm-related infections, these results suggest the need for more studies in order to evaluate Cath-A as a promising AMP with a potential clinical usefulness. Since the levofloxacin is a CLSI recommended antibiotic, we compared the eradication effects of Cath-A with levofloxacin in biofilm biomass.
Another important issue in this report was the method that was used for the production of Cath-A. The E. coli
expression system was applied so as to express recombinant Cath-A because of its cost-effectiveness and the over-construction of the desired product. The suitable expression vector (pET 32a) was used as it contains the Trx fusion protein in order to eliminate the toxicity of the peptide and to express the soluble peptide in a correct folding. The existence of the His-tag was an important factor in the purification process using the affinity chromatography from the HisTrap FF column. These properties made the purification easier and increased the product yield. The inserted extra enterokinase sequence at the N-terminus of rec-Cath-A facilitated the cleavage process and ended up being the desired sequence. In the present study, the time of expression after the IPTG induction played a critical role in producing rec- Cath-A. This is in accordance with the reports of other researchers [69
]. In contrast, increasing the expression time led to a reduction of the rec-Cath-A production after 6 h. The yield of the recombinant peptide was about 0.09 mg from 500 mL of bacterial culture. It was lower than the yield of other expressed AMPs in E. coli
]. This may have resulted in the cytolytic activity of the expressed rec- Cath-A or in the bacterial proteases’ effects on the peptide, which greatly decreased the yields of the products. The rec- Cath-A displayed antimicrobial activities on the MDR isolates. The impurities of the recombinant peptide may cause an increase in the concentration of MIC tests and result in twice as low antimicrobial activity compared to the synthetic peptide. It seems that other expression systems that have been used to express AMPs demonstrate a 100% in vitro activity when compared to their synthetic analogues [72