Quorum sensing inhibitory activity of sub-inhibitory concentrations of β-lactams

Introduction: The virulence factors of Pseudomonas aeruginosa are under the control of quorum sensing (QS) signals. Hence, interference with QS prevents its pathogenesis. Objective: The aim of the present research is to assess the influence of some β-lactam antibiotics on cell communication and the release of different virulence factors. Methods: The minimal inhibitory concentrations of ceftazidime, cefepime and imipenem were evaluated by microbroth dilu-tion method. The effect of sub-inhibitory concentration of the tested antibiotics on QS signals was investigated using reporter strain assay. In addition, different virulence factors (elastase, protease, pyocyanin and hemolysin) were estimated in the presence of their sub-inhibitory concentrations. Results: Low concentrations of ceftazidime, cefepime and imipenem caused significant elimination of the QS signals 3OH-C12-HSL and C4-HSL up to 1/20 MIC. Furthermore, low concentrations of the tested antimicrobials suppressed virulence factors elastase and hemolysin. Moreover, 1/20 of their MICs reduced elastase, protease, pyocyanin and hemolysin. Conclusion: Utilization of β-lactam antibiotics at low concentrations could be an effective approach for prevention and treat ment of P. aeruginosa infection.


Introduction
Pseudomonas aeruginosa is an opportunistic human pathogen with remarkable metabolic versatility.Infections with P. aeruginosa are common in compromised patients suffering from cystic fibrosis, severe burns, deep wounds, in addition to patients having urinary tract infections.P. aeruginosa produces various virulence attributes, including biofilm, toxins and enzymes such as pyocyanin, protease, elastase, and rhamnolipids 1 .P. aeruginosa exhibits its virulence behavior via quorum sensing (QS) 2 .The common QS systems in P. aeruginosahave been assigned las and rhl.QS circuits in P. aeruginosa are connected by signaling molecules called autoinducers.The las system is composed of the synthase gene lasI and its transcriptional regulatory protein LasR.Its autoinducer is called N-(3-oxododecanoyl) homoserine lactone (3OH-C12-HSL).Similarly, the rhl system consists of rhlI synthase, and its transcriptional regulatory protein LasR.Also, it possesses autoinducer molecule N-butyryl homoserine lactone (C4-HSL) 2,3 .When bacterial growth reaches a specific threshold, the signals acyl homoserine lactones (AHL) are released and stimulates the expression of virulence genes 4 .Both the las and rhl systems are coregulated, and las system is dominant over the rhl pathway.Hence, inhibition of these signaling molecules hinders the pathogenicity of P. aeruginosa, and could be used for prevention or treatment of its infections 5 .
Several plants have QS inhibiting activities.Medicinal plants 6 and edible plants 7 exhibited QSI effects.Also, Streptomyces coelicoflavus isolated from soil microbiota produced 1 H-pyrrole-2-carboxylic acid with QSI effect 8 .Synthetic molecules and peptides exhibited QSI activity 9 .Previous studies focused on the effect of some antimicrobials such as aminoglycosides and quinolones on QS of P. aeruginosa.Azithromycin showed significant elimination of QS and virulence factors of P. aeruginosa 10 .The present study focused on the effect of sub-inhibitory concentrations of some β-lactam antibiotics on QS of P. aeruginosa.Furthermore, virulence factors of P. aeruginosa were assessed in the presence of sub-inhibitory concentrations of the tested antibiotics.

Bacterial strains, growth media and conditions
The wild strain P. aeruginosa PAO1 was used for the assay of QSI effects of the tested antibiotics.Two reporter strains; P. aeruginosa pME3846 (rhlI-lacZ; Tc r ) and E. coli MG4/pKDT17 (lasB::lacZplac-lasR Ap r )2,3 were used for the assessment of rhlI and lasI/R, respectively in the presence and absence of the tested antibacterials.The QS deficient P. aeruginosa isolate PAO-JP2 double mutant ( lasI::Tn10,Tcr; rhlI::Tn5012, Hgr) was included as a negative control 11 .All bacterial cultures were grown in Luria Bertani medium (LB broth; tryptone 1%, yeast extract 0.5%, and NaCI 1%) at 37º C.

Determination of minimal inhibitory concentration
Minimal inhibitory concentrations (MICs) of the studied β-lactams: cefepime (Cfp), ceftazidime (Cft), and imipenem (Imp), were estimated by broth microdilution method (CLSI, 2014).Two-fold serial dilutions of each antibiotic were prepared and inoculated with 0.1 ml of PAO1 inoculum contained 5×10 6 CFU/ ml and incubated at 37 °C for 24 h.Values of MIC were recorded as the lowest concentration of the antibiotic at which there was no visible growth of the organism 12 .

Determination of the viable count of P. aeruginosa-PAO1
The viability of P. aeruginosa PAO1 wild type was examined in the presence of sub-inhibitory concentrations (1/4 MIC) of the tested β-lactams using pour plate counting method and cell proliferation was checked before supernatant collection 13 .Similarly, the viable count of the untreated cells was performed and compared to the treated cultures.
Preparation of the supernatant P. aeruginosa PAO1 was propagated in LB broth containing 1/4, 1/8 and 1/20 MIC of each antibiotic.P. aeruginosa PAO1 was also grown without antimicrobial agents as the positive control and PAO-JP2 was propagated under the same conditions as the negative control 14 .The supernatants of the treated and untreated Pseudomonas cultures were separated by centrifugation at 8.000 rpm for 10 min at 4 o C. Cell-free supernatants were then stored at -20 o C to be used for estimation of AHLs and assay of various virulence factors 15 .

Effect of β-lactams on QS signal molecules
The QS signals 3OH-C12-HSL and C4-HSL were detected in treated and untreated cultures of PAO1, respectively.The overnight growth of the reporter strains E. coli MG4 (pKDT17) and P. aeruginosa (pME3846) were diluted up to OD600 of 0.1.The previously prepared cell-free supernatant (1 ml) was mixed with 0.5 ml of E. coli MG4 and 1 ml P. aeruginosa pME3846.Cells were propagated till they reached 0.3-0.4 at OD600 then pelleted.β-galactosidase was estimated according to Miller assay method 16 .

Effect on virulence factors
The effect of the tested β-lactams on the virulence determinants of P. aeruginosa PAO1 was estimated using the prepared supernatants both in the presence and absence of tested β-lactams.

Evaluation of LasB elastase
The supernatant of P. aeruginosa was mixed with 10 mg of elastin Congo red (ECR, Sigma Chemicals, St. Louis, USA) in ECR buffer (100 mM Tris buffer, pH 7.2).The insoluble substrate was centrifuged and the absorbance of the supernatant was estimated at 495 nm 17 .

Total protease production
Total proteolytic effects of the treated and untreated cells were detected using modified skim milk method 18 .Cultures supernatants of PAO1 were mixed with skim milk (1.25%) at 1:2 ratio and reaction was incubated at 37°C for 30 min.The turbidity of the solution was determined at OD 600nm.The results were calculated as relative protease production to the untreated PAO1 strain.

Determination of hemolysin production
The hemolysin test was performed by incubating 700 μl of 2% erythrocytes suspension with 0.5 ml of PAO1 supernatant for 2 h at 37°C.The suspension was centrifuged and the produced hemoglobin was estimated by measuring the OD at 540nm.The negative control; RBCs in LB broth was evaluated under the same conditions.The total RBCs lysed were detected using 0.1% SDS 19 .
Media without antibiotics was also inoculated with PAO1 and propagated as a positive control.The culture was centrifuged and pyocyanin was extracted using chloroform in acidic pH and the red layer was separated.Its absorbance was measured at 520 nm and pyocyanin concentration was expressed as μg/ml by multiplying the OD520 by 17.072 20 .

Statistical analysis
The mean of three independent experiments and the standard deviation of the mean were calculated by Excel data package.P. aeruginosa treated with β-lactams were compared to the untreated control using GraphPad Instate software (version 3.05) and Tukey-Kramer multiple-comparison test.Significant difference between treated and untreated culture was assigned when the probability value was P< 0.05 or P< 0.01.

Results
Growth inhibitory concentrations for P. aeruginosa PAO1 Table (1) showed the MIC values of tested β-lactams and the mean value for their sub-inhibitory concentrations used for testing QSI effect and virulence factors.

Effect of sub-inhibitory concentrations of the tested β-lactams on the growth of P. aeruginosa PAO1
Viable counts of P. aeruginosa PAO1 treated with sub-inhibitory concentrations of cefepime, ceftazidime, and imipenem was performed using pour plate method.The tested concentrations (1/4 MIC) did not alter bacterial growth compared to the control untreated cultures and the same bacterial count of untreated PAO1 (161±21X10 7 CFU/ml).
All tested antibiotics produced no effect on both reporter strains.

Discussion
P. aeruginosa is one of the main causes of the persistent urinary tract, respiratory tract, burn and wound infections 21 .Pathogenesis of P. aeruginosa is attributed to secreted virulence factors such as elastase, proteases, and pyocyanin.Its pathogenesis is also disseminated through bacterial adhesion and biofilm formation 5 .QS is the key regulator for Pseudomonas virulence and biofilm formation.Hence, reduction in bacterial communication by QS inhibitors results in reduced levels of virulence factors 22 .Therefore, targeting QS cascade developed a new therapeutic approach for control of Pseudomonas infections.This study showed that the sub-inhibitory concentrations of the three tested β-lactams exhibited significant elimination of QS signals.Quantitative assessment of both QS signals has clearly indicated that anti-QS activity of the tested drugs is concentration dependent.Regarding the autoinducers 3OH-C12-HSL and C4-HSL, they significantly decreased (P<0.01) by sub-inhibitory concentrations of cefepime, ceftazidime and imipenem compared to the untreated PAO1 strain (Figs. 1 and 2).In line with these findings, certain macrolide antibiotics (azithromycin) are capable of repressing the synthesis of P. aeruginosa AHLs signal molecules when applied at sub-MICs 23 .Furthermore, sub-inhibitory concentrations of tobramycin, ciprofloxacin, and ceftazidime were effective in lowering the AHL levels in P. aeruginosa 24 .The QSI of some antimicrobials is attributed to the alteration in membrane permeability, and subsequent elimination of the flux of N-3-oxo-dodecanoyl-l-homoserine lactone 25 .Also, efflux inhibitor phenylalanine arginyl β-naphthylamide caused significant elimination of QS signals and related virulence factors among multidrug resistant P. aeruginosa clinical isolates 26 .
To confirm that the elimination of QS signals and virulence was not related to the antimicrobial activities of the tested β-lactams, viable counts of the treated cells were performed and our data have shown no change in the cell growth.Bacterial growth in the presence of Sub-MICs of tested β-lactams produced viable bacterial count similar to the positive control (untreated P. aeruginosa PAO1).
The effect of sub-MIC concentrations on P. aeruginosavirulence has been verified.One of the main pathogenic factors in P. aeruginosa is LasB elastase.LasB elastase and proteases interfere with host defense mechanisms and destroy tissue components 27 .Most of sub-MICs of the tested compounds resulted in a significant decrease in LasB elastase and proteases enzymes (P<0.01 and P<0.05) (Figs. 3-4).The inability of PAO1 strain to produce elastase and protease has been linked with their reduced level of associated QS molecules released under the influence of the assayed antibiotics.Much lower concentration (1/20 MIC) of some of these antibiotics did not affect bacterial QS signaling network and hence, showed no remarkable effect on elastase and protease activities.QS-deficient strains were non-virulent and lose the ability to produce elastase and protease 28 .
Hemolysin is another aspect of P. aeruginosa virulence which enhances its pathogenicity and helps in the sur-vival of pathogen by inhibiting the host defense factors.
Significantly reduced levels (P<0.01) of hemolysin were observed after treating P. aeruginosa PAO1 with sub-inhibitory concentrations of cefepime, ceftazidime, and imipenem (Fig. 5).Similarly, a low hemolytic activity of P. aeruginosa was reported with sub-inhibitory concentrations of erythromycin 29 , eugenol 30 and the 7-fluoroindole 17 .
On the same instance, QS system coordinates the release of secondary metabolites such as pyocyanin 15 .Direct antagonists of QS in P. aeruginosa have a great impact on the production of pyocyanin 31 .In this study, the level of pyocyanin was significantly reduced (P<0.01)with sub-MICs of cefepime, ceftazidime and imipenem (Fig. 6).
These results indicated that controlling the production of QS molecules hinders most of the associated virulence agents.Also, inhibition of AHL signaling in Pseudomonas caused a reduction in the related virulence factors and attenuation of this pathogen when tested on animal models of pneumonia.On the same instance, synergistic effect has been achieved upon treating mice with both the QSI furanone C-30 and tobramycin, with increase in the clearance of P. aeruginosa in a foreign-body infection model 32 .

Conclusion
The fact that these antibiotics are already approved drugs for human use is a significant benefit in the future application and development of anti-pathogenic drugs.Hence, further investigations are necessary to detect anti-quorum sensing potential of antimicrobial combinations.Also, further in-vivo studies are required to confirm their possible utilization as anti-pathogenic agents for competing with P. aeruginosa infections.