Cephalosporins target quorum sensing and suppress virulence of Pseudomonas aeruginosa in Caenorhabditis elegans infection model

Pseudomonas aeruginosa utilizes a chemical social networking system referred to as quorum sensing (QS) to strategically co-ordinate the expression of virulence factors and biofilm formation. Virulence attributes damage the host cells, impair the host immune system, and protect bacterial cells from antibiotic attack. Thus, anti-QS agents may act as novel anti-infective therapeutics to treat P. aeruginosa infections. The present study was performed to evaluate the anti-QS, anti-biofilm, and anti-virulence activity of β-lactam antibiotics (carbapenems and cephalosporins) against P. aeruginosa. The anti-QS activity was quantified using Chromobacterium violaceum CV026 as a QS reporter strain. Our results showed that cephalosporins including cefepime (CP), ceftazidime (CF), and ceftriaxone (CT) exhibited potent anti-QS and anti-virulence activities against P. aeruginosa PAO1. These antibiotics significantly impaired motility phenotypes, decreased pyocyanin production, and reduced the biofilm formation by P. aeruginosa PAO1. In the present study, we studied isogenic QS mutants of PAO1: ΔLasR, ΔRhlR, ΔPqsA, and ΔPqsR and found that the levels of virulence factors of antibiotic-treated PAO1 were comparable to QS mutant strains. Molecular docking predicted high binding affinities of cephalosporins for the ligand-binding pocket of QS receptors (CviR, LasR, and PqsR). In addition, our results showed that the anti-microbial activity of aminoglycosides increased in the presence of sub-inhibitory concentrations (sub-MICs) of CP against P. aeruginosa PAO1. Further, utilizing Caenorhabditis elegans as an animal model for the in vivo anti-virulence effects of antibiotics, cephalosporins showed a significant increase in C. elegans survival by suppressing virulence factor production in P. aeruginosa. Thus, our results indicate that cephalosporins might provide a viable anti-virulence therapy in the treatment of infections caused by multi-drug resistant P. aeruginosa.


INTRODUCTION
5 based on hydrogen bond interactions of the carbonyl group with Trp84, the amide carbonyl group 186 with both Tyr80 and Ser155, and the secondary amine with Asp97 ( Fig 1J). The last carbon on 187 the alkane tail of C10-HSL formed an alkyl interaction with Met72 and a -alkyl interaction with 188 Tyr88. Of the inhibitors, CT displayed the most favorable interactions with the CviR receptor, 189 with a predicted affinity of -6.5 kcal mol -1 . CT formed four hydrogen bonds; one between Met72 190 and the carbonyl on the beta-lactam ring, one between Ile69 and the carboxylic acid, and two more 191 hydrogen bonds with Tyr88 and Asn92 from the amine connected to the aromatic ring (Fig 1M). 192 A -sulfur interaction was formed between Met72 and the aromatic ring, an alkyl interaction 193 between Ala94 and the non-aromatic ring thioether, as well as a -alkyl interaction with the 194 aromatic ring Ala94. In addition to this, an unfavorable interaction was formed between Ala94 195 and the proton of the amine connected to the aromatic ring, which may be the reason for the slightly 196 lower predicted energy than the natural ligand. 197 198 CP showed the second highest predicted binding affinity of -6.3 kcal mol -1 with the CviR receptor.

199
CP formed -alkyl interactions with Met72 and Ala94 and an amide- stacking interaction with 200 Gln70 (Fig 1K). Two hydrogen bonds were formed between Ile69 and the proton of the primary 201 amine connected to the aromatic ring, and between Met72 and the nitrogen in the methoxy-amino 202 group. Alkyl interactions were formed with Met72 and Val75 residues. In addition, an unfavorable 203 donor-donor interaction was detected between the proton of the carboxylic acid and Ser89.

205
Among the three inhibitors, CF showed the lowest affinity for the CviR receptor (-6.1 kcal mol -1 ).

206
CF formed a hydrogen bond with Ser89 from the primary amine connected to the aromatic ring, 207 and another with Met72 from a carboxylic acid group. Near the carboxylic acid group, four alkyl 208 interactions were formed with the neighboring carbon atoms (Fig 1L). and CP were found to be 256 µg/mL, 2 µg/mL, 32 µg/mL, 1 µg/mL, 32 µg/mL, 4 µg/mL, and 2 216 µg/mL (Supplementary Fig 6). Interestingly, the OD600 values at sub-MIC of each antibiotic were 217 found to be significantly lower (p<0.001) than the control group (PAO1) (Supplementary Fig   218   7A-7H). This indicated that growth was influenced by antibiotics. Surprisingly the growth was 219 also significantly affected in oxacillin antibiotics that showed no anti-microbial activity against 220 PAO1 (512-0.5 µg/l) (Supplementary Fig 6E). These results were encouraging and indicated that 221 antibiotics were targeting alternate molecular pathways influencing the growth of P. aeruginosa. 222 Further, we measured growth curves of PAO1 in the presence and the absence of sub-MIC (MIC, 223 MIC/2, and MIC/4) of each antibiotic (Fig 2A-2C). We also compared the growth of PAO1 with 224 QS mutant strains as shown in Fig 2D.

226
Effect of cephalosporins on pyocyanin production. In the present study, the antibiotic treatment 227 showed significant inhibition of pyocyanin production at a sub-MIC concentration as compared to 228 control (Fig 3A and 3B). PAO1 showed high pyocyanin pigment production OD690 nm (1.67 + 0.19) 229 and with CT (1.02 + 0.18), CF (0.77 + 0.27), CP (0.48 + 0.17), pyocyanin production was 230 significantly reduced (Fig 3A and 3B). These results collectively suggested that CP, CF, and CT 231 7 biofilms of PAO1 of the control group showed the formation of a thick layer of bacterial cells 278 embedded in an exopolysaccharide matrix (Fig 6Bi). Biofilms appeared to be complex in structure 279 with the presence of long polymer threads, mucilaginous matrix, and high cellular growth. Live 280 cell counts were higher on day 3 in control groups (8.28±1.13 log cfu) (Fig 6B). The CP-treated 281 catheter surface showed low cell counts attached to the urinary catheter surface at day 3 indicating 282 the potent anti-adherence ability of CP (Fig 6Bii). The bacterial live cell count on day 3 CP treated 283 urinary catheters was lower (5.01 + 0.56 log cfu) as compared to the control live cell count (Fig   284   6B). CF and CT antibiotics showed thin biofilm formation on the catheter surface (Fig 6Biii and   285 Biv). The bacterial cells appeared to be longer in size as compared to the control. These results 286 indicate that the effect of antibiotic treatment altered the cell physiology and growth cycle of P. 287 aeruginosa. Log cfu count of the treated groups were significantly reduced (p<0.05) as compared 288 to the PAO1 control (CT 6.0±0.56 log cfu and CF 6.6±0.73 log cfu) (Fig 6B).  biofilms of the PAO1 control group showed thick biofilm and completely covered with a thick 290 exopolysaccharide layer (Fig 6Ci). On day 5, the biofilms started to construct mushroom-shaped 291 structures. At this stage, biofilms might be completely resistant to antimicrobial agents due to the 292 protection provided by the exopolysaccharide layer. These clusters of live bacterial cells help in 293 the formation of the three-dimensional architecture of bacterial biofilm. As expected, the biofilm 294 showed a high live cell count in five-day-old biofilms (10.05±0.86 log cfu) (Fig 6C). In CP treated 295 groups, the biofilm appeared to be defective (Fig 6Cii) and the log cfu count of the live cell was 296 significantly lower indicating the potent antibiofilm activity of CP (Fig 6C). In CT and CF, treated 297 biofilms were condensed and covered with thick polysaccharide matrix on the catheter surface 298 (Fig 6Ciii and Civ). In CF treated biofilms, bacterial cells appeared long chains embedded in the 299 polysaccharide matrix. However, there was no significant difference in the log cell count of live 300 cells in CF treated biofilms as compared to the control group. CT treated biofilms showed a 301 significant difference in the log cfu count of bacterial cells (7.01±0.19 log cfu) (Fig 6C). 302 Moreover, on day-7 control biofilms appeared highly complex three dimensional with numerous 303 mushroom-shaped secondary structure on the biofilm surface (Fig 6Di). In the untreated control 304 group, biofilms showed a larger assembly of multi-mushroom shaped structures, and the log cfu 305 count of live cells in 7-day-old-biofilm was found to be very high (12.60±0.75 log cfu) (Fig 1D). 306 The seven-day-old CP treated biofilms were still defective in biofilm architecture. The mushroom-307 shaped structures were absent and individual cells can be visualized on the catheter surface. In 308 treated biofilms, bacterial cells appeared as long thread shaped and produced significantly less 309 polysaccharide with low log cfu count of 7.81±0.65 (Fig 6D). CT and CF biofilms were covered 310 with mushroom-shaped structures and few individual cells were visible indicating the thick three-311 dimensional biofilm formation. Live cell count of the catheter surface of these antibiotic-treated 312 groups was found to be non-significant as compare to control indicating the cells have counteracted 313 the effect of these antibiotics using complex exopolysaccharide shield (Fig 6D).

315
Synergistic effect of cefepime and aminoglycosides against P. aeruginosa PAO1. After 316 determining the anti-QS and anti-virulence effect of cephalosporin antibiotics against PAO1, we 317 were interested in testing the synergistic activity of CP with aminoglycosides against P. 318 aeruginosa PAO1. First, the antimicrobial efficacy of aminoglycosides was tested against P. 319 aeruginosa PAO1. Kanamycin sulfate showed poor antibacterial activity against P. aeruginosa 320 PAO1 (MIC: 64 µg/mL), however, in presence of CP, the MIC of kanamycin has significantly 321 decreased to 16 µg/mL (Fig 7A). Streptomycin sulfate showed potent efficacy against PAO1 and 322 the MIC was found to be 8 µg/mL (Fig 7B). In synergy with CP, the MIC of streptomycin was 323 significantly reduced to 2 µg/mL. Similarly, the MIC of neomycin was found to be 16 µg/mL and 324 in combination with CP, the MIC was decreased to 8 µg/mL (Fig 7C). In addition, gentamicin and 325 tobramycin were found to be highly effective antibiotics against PAO1 with the MIC values of 2 326 µg/mL (Fig 7D and 7E). However, CP also showed a synergistic effect with these antibiotics and 327 the MIC values were decreased to 1 µg/mL. In literature, the anti-QS compound has shown to 328 potential the anti-microbial activities of various antibiotics. The anti-QS compounds target various 329 pathways that silence the expression of virulence factors. Major virulence factors including 330 lipopolysaccharide, alginate, and extracellular matrix proteins help in the development of 331 antimicrobial resistance and blocking QS pathways may lead to increased susceptibility of 332 antimicrobial drugs due to suppressed virulence state of the pathogen. Our results showed that the 333 CP at a sub-inhibitory concentration in combination with major aminoglycosides led to the 334 increased antimicrobial potency of aminoglycosides due to its potent anti-virulence activity. This 335 combination needs to be tested in animal models to validate its effectiveness against P. aeruginosa 336 and could be established as a clinically useful combinational therapy to treat biofilm-associated P. 337 aeruginosa infections.

339
Molecular docking of cephalosporins with P. aeruginosa quorum-sensing receptors. To 340 compare cephalosporin binding to the LasR interactions with the natural ligand, we first docked 341 3-oxo-C12HSL to its binding pocket in the LasR receptor. The predicted binding affinity was -8.0 342 kcal mol -1 . 3-oxo-C12HSL formed three hydrogen bonds, one with Ser129 and the amide carbonyl, 343 and two more with Asp73, Thr75, and the proton of the secondary amine (Fig 8A). A hydrophobic 344 interaction connected Tyr56 and the ring carbon neighboring the secondary amine, supported by 345 two alkyl interactions between the carbon at the end of the hydrophobic tail with Leu40 and Ala50.

346
Next, the three selected antibiotics were docked to the same ligand-binding pocket of LasR. CP 347 formed two hydrogen bonds, one between the carboxyl group and Asp65 and another between 348 Ala50 and the proton of the secondary amine connected to the beta-lactam ring (Fig 8B). A -349 sulfur interaction was formed between Phe167 and the sulfur atom of the aromatic ring, an 350 electrostatic interaction was formed between Asp65 and the charged nitrogen atom, a carbon-351 hydrogen bond was formed between Asn49 and the beta-lactam carbonyl, and an alkyl interaction 352 was formed between Ile52 and the thioether group. CP showed a comparative docking score of -353 5.6 kcal mol -1 . CF formed three hydrogen bonds; one between Gly54 and a carboxyl group, and 354 two between the primary amine with Tyr56 and Asn55. Lys16 formed a carbon-hydrogen bond 355 with the carboxyl group and an alkyl interaction with a nearby methyl group (Fig 8C). A pi-alkyl 356 interaction was formed between Ala58 and the aromatic ring, and an unfavorable electrostatic 357 interaction was formed between Glu62 and the carboxyl nearest to the beta-lactam ring. CF showed 358 a comparatively low docking score of -5.9 kcal mol -1 . CT formed two hydrogen bonds; one 359 between Asn49 and the primary amine and another between Lys16 and the non-aromatic ring 360 thioether (Fig 8D). A pi-sulfur interaction was formed between Phe167 and the aromatic ring 361 thioether. Carbon hydrogen bonds were formed between Ala58 and the methyl group connected 362 9 last carbon in the hydrophobic tail and Leu189, Val170, Trp234, Val211, Ile236 of PqsR receptor 370 (Fig 8E). The natural ligand showed a docking score of -7.4 kcal mol -1 . CP formed two hydrogen 371 bonds from its primary amine to Arg209 and Leu208. A carbon-hydrogen bond was formed 372 between the methoxyl group and Thr265 (Fig 8F). Two - interactions were formed from the 373 aromatic ring to Leu208 and Ile236. An alkyl interaction and a -alkyl interaction were formed 374 from the thioether to Ile263 and Tyr258, respectively. The docking score of CP was found to be -375 6.7 kcal mol -1 . Similarly, CF formed a hydrogen bond between its primary amine and Pro210 as 376 well as a carbon-hydrogen bond from Leu207 to the six-membered ring (Fig 8G). An unfavorable 377 interaction was also formed between Glu259 and the carboxyl nearest the beta-lactam ring. CF 378 showed the docking score of -6.2 kcal mol -1 . CT formed four hydrogen bonds; one between its 379 primary amine and Glu151, one between Leu207 and the carboxyl group, one between Ile236 and 380 the proton on the triazine ring, and lastly one between Leu197 and a carboxyl on the triazine ring 381 (Fig 8H). The docking score of CT was -6.7 kcal mol -1 . These conformations would obstruct the achieved 0% survival. All worms were found dead after 72 h (Fig 9F). Interestingly, the sub-397 inhibitory concentration of antibiotic treatment showed reduced virulence in C. elegans. CP 398 treatment showed a significant increase in the percentage survival of C. elegans. At 12, 24, 48 and 399 72h the % survival was 95.6 + 2.51%, 92.33 + 2.50%, 83.33 + 4.16%, and 65.33 + 5.03% (Fig   400   9F). Similarly, CF treated supernatant also showed a significant increase in worm survival. At 12, 401 24, 48 and 72 h the % survival was found to be 93.6 + 1.52%, 85.33 + 5.03%, 77.0 + 5.56% and 402 58.0 + 4.30% (Fig 9F). CT showed similar anti-virulence effect and treatment groups showed 403 reduced mortality. At 12, 24, 48 and 72 h, the percentage survival was found to be 92.0 + 2.60%, 404 84.33 + 5.56%, and 48.66 + 8.08% (Fig 9F). All three antibiotics showed a significant reduction 405 in the C. elegans mortality indicating the potent antivirulence effect of a sub-inhibitory 406 concentration of cephalosporin antibiotics. C. elegans was found to be healthy and inflammatory 407 damage of intestinal tissue was significantly reduced indicated the strong antivirulence effect of 408 cephalosporins. Subsequently, to prove antivirulence activity is related to QS inhibition of P. 409 aeruginosa, we tested this hypothesis by evaluating the effect of QS mutant strains of P. 36.66 + 5.77% survival rates of C. elegans (Fig 9G). We have also used an uninfected group with 417 E.coli OP50 to check the C. elegans mortality and no mortality was observed until 72 hr. The result 418 of our experiment showed that the sub-MIC concentration of antibiotics suppressed the virulence 419 factors of P. aeruginosa and led to an increase in the survival of C. elegans. Finally, we have 420 compared the percentage mortality of all the groups with the control PAO1 group and all the groups 421 have shown a significant reduction in the percentage mortality at 72 hr (Fig 9H). Additionally, 422 mortality rates were higher in the QS mutant strains as compared to the antibiotic-treated groups 423 (Fig 9G). This indicates that the single QS mutation does not completely makes the bacteria 424 avirulent, therefore, the therapies targeting multiple QS systems will be useful as antivirulence 425 drugs. This also indicates that cephalosporins might have additional alternative antivirulence 426 mechanism apart from anti-QS activity that may target virulence of P. aeruginosa. In the future, it 427 might be interesting to investigate the antivirulence studies at the molecular level and validate the 428 CP, CF, and CT as potential antivirulence therapies to treat P. aeruginosa infections.  [64] might also play a key role for their antimicrobial activity.

501
In a typical agar well diffusion assay, the antibiotics diffuse in media and its concentration 502 decreases as it diffuse from wells towards the periphery. At the interface between bacterial growth 503 and no growth, there is a zone where antibiotic is present in sub-inhibitory concentrations. The Sub-MIC is not sufficient to kill the bacteria; however, it can provide useful information on the 505 alternative activity of antibiotics such as anti-QS activity. We analyzed our zones of sub-MIC 506 concentrations for each antibiotic and found that the three major cephalosporins showed a large 507 zone of pigment inhibition at the interface of growth and no growth indicating their potential anti-508 QS activity against C. violaceum CV026. The presence of the zone of pigment inhibition indicated 509 that the cephalosporin antibiotics were interfering with the activation of the QS pathway in the 510 CV026 strain (Fig 1A-C). The size of zones for the antibiotics was as follows (large to small) 511 CP>CF>CT>EP>DP>MP>IP>OX. There was clear evidence that the carbapenem (MP, IP, DP, 512 and EP) showed significantly low anti-QS activity as compare to cephalosporins (CP, CF, and 513 CT). The anti-QS activity trend was completely reversed to the anti-microbial activity trend. We 514 hypothesized the main two reasons to explain this trend (1) the small zone of anti-QS activity for 515 carbapenem might be because of their potent anti-microbial activity as compare to the 516 cephalosporins; (2) the potent anti-QS activity of cephalosporin as compared to the carbapenems.

517
To quantify the anti-QS effect among cephalosporin antibiotics (CP, CF, and CT) we used OD 518 based microtiter plate assay. First, we quantified MICs of these antibiotics and MIC value showed 519 the tread of CP>CT>CF. This indicates that CP is required in high concentration as compared to 520 CT and CF to kill C. violaceum. Further, we tested sub-MICs to find out the concentration that with Met72 and Tyr88 (Fig 1J-1M). The active site is not fully accessible to cephalosporins due 539 to their relatively large size. However, molecular docking revealed that the cephalosporins might 540 have the affinity to bind with amino acids at the edge of the natural binding pocket including Met72 541 and Tyr88 (Fig 1J-1M). This binding might block ligand binding or may induce conformational 542 changes to block activation of CviR. Molecular docking results showed that all three antibiotics 543 formed H-bonds with Met72. CT also formed hydrogen bonds with Tyr88 and two more amino 544 acids. However, CF and CT also showed unfavorable interactions with Ser89 and Ala94. The 545 docking score predicted the affinity of cephalosporins against CviR (higher to lower) as 546 CT>CP>CF. Our experimental anti-QS trend followed the sequence CP>CT>CF. In both cases, 547 CF showed the lowest activity, supporting the conclusion that CF binding is the weakest of the was absent for QS-mutant strains (Fig 2D). We fitted the growth curves with the equation [ (1)

562
Equation (1) fitted (solid lines) data points (symbols) well (Fig 2A-2D). Typically, a monophasic 563 growth described by a logistic equation is observed in a bacteria culture. There are three distinct 564 phases: (1) a lag phase with very slow growth when the bacteria undergo preparatory steps for the 565 next phase, (2) a log phase of exponential growth when the bacteria divide at a constant rate, (3)  pathways resulting in the observed biphasic growth (Fig 2A-2C). This conclusion is supported by 573 the absence of the second growth phase in all QS-mutant strains of PAO1 (Fig 2D). An analysis to be 2 µg/mL, 4 µg/mL, 32 µg/mL respectively, i.e., CP is the most potent among the three against 584 PAO1 (Supplementary Fig 6). Despite the low MIC, sub-MIC concentrations did not delay the 585 start of the exponential log phase of PAO1 growth, suggesting that CP induced less environmental 586 stress to PAO1 leading to a shorter preparatory lag phase. 1/2 th MIC concentration was 587 significantly affecting the growth of the cells and delaying the log phase, while 1/4 th MIC did not 588 affect the growth profile of the bacteria, and cells were effectively growing in presence of these 589 concentrations of antibiotics (Fig 3A-3F). Therefore, 1/4 th MIC concentration of each antibiotic 590 was selected to test anti-virulence and anti-biofilm activities against PAO1. These findings are in 591 agreement with previous reports that show these antibiotics alter gene expression induced 592 mutations and alter growth profiles at sub-MICs leading to growth inhibition of bacteria [73,74].

594
Pyocyanin production was blocked by sub-inhibitory concentrations of cephalosporins.

595
Pyocyanin is a blue colored secondary metabolite and major virulence factor responsible for the 596 extreme toxicity of P. aeruginosa against host cells [75]. Previous research has shown that the 597 pyocyanin deficient phnAB and PhzB1 mutants produce a low level of pyocyanin leading to 598 reduced mortality in the burn wound model of P. aeruginosa [76,77]. Pyocyanin also induces pro- results showed that all three antibiotics significantly suppressed the pyocyanin production in vitro 603 and their ability in the suppression of pyocyanin production was as follows: CP>CF>CT (high to 604 low) (Fig 3A and 3B). CP showed the highest effect as compared to CT and CF. Previously anti-

605
QS effect of macrolide antibiotics (azithromycin and erythromycin) has been related to pyocyanin 606 inhibitory effect [80][81][82]. The in vitro anti QS activity followed the pattern as follows: CP>CF>CT 607 (high to low) and in vitro pyocyanin production also followed the same pattern (CP>CF>CT). This 608 indicates that the QS inhibitory potential of cefepime targeted the pyocyanin production in P. Swarming motility is also a flagella-dependent movement of cells to spread as a biofilm over biotic 630 and abiotic surfaces [86]. Previous research has shown that QS controls biofilm development by 631 regulating swarming motility phenotypes [87]. Our results also showed that swarming motility is 632 dependent on las and rhl signaling systems and independent of the PQS signaling system.

634
However, there was a significant difference in the swarming motility of ∆LasR and ∆RhlR mutant 635 strains with and without cephalosporins. This indicates that cephalosporins might also have a direct 636 effect on the flagellar motility. Moreover, the inhibition of swarming motility also provides strong 637 evidence that cephalosporins may interfere with the QS communication of P. aeruginosa.

639
Twitching motility is a flagella-independent bacterial motion on moist surfaces by extension and pattern of twitching motility inhibition by cephalosporins was similar to swimming and swarming 645 motility inhibition pattern (CP>CF>CT) (Fig 4Ci and Fig 5C). There was also a significant 646 difference in the twitching motility of ∆LasR and ∆RhlR mutant strains with and without 647 cephalosporins suggesting their alternate inhibitory mechanisms. In contast, CT showed no effect 648 on twiching motlity phenotypes of PAO1 or its isogenic mutant. This indiates that the  Fig 6). This pattern was similar to our anti-QS activity pattern. In addition, despite 665 the low anti-QS activity, the inability of CT to inhibit twitching motility might also contribute to 666 its low antibiofilm efficacy. CP was the most effective antibiofilm agent and significantly inhibited 667 seven-day-old biofilms. CF was ineffective against day-five and CT was ineffective against day- [97]. The synergistic activity of gentamicin and ciprofloxacin has also been reported against P.

687
aeruginosa [98]. In addition, CP has shown synergistic activity with amikacin against Gram-688 positive and Gram-negative pathogens [99,100]. Our results showed that CP has significantly 689 decreased the MIC of aminoglycosides (Fig 7). There was 75% decrease in kanamycin MIC (from 690 691 Neomycin, gentamicin, and tobramycin showed a 50% decrease in MIC values in presence of 692 cefepime. Our results suggested that although CP enhanced antimicrobial activity of all the tested 693 aminoglycosides however, it worked better with kanamycin and streptomycin (Fig 7). Further 694 research is required to validate its efficacy in animal models to confirm the in vitro results.

696
Cephalosporins may bind to P. aeruginosa QS receptors to inhibit ligand binding. The bond with leu208 and -interactions with Ile236 (Fig 8) showed the highest protective effect in terms of C. elegans survival as compared to CF and CT.

746
The high protective effect of CP may be linked with its high anti-QS activity and anti-virulence In the present study, we have shown that cephalosporins can be used as QS inhibitors. We proved and OD was adjusted to 0.1 (OD600). Stock cell suspension (10 µL) was used as inoculum for 831 each well. C6-HSL at the final concentration of 10 µg/mL was used for pigment production in 832 each well. Antibiotics were serially diluted in the well. After adding C6-HSL and CVO20, the 833 plate was incubated at 37 o C for 24 h. After incubation, the OD567 was measured using a plate 834 reader. We have tested sub-MICs of CP, CF, and CT using a microtiter plate assay. The minimum 835 inhibitory concentration was determined using NCCLS (2002) Table 1. Comparative analysis of molecular interaction of natural ligands and antibiotics with receptor proteins. Amino acids found only in the natural binding pocket are highlighted in red, while amino acids found in the natural and the antibiotic binding pockets are highlighted in green. Amino acids only found in inhibitor binding pockets are shown in black.