Broad-spectrum inhibition of AHL-regulated virulence factors and biofilms by sub-inhibitory concentrations of ceftazidime

Abstract

Quorum sensing (QS) in bacteria is a density dependent communication system that regulates the expression of genes, including production of virulence factors in many pathogens. The emergence of antibiotic resistance among pathogenic bacteria represents a major threat in both hospitals as well as environmental settings. Interference of quorum sensing (QS)-regulated virulence factors and biofilms is a recognized anti-pathogenic therapy. Safe, stable and effective anti-QS agents are needed to combat diseases caused by multidrug-resistant bacteria. The present study was performed to assess the inhibitory effect of third generation antibiotic ceftazidime against Gram-negative bacterial pathogens. Sub-MICs of ceftazidime demonstrated dose dependent inhibition of QS regulated virulence traits and biofilm formation in various strains of Chromobacterium violaceum (CV12472 and CVO26), Pseudomonas aeruginosa (PAO1 and PAF79) and Aeromonas hydrophila (WAF38). β-galactosidase assay revealed ceftazidime inhibited the las and pqs QS systems in P. aeruginosa. Alongside, in vivo studies demonstrated enhanced survival of Caenorhabditis elegans after the treatment with the drug. Molecular docking analysis showed the high binding affinity of ceftazidime which represents its QS inhibitory activity. By highlighting the broad spectrum anti-quorum sensing and biofilm inhibiting activities against 3 different bacterial pathogens, ceftazidime seems a more potent candidate in counteracting the infections caused by drug resistant bacteria.

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Introduction

Bacterial phenotypes such as virulence, secondary metabolite production and biofilm maturation are controlled by cell-to-cell communication, a process commonly known as quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are employed as QS signal molecules in many Gram-negative bacteria. AHL mediated QS systems are composed of two components: a signal (AHL) generator (LuxI homologue) and a response regulator (LuxR homologue) which can bind with the AHLs to form AHL-receptor complexes that regulate the transcription of target QS-regulated genes.¹

Pseudomonas aeruginosa QS systems comprise two AHL-mediated LasR/I and RhlI/R and one quinolone based PQS system that work in a hierarchical manner.^2,3 The Las, Rhl and PQS systems regulate multiple genes in P. aeruginosa, which include virulence, drug resistance and programmed cell death.⁴ P. aeruginosa secretes a range of virulence factors, such as elastase,⁵ quorum sensing molecule Pseudomonas quinolone signal (PQS),⁶ pyocyanin,⁷ rhamnolipids,⁸ and siderophores (pyoverdine and pyochelin).⁹ It also produces several adhesion factors like exotoxin A, phospholipase C (used for hemolysis), and exoenzyme S.¹⁰

Quorum sensing in bacteria has been considered as an antinfective drug target as it's inhibition attenuates bacterial virulence and may help to control infections. Quorum sensing interference is achieved either by degrading QS signals or by interrupting the perception of signal molecules by receptor proteins.¹¹ Since the discovery of halogenated furanones as quorum-sensing inhibitors by Givskov et al.¹² a variety of synthetic and natural agents have been studied for their QS inhibitory potential both in vitro and in vivo.¹³ Azithromycin (AZM), an azalide (a sub-class of macrolide antibiotics), is one such agent that possesses anti-quorum sensing and anti-biofilm activity but does not show significant bactericidal activity against P. aeruginosa at therapeutic concentrations.¹⁴ Several studies have suggested that AZM positively influences the clinical outcome in patients suffering from chronic P. aeruginosa infections as seen in diffuse panbronchiolitis, cystic fibrosis and chronic pulmonary disorders.^15,16 The antibiofilm activity of AZM in vitro and its therapeutic potential against P. aeruginosa experimental UTI induced with planktonic cells has also been reported.¹⁴ Certain other antibiotics like ciprofloxacin, tobramycin and doxycycline have also been reported to inhibit QS at their respective sub-MICs.^16–18

Ceftazidime is a third generation semisynthetic, broad-spectrum cephalosporin antibiotic for parenteral administration. Like other third-generation cephalosporins, it has broad-spectrum activity against Gram-positive and Gram-negative bacteria. However, unlike most third-generation agents, it is active against Pseudomonas aeruginosa.¹⁹ Therefore, the present study was aimed to identify ceftazidime, one of the most effective antibiotics against P. aeruginosa, as a broad spectrum QS and biofilm inhibitor in vitro and to further evaluate its in vivo efficacy in a C. elegans nematode model.

Methods

Bacterial strains and growth conditions

C. violaceum 12472 is a wild-type strain that produces a QS regulated purple coloured pigment, violacein in response to cognate C₄ and C₆ acyl homoserine lactone molecules. Chromobacterium violaceum CVO26 is a Tn5 mutant strain that only produces violacein when short-chain autoinducers are added.²⁰ P. aeruginosa PAO1 is an opportunistic pathogenic bacteria and many of its virulence factors and traits are QS controlled. A clinical laboratory strain of Pseudomonas aeruginosa (PAF-79) and Aeromonas hydrophila (WAF38) were also included in this study. All strains were maintained on Luria Bertani or LB broth (15.0 g tryptone, 0.5% yeast extract, 0.5% NaCl) solidified with 1.5% agar (Hi-media). C. violaceum 12472, C. violaceum CVO26 and P. aeruginosa (PAO1 and PAF-79) strains were cultivated at 28 °C and 37 °C respectively.

Determination of minimum inhibitory concentration (MIC) of ceftazidime

The MICs of ceftazidime against the bacterial pathogens were determined using the CLSI macrobroth dilution method.²¹ MIC is defined as the minimum concentration of ceftazidime at which there was no visible growth of the test strains. Concentrations below the MICs were considered sub-inhibitory and were further used to study the anti-QS and biofilm inhibitory properties.

Violacein inhibition assay

Biosensor strain Chromobacterium violaceum CVO26 was incubated for 16–18 h (OD_{600 nm} = 0.1) and inoculated to in Erlenmeyer flasks containing Luria broth (LB), LB supplemented with C₆-HSL (10 μM l⁻¹) and LB supplemented with C₆-HSL and test agent. The flasks were incubated at 27 °C with 150 rev per min agitation for 24 h in a shaking incubator.²²

Violacein production by Chromobacterium violaceum (CVO26) in presence of ceftazidime was studied using method described by Castillo-Juarez et al.²³

Effect on virulence factors production

Effect of sub-MICs of ceftazidime on virulence factors of P. aeruginosa and A. hyrophila such as LasB elastase, protease, pyocyanin, chitinase, swarming motility, EPS extraction and quantification was determined using protocols described previously.²⁴

Assay for biofilm inhibition

The effect of antibiotic on biofilm formation was measured using the polyvinyl chloride biofilm formation assay.²⁵ Briefly, overnight cultures of PAO1, PAF79 and WAF38 were re-suspended in fresh LB medium in the presence and the absence of ceftazidime and incubated at 30 °C for 24 h. The biofilms in the microtiter plates stained with a crystal violet solution and quantified by solubilizing the dye in ethanol and measuring the absorbance at OD₄₇₀.

Assessment of biofilm metabolic activity (XTT reduction assay)

XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay was performed following the method used by Sabaeifard-Parastoo et al.²⁶ with some modifications. Fresh XTT solution was made by dissolving 4 mg XTT (Sigma) in 10 ml normal saline solution (NSS) (37 °C) before each assay. Filter-sterilized solution is then supplemented with 100 μl menadione solution, containing 55 mg menadione (Sigma) in 100 ml acetone. 100 μl of XTT-menadione solution and 100 μl of fresh LB medium supplemented with 0.2% glucose were added to each well. Plates were incubated in the dark for 3 h at 37 °C and 120 rpm. After 3 h of incubation, 200 μl of well contents were transferred to a new flat-bottomed microplate and the absorbance was measured at 490 nm with microplate spectrophotometer.

Analysis of lasB and pqsA transcriptional activity in E. coli

lasB and pqsA transcriptional activity in E. coli MG4/pKDT17 and E. coli pEAL08-2 in was measured using the β-galactosidase assay described by Pearson et al.²⁷ and Cugini et al.²⁸

Caenorhabditis elegans slow killing assay

The method described by Husain et al.²⁴ was adopted to study the in vivo efficiency of ceftazidime in C. elegans nematode infection model.

Caenorhabditis elegans paralytic assay

Method of Adonizio et al.²⁹ was adopted for this assay. Briefly, brain heart infusion agar (BHI) plates supplemented with or without ceftazidime were seeded with 10 ml of an overnight culture of PAO1 and incubated at 37 °C for 24 h to form lawns of bacteria. Caenorhabditis elegans were washed and suspended in a minimal volume of M9 buffer (pH 6.5). Droplets containing 20–30 adult nematodes were placed onto the bacterial lawns and the plates were incubated at room temperature (21–23 °C). Worms were evaluated for viability every hour for a total of 4 hour.

In silico analysis

Preparation of enzyme. The 3D crystal structure of LasR receptor protein (PDB ID code 2UV0) was retrieved from Research Collaboratory for Structural Bioinformatics (RCSB) protein databank. All the water molecules and hetero atoms were removed. Due the unavailability of the 3D structure of RhlR, the three dimensional structure was modelled. The 241 amino acid long sequence of transcriptional regulator RhlR from Pseudomonas aeruginosa PAO1 was retrieved from NCBI protein database (accession no. NP_252167). Blastp search was performed against the sequence with known structures available with rcsb protein data bank. X-ray structure of regulatory protein SDIA from E. coli (pdb id: 4LFU) was taken as a template for modeling the structure of RhlR. Modeller 9v14 program was used to generate the model structure of RhlR.³⁰ The best model was selected on the basis of their dope score. The quality of the modeled structure was validated using procheck.

Molecular docking. The 3D structure of ceftazidime, 3-oxo-C₁₂-HSL and C₄-HSL were retrieved from pubchem compound database (CID 5481173 and CID 127864 and CID 44602431). AutoDock Tools 4.0 (ref. 31) was used to carry out docking of ceftazidime within the binding site of LasR and RhlR. Further the binding efficacy of ceftazidime was compared with 3-oxo-C₁₂-HSL and C₄-HSL, the natural inhibitors for LasR and RhlR respectively. Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was used as a parameter for the molecular docking. The grid box dimension was set to 60 × 60 × 60 Å around active site of LasR and RhlR making sure that ceftazidime, 3-oxo-C₁₂-HSL and C₄-HSL can freely rotate inside the grid. The number of docking runs was set to 15. The final poses were selected on the basis of their binding energies. The final complexes were subsequently visualized using PyMol.³²

Statistical analysis. All experiments were performed in triplicates and the data obtained from experiments were presented as mean values and the difference between control and test were analyzed using student's t test as described previously by Abraham et al.²²

Results

MIC of ceftazidime was assessed for all the test organisms (CVO26, PAO1, PAF79, and WAF38). The MICs were found to be 0.5, 1, 4 and 1 μg ml⁻¹ against CVO26, PAO1, PAF79, and WAF38, respectively. Concentrations below the MIC level were considered as sub-MICs and all the further assays were carried out at sub-MICs of ceftazidime.

Effect on violacein production

In Chromobacterium violaceum CVO26, a gradual decrease in the production of violacein content was observed when treated with the increasing sub-MICs of ceftazidime (Fig. 1a and b). Maximum of 74.6% inhibition in violacein production was observed at the concentration of 0.25 μg ml⁻¹ (Fig. 1a). The reduction in violacein production was also statistically significant at lower concentrations (0.006 and 0.125 μg ml⁻¹). No significant reduction in growth of CVO26 was observed at all the tested concentrations.

Fig. 1 (a) Quantitative assessment of violacein inhibition in CVO26 by sub-MICs of ceftazidime. Data are represented as percentage of violacein inhibition. All of the data are presented as mean ± SD. *, significance at p ≤ 0.05, **, significance at p ≤ 0.005. (b) Inhibition of violacein by sub-MICs of ceftazidime, (1) 0.06 μg ml⁻¹, (2) 0.125 μg ml⁻¹, (3) 0.25 μg ml⁻¹ in C. violaceum CVO26.

Effect on quorum sensing regulated virulence factors/traits

The effect of ceftazidime in reducing the production of QS-dependent total protease and LasB elastase activity in PAO1 was assessed. The cell-free supernatant of ceftazidime-treated PAO1 exhibited a significant reduction in the azocasein-degrading protease activity (55.7%), elastin-degrading elastase activity (62.8%), chitinase activity (63.8%), pyocyanin production (61.1%) and EPS production (58.9%) at 0.5 μg ml⁻¹ concentration over untreated control (Fig. 2A). The addition of ceftazidime showed a dose dependent decrease in the swarming motility of PAO1 and significant reduction in the migration ability of the pathogen was recorded at all tested concentrations. The maximum inhibition of 81.7% in swarming behavior was registered at 0.5 μg ml⁻¹ concentration.

Fig. 2 Effect of ceftazidime on quorum sensing regulated factors in P. aeruginosa (A) PAO1 and (B) PAF79 at sub-MICs. The data represents mean values of three independent experiments. *, significance at p ≤ 0.05, **, significance at p ≤ 0.005, *** significance at p ≤ 0.001.

Similar concentration dependent decrease in the QS regulated virulence factors was also observed in the clinical strain of P. aeruginosa PAF79. Elastase activity was decreased significantly at all tested concentrations. Ceftazidime (0.25–2 μg ml⁻¹) exhibited 36.8–65.7% reduction in elastase activity as compared to the control. Total protease activity, chitinase activity and pyocyanin production was reduced significantly (p ≤ 0.05) only at 2 μg ml⁻¹. EPS production in the test pathogen was decreased by 52.7% and 60.2% at 1 and 2 μg ml⁻¹ treatment respectively. Swarming ability of the pathogen was also impaired significantly (47.2–76.6%) over control at above tested concentrations (Fig. 2B).

The effect of sub-MICs of ceftazidime was also assessed against A. hydrophila WAF38 (Fig. 3). Concentration dependent decrease in the virulence factors (total protease and EPS production) was recorded. Most effective concentration was found to be 0.5 μg ml⁻¹ for significant reduction in total protease activity (56.9%) and EPS production (60.2%) was recorded.

Fig. 3 Effect of sub-MICs of ceftazidime on inhibition of quorum sensing regulated virulence factors in Aeromonas hydrophila WAF-38. The data represents mean values of three independent experiments. *, significance at p ≤ 0.05, **, significance at p ≤ 0.005.

Effect on biofilm formation

Significant (p ≤ 0.005) decrease in biofilm formation was observed in the tested bacterial strains when grown in the presence of ceftazidime. In PAO1, a significant reduction of 44.4% and 70% was observed at 0.25, 0.5 μg ml⁻¹ concentrations. In a similar manner biofilm forming ability of PAF79 was also reduced significantly at 1 and 2 μg ml⁻¹ with a maximum of 65.3% reduction in biofilm formation as depicted in Fig. 4. Biofilm formation in the A. hydrophila WAF38 was also reduced considerably in a concentration dependent manner ranging from 30.9–65.2% at the tested sub-MICs as shown in Fig. 3.

Fig. 4 Effect of ceftazidime on biofilm formation in P. aeruginosa PAO1 and P. aeruginosa PAF79 at respective sub-MICs.

Effect on metabolic activity (XTT reduction assay)

XTT reduction assay revealed the metabolic activity of P. aeruginosa (PAO1 and PAF79) and A. hydrophila WAF38 biofilms against the respective sub-MICs of ceftazidime after 24 h of incubation at 37 °C. Results clearly indicate that the metabolic activity of pathogens against ceftazidime was concentration dependent and metabolic activity in biofilms was higher at lower ceftazidime concentrations. These results suggest that in addition to reducing biofilm biomass, ceftazidime had a significant effect on decreasing the metabolic activity (Fig. 5).

Fig. 5 Metabolic activity of biofilms formed by test bacteria at their respective subinhibitory concentrations of ceftazidime using XTT reduction assay. (A) PAO1; (B) PAF79; (C) WAF38. Mean value of triplicate independent experiments and SDs are shown.

Effect of ceftazidime on β-galactosidase activity

The addition of sub-MICs concentrations of ceftazidime exhibited a concentration dependent decrease on β-galactosidase activity. Significant reduction in β-galactosidase activity in E. coli MG4/pKDT17 was recorded at sub-MICs. Untreated control produced 538 miller units (MU) whereas at 0.03, 0.06, 0.125 and 0.25 μg ml⁻¹ concentration of ceftazidime 471, 299, 236 and 194 miller units AHL (Fig. 6A). The reduced AHL levels point to the fact that inhibition of lasB promoter activity involves LasR controlled transcription.

Fig. 6 Effect of ceftazidime on las and pqs systems. (A) β-Galactosidase activity was measured in the E. coli MG4/pKDT17 with and without sub-MICs of ceftazidime. (B) β-Galactosidase activity was measured in the E. coli pEAL08-2 with and without ceftazidime. All of the data are presented as mean ± SD. *, significance at p ≤ 0.05, **, significance at p ≤ 0.005, ***, significance at p ≤ 0.0001.

Further, pyocyanin production in P. aeruginosa PAO1 is mainly regulated by the PQS system. Therefore, E. coli pEAL08-2 strain containing the pqsA promoter fused to lacZ was used to determine whether the inhibition of pyocyanin production was directly due to the effects of PQS system. The addition of ceftazidime reduced the β-galactosidase luminescence in E. coli pEAL08-2 by up to 75% at 0.25 μg ml⁻¹ (Fig. 6B), which proved that ceftazidime inhibits PQS-stimulated transcription.

C. elegans paralytic assay

The paralytic assay revealed that after 4 h around 82% worms survived on PAO1 plates that contained 0.5 μg ml⁻¹ of ceftazidime (Fig. 7A). On the other hand, more than 50% worms died in the span of 2 h after transfer to untreated PAO1 plates and all worms were dead after 4 h.

Fig. 7 Nematode survival curves at sub-MIC (0.25 μg ml⁻¹) of ceftazidime. (a) Paralytic and (b) slow-killing assays for Caenorhabditis elegans.

C. elegans low-killing assay

The anti-infection potential of the sub-MIC of ceftazidime was assessed using a slow killing assay of C. elegans by PAO1 in a 24-well microtitre plate at sub-MIC concentrations of the drug. Complete (100%) mortality of the P. aeruginosa PAO1 preinfected C. elegans was observed within 72 h. However, C. elegans preinfected with PAO1 and treated with doxycycline (4 μg ml⁻¹) and ceftazidime (0.5 μg ml⁻¹) separately displayed enhanced survival rate of 55% and 61% respectively (Fig. 7B).

In silico study

Molecular modelling of RhlR. In the present study we modelled the three dimensional structure of RhlR by using the structural information from the earlier resolved X-ray structure of regulatory protein SdiA of E. coli (pdb id: 4LFU) with considerable similarity.³³ The overall quality of the modeled RhlR structure was validated using PROCHECK, and it provides an idea of the stereo chemical quality of the protein and is based on the inspection of psi/phi Ramachandran plot which showed 96.7% of the residues in favored region, 3.3% residues in the allowed regions, no residues in outlier region (disallowed region).

Molecular docking. The complementary application of molecular docking analysis was further carried out for better understanding the interaction of ceftazidime against LasR and RhlR. The structure–activity relationship of ceftazidime was assessed against LasR and RhlR binding sites. As a reference model, the natural inhibitors, 3-oxo-C₁₂-HSL and C₄-HSLwere docked in ligand binding domain of LasR and RhlR respectively. The molecular docking scores of all the compounds are depicted in Table 1. Molecular docking scores studies revealed that ceftazidime was active against both RhlR and LasR and showed binding affinity of −7.54 and −7.31 kcal mol⁻¹ respectively. It was found that ceftazidime binds RhlR with greater strength than C₄-HSL (ΔG −5.44 kcal mol⁻¹). Ceftazidime was found to be equally effective against LasR as compare to its natural counterpart (3-oxo-C₁₂-HSL), which shows binding free energy of −9.38 kcal mol⁻¹. The role of some important amino acid residues of LasR as well as RhlR playing an important role in accommodating ceftazidime and its natural counterpart has also been revealed. It has been investigated that L36, A50, I52, Y56, W60, Y64, D73, V76, C79, W88, L125 and G126 are the key residues that take part in accommodating ceftazidime and 3-oxo-C₁₂-HSL within the active site of LasR. Among them, W60, R61 were the common residues involved in hydrogen bonding with both the selected compounds. Similarly, in the case of RhlR, Y64, L69, Y72 and L107 were the common amino acids found to be involved in the binding of ceftazidime and C₄-HSL. The importance of these active site residues of LasR and RhlR has been discussed in previous studies.³⁴ The molecular docking scores of ceftazidime, 3-oxo-C₁₂-HSL and C₄-HSL against LasR and RhlR are shown in Table 1 and Fig. 8 and 9.

Table 1 Binding efficacy of ceftazidime and 3-oxo-C₁₂-HSL against both the selected target enzymes and the amino acid residues involved in their complex formation

Enzyme	Compounds	Autodock binding free energy (kcal mol^−1)	Residues involved
			Hydrogen bond formation	Hydrophobic interactions
LasR	Ceftazidime	−7.31	W60, R61, Y64, V76	L36, Y47, A50, I52, Y56, W60, Y64, A70, D73, V76, C79, W88, F101, L110, L125, G126
	3-Oxo-C12-HSL	−9.38	W60, R61, Y64, D73, S129	L36, G38, A50, I52, Y56, Y64, T75, V76, C79, W88, L125, G126
RhlR	Ceftazidime	−7.54	T58, Y72, D81	T58, V60, Y64, L69, Y72, Q73, D81, A83, W96, F101, L107
	C4-HSL	−5.44	W68	Y64, W68, L69, Y72, Y77, L107, E110, A111, W114

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Fig. 8 Molecular docking analysis of (A) LasR with a 3-oxo-C₁₂-HSL, (B) RhlR with C₄-HSL.

Fig. 9 Molecular docking analysis. (A) LasR with ceftazidime (B) RhlR with ceftazidime.

Discussion

The present study demonstrated that the antibiotic, ceftazidime, a third generation cephalosporin interferes with the AHL regulated production of violacein in both strains of Chromobacterium violaceum (CV12472 and CVO26) at sub MICs. Ceftazidime at tested sub-MICs reduced the violacein production in CVO26 in dose dependent manner without significant inhibition of the growth. Similar reduced violacein production was observed with doxycycline at sub-MICs.¹⁸ Further studies were conducted on strains of Pseudomonas aeruginosa (PAO1 and PAF79) and Aeromonas hydrophila WAF38. It has been established that the virulence of Pseudomonas aeruginosa is regulated by multiple signaling systems, signals that are quorum-sensing dependent.³⁵ Virulence factors like enzymes, swarming motility, EPS production and biofilm mode of growth under QS regulation help to evade antibiotic shock and host defense mechanisms.¹¹

The production of virulence enzymes (LasB, protease and chitinase) was significantly reduced after treatment with ceftazidime. In earlier studies, a similar effect in the reduction of QS-dependent virulence enzyme production was observed in PAO1 when treated with compounds like azithromycin,¹⁵ salicylic acid, nifuroxazide, chlorzoxazone,³⁶ and naringenin.³⁷ In cystic fibrosis patients, pyocyanin, a secondary metabolite whose expression is under the control of QS, along with its precursor molecule, leads to severe toxic effects.³⁸ Therefore, the effect of ceftazidime in inhibiting the pyocyanin production was assessed and a considerable decrease was recorded without any impairment of the growth of the test organisms. The results obtained in this present study are in accordance with the findings of an earlier report wherein the anti-QS activity of compounds such as V-06-018 and PD12 was demonstrated.³⁹ Inhibition of pyocyanin production in PAO1 by V-06-018 was about 90%, and inhibition by PD12 was about 40%. In another study, N-decanoyl cyclopentylamide at 250 μM concentration reduced pyocyanin production by 36% over untreated control.⁴⁰

Swarming motility and EPS production are highly essential for the successful development and maturation of biofilms, hence any interference with the motility or EPS production will impair the biofilm mode of growth and will also lead to reduced resistance of sessile cells to antibiotic treatment or to host immune responses. The results of the present study showed a significant reduction in the flagellum-driven swarming motility of the test pathogens. Similarly, swarming motility of PAO1 was impaired by 67% and 74% upon treatment with sub-MICs of azithromycin¹⁵ and doxycycline,¹⁸ respectively. Salicylic acid³⁶ a compound produced by plants was also found effective in reducing swarming motility of S. liquefaciens, which uses small chain length (C₄- and C₆) AHL similar to P. aeruginosa for QS-regulated swarming. Further, the observed results also indicated the potential of the tested antibiotic in inhibiting EPS production in P. aeruginosa and A. hyrophila. Moreover, the compound at the tested concentrations showed no growth inhibitory effect.

Biofilm is a complex aggregation of microorganisms which is resistant to drugs leading to severe persistent infections.⁴¹ Therefore, biofilm is considered as one of the potential drug target to control chronic and persistent infections including cystic fibrosis. In this study statistically significant reduction of biofilm formation was observed on the treatment with sub-inhibitory concentrations of ceftazidime. Our study is in concordance with previous reports that showed the disruption of biofilms in P. aeruginosa PAO1 by agents like, tobramycin,¹⁷ and 2,5-piperazinedione.⁴² A new cephalosporin CXA-101 and doxycycline have also been reported to inhibit biofilm in P. aeruginosa.^18,43 The results of the effect of ceftazidime on metabolic activity do not show a correlation with the biofilm biomass. Similar observations were reported by Sandasi et al.⁴⁴ with plant extracts against Listeria monocytogenes.

The addition of sub-inhibitory concentrations of ceftazidime decreased β-galactosidase activity in E. coli MG4/pKDT17 ranging from 15–65% (p ≤ 0.005). The results of the assay demonstrated that the reduced production of AHL under the effect of sub-MICs of ceftazidime inhibits Las-controlled transcription. The dependence of lasB–lacZ expression on the autoinducer concentration was previously shown by Pearson et al.²⁷ The regulation is, therefore, sensitive to the autoinducer (AHL) concentration. The findings of the assay are in agreement with the above observations as reduced β-galactosidase activity is suggestive of reduced AHL levels and, therefore, reduced expression of the lasB gene. Similarly, ceftazidime also reduced both the pyocyanin production of PAO1 and the transcriptional activation of pqsA in E. coli, which indicates that ceftazidime inhibits the pqs system. Considering the las and pqs systems regulate the expression of numerous virulence-related genes, ceftazidime could be used to inhibit these two systems leading to significantly decreased virulence of P. aeruginosa.

In this study, the C. elegans nematode model was used for the assessment of the anti-infective potential of the ceftazidime against PAO1 infection. The advantage of using the C. elegans live nematode model is that both the efficiency and the host toxicity of a particular anti-infective agent can be tested in parallel. Mortality of nematode by PAO1 in paralytic assay is attributed to QS controlled cyanide production that leads to cyanide asphyxiation and paralysis.⁴⁵ Thus from the results of the paralytic assay it can suggested that the antibiotic was interfering with the production of cyanide directly or indirectly via QS genes. The slow killing assays demonstrated an enhanced survival of PAO1-preinfected C. elegans when maintained with 0.5 μg ml⁻¹ concentration of ceftazidime. Slow killing of the nematode occurs due to ingestion and subsequent infection by the pathogen PAO1 over a period of 60 h.⁴⁶ Since lasR and gacA are required for the infection, the enhanced survival of the nematode after addition of ceftazidime suggests that drug effects the expression of the lasR or gacA genes. Musthafa et al.⁴² observed a similar effect in the increased survival of C. elegans against PAO1 infection after treatment with sub-MICs of 2,5-piperazinedione.

In silico studies

The las and rhl quorum-sensing systems regulate the production of several extracellular virulence factors, including elastase, exoproteases, siderophores, exotoxins, rhamnolipid and several secondary metabolites, and participate in the development of biofilms.^47–49 So LasR and RhlR were taken as a molecular target for ceftazidime. Due to the unavailability of the X-ray structure of RhlR we generated the three dimensional structure of this enzyme using homology modelling approach. Homology modeling approach is based on the assumption that if the sequences of two proteins are related they will have similar structure as the structures are more stable than sequences.³³ The best model was selected on the basis of dope score.⁵⁰ Ramachandran plot provide an idea of the stereochemical quality of the protein and is based on the inspection of psi/phi. The model was selected only if it passes the stereochemical quality check.⁵¹ Here in this study the molecular docking analysis was carried out to investigate the inhibitory activity of ceftazidime against LasR and RhlR and to further gain insight into the binding of ceftazidime within the active site of both these enzymes.

It has been reported earlier that LasR of P. aeruginosa triggers the expression of virulence, biofilm and other production factors when is present in combination with 3-oxo-C₁₂-HSL (a natural ligand derived from LasI). Inhibition of LasR/RhlR results in the reduced production of the QS dependent factors in P. aeruginosa which further disrupts its formation.^36,50 Previous findings shows that C₄-HSL and 3-oxo-C₁₂-HSL modulates the expression of genes regulated by LasR and RhlR respectively.^52,53 In the light of these previous findings we considered C₄-HSL and 3-oxo-C₁₂-HSL as a control to compare the affinity of ceftazidime. Our molecular docking scores suggests binding efficiency of ceftazidime was higher against RhlR as compare to it natural counterpart (C₄-HSL), while it shows moderate activity against LasR. This further supports our findings that ceftazidime can act a therapeutic agent against QS. Hence, it is envisioned that ceftazidime has potential to inhibit both the therapeutic target involved in QS.

Conclusion

The present investigation highlights the broad spectrum anti-quorum sensing and biofilm inhibiting property of ceftazidime in tested pathogens. In vitro and in vivo QS inhibitory property of the antibiotic indicates its propitious anti-infective drug against infection-causing bacteria. Further, molecular studies are needed to unearth the exact mechanisms of action of ceftazidime.

Conflicts of interest

All contributing authors declare no conflicts of interest.

Acknowledgements

The authors extend their appreciation to the Deanship of Scientific Research at KSU for funding this work through research group project number RGP-215. We also acknowledge the support of Prof. Rodolfo Garcia-Contreras for editing the manuscript.

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