Non-steroidal anti-inflammatory drugs and sodium ascorbate potentiate the antibiotic activity against Pseudomonas aeruginosa biofilms

 

Hisham A. Abbas, Fathy M. Serry, Eman M. EL-Masry

Department of Microbiology and Immunology-Faculty of Pharmacy-Zagazig University- Zagazig-Egypt

*Corresponding Author E-mail: h_abdelmonem@yahoo.com

 

ABSTRACT:

This study aimed to investigate the possible synergistic effects of three Non-steroidal anti-inflammatory drugs;  piroxicam, ketoprofen, diclofenac sodium in addition to sodium ascorbate with ciprofloxacin, cefoperazone, tobramycin, amikacin and imipenem against established biofilms formed by five clinical isolates of Pseudomonas aeruginosa. The biofilm inhibiting effect of the antibiotics was investigated by determination of minimum regrowth concentration (MRC). Complete or partial synergism was found with 87% of the isolates. The highest synergistic effect was exerted by piroxicam with each of tobramycin and amikacin. Complete synergy was also observed for piroxicam with ciprofloxacin and cefoperazone, sodium ascorbate with cefoperazone, tobramycin, ciprofloxacin and amikacin, ketoprofen with tobramycin, imipenem and amikacin and diclofenac with each of tobramycin and amikacin. Partial synergy was found for imipenem with each of piroxicam, diclofenac and sodium ascorbate, cefoperazone with each of diclofenac sodium and ketoprofen. No synergy was observed with ketoprofen when combined with ciprofloxacin. Piroxicam was more synergistic than other tested agents, followed by sodium ascorbate, ketoprofen and diclofenac sodium. Piroxicam decreased the MRC of antibiotics by up to 128-1024 folds, sodium ascorbate by up to 4-256 folds, diclofenac sodium by up to 4-128 folds, while the MRCs were decreased by up to 2-128 folds in the presence of ketoprofen.

 

Comparing the synergy of antibiotics when combined with the tested agents, the aminoglycosides amikacin and tobramycin were generally more potentiated than ciprofloxacin, cefoperazone and imipenem. With piroxicam, amikacin and tobramycin were more augmented than ciprofloxacin, cefoperazone and imipenem. Combined with sodium ascorbate, cefoperazone was more potentiated than amikacin, tobramycin, imipenem and ciprofloxacin. Higher synergistic effect of diclofenac sodium and ketoprofen was demonstrated with tobramycin and amikacin, while imipenem, cefoperazone and ciprofloxacin were less augmented.

 

Our results suggest the use of antibiotics in combination with sodium ascorbate and NSAIDs to increase the efficacy of antibiotics against biofilm infections.

 

KEY WORDS: NSAIDs, sodium ascorbate, antibiotic synergy, biofilm, Pseudomonas aeruginosa


 

INTRODUCTION:

Pseudomonas aeruginosa is a gram-negative bacterium that represents a main cause of opportunistic hospital-acquired infections.1 For survival of bacteria in natural habitats and during infection, they grow as biofilms. P. aeruginosa is one of the bacteria that have the ability to form biofilms. In biofilms, bacteria are present as sessile communities that are attached to a surface and enclosed in a matrix composed of polysaccharides in addition to protein, and DNA.2, 3

 

Growth of P. aeruginosa in the biofilm mode greatly increases their resistance to antibiotics (up to 100-1000 folds increase in their minimum inhibitory concentrations) and enable it to evade the host immunity.4, 5, 6 Moreover, the antimicrobial resistance of P. aeruginosa biofilms seems to be multifactorial.7, 5 Differential growth and metabolism of biofilm sub-populations due to difference in oxygen and nutrients availability contributes to resistance.8 Oxygen limitation and presence of persister cells in biofilms has a marked effect on increasing the antimicrobial resistance of P. aeruginosa biofilms.9,10 In addition, the matrix diffusion barrier and the interaction between the matrix material and the antimicrobial agents are other mechanisms of biofilm resistance.11 Furthermore, antibiotic resistance of biofilms is affected by quorum sensing.12 The high antimicrobial resistance of biofilms makes the treatment of infections caused by biofilm-forming bacteria difficult.13

 

Non-steroidal anti-inflammatory drugs (NSAIDs) were found to have good antimicrobial activities.14 Diclofenac sodium exerted a potent antimicrobial activity against E. coli, P. aeruginosa, S. typhi, and C. albicans.15 Annaduri et al.14 reported that diclofenac sodium has antibacterial activity against most bacteria. This antimicrobial activity may be due to interference with bacterial DNA synthesis.16 Diclofenac sodium showed inhibitory activity against biofilm formation.15 Abbas et al.17 reported that diclofenac sodium, piroxicam and ketoprofen possess antibiofilm activities against P.aeruginosa biofilms. The biofilm inhibiting activity of NSAIDs may be attributed to inhibition of prostaglandin synthesis.18

 

Ascorbic acid may inhibit biofilm formation by different mechanisms. Ascorbic acid and sodium ascorbate could inhibit quorum sensing in Clostridium perfringens.19 Furthermore, ascorbic acid could inhibit efflux pumps in E. coli.20 The inhibitory effect on efflux pumps and quorum sensing may account for the biofilm inhibitory activity of ascorbic acid.

 

The aim of this study is to study the possible synergic interaction between antibiotics and sodium ascorbate and non-steroidal anti-inflammatory agents against established P. aeruginosa biofilms.

 

MATERIALS AND METHODS:

Bacterial strains:

Five isolates of Pseudomonas aeruginosa were obtained by endotracheal aspiration from patients hospitalized in intensive care unit in Zagazig university Hospitals.

 

Biofilm formation assay:

Biofilm formation assay was performed following the method described by Stepanovic et al. 21  Briefly, bacterial strains were grown for 24 h on tryptone soya agar plates and were inoculated, each into 5 ml of tryptone soya broth supplemented with 1% glucose (TSBglu) in Falcon tubes and the turbidity was adjusted to match the turbidity of 0.5 McFarland standard. Negative control tubes with TSBglu only were used.  Following incubation of the broth tubes for 24 h at 37 ºC; the contents of each tube was emptied by aspiration, and sterile saline was used to wash the tubes three times. For removal of any non-adherent bacteria, the tubes were vigorously shaken and 99% methanol was added and left for 15 minutes to fix the adherent cells. The tubes were then decanted, left to be air-dried and stained with 2% Hucker crystal violet for 5 minutes. After removing the dye solution with water, the tubes were air dried and the attached dye was re-eluted using 33% (v/v) glacial acetic acid. The biofilm was quantified by reading the optical density (OD570 nm) using Spectrophotometer (UV-1800 Shimadzu, Japan). To categorize the test strains according to the measured ODs of their bacterial biofilms, the cut-off OD (ODc), which is three times standard deviations above the mean OD of the negative control was calculated. The isolates were either non-biofilm forming (OD ≤ ODc), weak biofilm forming (OD > ODc, but ≤ 2x ODc), moderate biofilm forming (OD >2x ODc, but ≤ 4x ODc), or strong biofilm forming (OD > 4x ODc). The test was made in triplicates, repeated three times and the mean optical densities were calculated.

 

Determination of minimum inhibitory concentration (MIC):

The minimum inhibitory concentrations (MICs) of the antibiotics and the tested agents were determined by the agar dilution method according to Clinical Laboratory and Standards Institutes Guidelines (CLSI). 22 over night cultures of the bacterial strains were standardized to achieve a turbidity matching that of 0.5 McFarland standard and diluted with sterile saline so that the cell density was approximately 107 CFU/ml. A standardized inoculum was deliveded to the surface of Mueller-Hinton agar plates with different antibiotic dilutions to achieve a final inoculum of approximately 104 CFU per spot on the agar. Antimicrobial-free plates were used as growth control. To allow the liquid to be absorbed into the agar, the inoculated agar plates were allowed to stand at room temperature. The plates were inverted and incubated at 35-37 °C for 16–20 h. The MIC was considered as the lowest concentration of antimicrobial agent that could inhibit growth.

 

Determination of minimum regrowth concentration:

The minimum regrowth concentrations (MRCS) of antibiotics, sodium ascorbate and tested anti-inflammatory agents were determined according to the method described by Černohorská and Votava. 23 bacterial inocula were prepared in TSB to have a turbidity that matches that of 0.5 McFarland standard. Aliquots of 75 μl of the inoculated medium were placed in the wells of the polystyrene microtiter plates, and the plates were incubated for 24 h at 37°C. The wells were washed aseptically three times with PBS to remove the non-adherent cells. The plates were inverted, dried and volumes of 100 μl of appropriate two-fold dilutions of the tested agents in Mueller–Hinton broth were placed into the wells with established biofilms. The microtiter plates were incubated for 18–20 h at 37 ºC and the wells were washed three times with PBS under aseptic conditions. The wells were filled with 100 µl TSB and incubated for 24 h at 37 ºC and the minimum regrowth concentration (MRC) was determined. MRC of each tested agent is defined as the minimum concentration of the tested agent which inhibits regrowth of the biofilm cells. Each experiment was repeated thrice with positive and negative controls included in all experiments.

 

The synergistic effect of the combinations of antibiotics with sodium ascorbate or anti-inflammatory agents on the established biofilms

 

The potentiating effect of the tested antibiofilm agents on the antibiotic activity against established biofilms were determined by the method described by Černohorská and Votava. 24 five antibiotics were used; ciprofloxacin, amikacin, tobramycin, cefoperazone and imipenem. Volumes of 100 μl of the individual dilutions of the respective antibiotics and sodium ascorbate or tested NSAIDs in Mueller Hinton broth (50 μl of antibiotic and 50 μl of sub-MRCs of sodium ascorbate or NSAIDs) were placed into the microtiter plate wells with established biofilms. The plates were incubated for 18–20 h at 37 ºC and the wells were washed aseptically three times with PBS. The wells were filled with 100 µl TSB and incubated for 24 h at 37 ºC and the MRCs of antibiotics were determined. Each experiment was repeated thrice with positive and negative controls included in all experiments.

 

RESULTS:

Biofilm formation assay

According to the criteria proposed by Stepanovic et al.21, strong biofilm formation capability was found with the five clinical Pseudomonas aeruginosa isolates.

 

Antibiotic susceptibility of planktonic and adherent cells of Pseudomonas aeruginosa isolates

 

Biofilm formation increased antibiotic resistance of the 5 clinical P. aeruginosa strains by 8 to 8192 folds (Table 1).

 

Table 1. Comparative determination of antibiotic susceptibility against biofilm and planktonic cells of Pseudomonas aeruginosa.

Isolate

MRC/MIC ratio

Ami

kacin

Tobra

mycin

Cipro

floxacin

Imipenem

Cefo

perazone

P1

P2

P3

P4

P5

 

64

64

256

256

64

1024

1024

4096

2048

8192

 

32

64

8

512

128

 

128

256

1024

2048

512

4096

8192

4098

1024

512

 

 

Antibiofilm activity of sodium ascorbate and NSAIDs:

Sodium ascorbate, piroxicam, ketoprofen and diclofenac sodium showed direct antibiofilm activities. All agents could inhibit the regrowth of biofilm cells. Sodium ascorbate showed that activity at concentrations ranging between 80 and 320 mg/ml. Non-steroidal anti-inflammatory agents showed higher activity; the regrowth inhibiting activity was found for Ketoprofen at concentration of 6.25 mg/ml, for diclofenac sodium at 3.125-12.5 mg/ml and piroxicam at 10 mg/ml (Table 2).

 

Table 2. Biofilm minimum regrowth concentrations of anti-inflammatory agents and sodium ascorbate.

Isolate

MRC (mg/ml)

Piroxicam

Diclofenac sodium

Keto

profen

Sodium ascorbate

P1

P2

P3

P4

P5

10

10

10

10

10

3.125

6.25

6.25

6.25

12.5

6.25

6.25

6.25

6.25

6.25

320

160

320

80

320

 

Synergic interaction between antibiotics& NSAIDs and sodium ascorbate:

NSAIDs and sodium ascorbate augmented the activity of antibiotics against established biofilms (Tables 3& 4). Piroxicam decreased the resistance of biofilm cells to the tested antibiotics by up to 128-1024 folds, ketoprofen by up to 2-128 folds, and diclofenac sodium by up to 4-128 folds while sodium ascorbate increased the susceptibility of biofilm bacteria by up to 4-256 folds. The synergistic effect of piroxicam was demonstrated with tobramycin and amikacin in all isolates, with ciprofloxacin in 80% of isolates and with cefoperazone and imipenem in 20% of isolates. On the other hand, ketoprofen potentiated the antibiotic action on biofilms in 60% of isolates for amikacin and tobramycin, 20% of isolates for imipenem, while no synergistic effect of ketoprofen was observed when combined with ciprofloxacin and cefoperazone. The synergistic effect of diclofenac sodium was obtained with amikacin in 80% of isolates, with ciprofloxacin in 20% of isolates, while no synergy was found with cefoperazone, imipenem and tobramycin, while sodium ascorbate showed synergy with cefoperazone in all isolates, with ciprofloxacin, amikacin and tobramycin in 20% of isolates, while no synergy was found with imipenem.

 

Table 3. Effect of piroxicam and ketoprofen on the susceptibility of biofilm bacteria to antibiotics.

Antibiotic

Sub-MRC of antibiofilm agent

Folds decrease in MRC of antibiotic for isolate

P1

P2

P3

P4

P5

Pira

Ketb

Pira

Ketb

Pira

Ketb

Pira

Ketb

Pira

Ketb

Ciprofloxacin

 

Cefoperazone

 

Tobramycin

 

Amikacin

 

Imipenem

½

¼

½

¼

½

¼

½

¼

½

¼

512

16

128

8

128

32

256

128

512

8

2

ND*

2

2

8

2

128

8

2

2

256

32

128

2

4

4

32

8

128

2

2

ND

2

2

8

2

128

8

2

2

128

128

64

2

256

256

1024

256

4

2

2

ND

2

ND

32

16

32

2

8

2

512

64

8

2

128

128

128

64

64

2

2

ND

4

2

8

8

16

8

8

2

64

2

64

2

256

64

64

8

64

2

2

ND

2

2

32

8

8

ND

8

4

Pira, piroxicam; Ketb, ketoprofen; ND*, No decrease in MRC of antibiotic

 

Table 4. Effect of diclofenac sodium and sodium ascorbate on the susceptibility of biofilm bacteria to antibiotics.

Antibiotic

Sub-MRC of antibiofilm agent

Folds decrease in MRC of antibiotic for isolate

P1

P2

P3

P4

P5

Dcc

Ascd

Dcc

Ascd

Dcc

Ascd

Dcc

Ascd

Dcc

Ascd

Ciprofloxacin

 

Cefoperazone

 

Tobramycin

 

Amikacin

 

Imipenem

½

¼

½

¼

½

¼

½

¼

½

¼

4

2

128

2

2

2

2

ND

4

2

2

ND*

32

32

2

2

4

2

2

2

2

ND

64

ND

16

4

8

2

16

2

2

ND

256

32

2

2

2

2

2

2

2

ND

128

2

2

2

8

2

4

2

2

2

32

4

4

2

4

2

2

2

2

ND

64

2

2

2

8

4

32

2

4

4

64

64

8

8

16

16

2

2

4

4

128

2

2

2

8

2

32

2

2

2

128

128

2

2

2

2

4

2

Dcc, diclofenac sodium; Ascd, sodium ascorbate; ND*, No decrease in MRC of antibiotic


 

DISCUSSION:

Biofilms account for high percentage of nosocomial infections. Bacterial cells within biofilms are markedly resistant to antibiotics and hence biofilm infections are very difficult to eradicate.11, 25 To determine the effect of biofilm on resistance to antibiotics, the antibiotic susceptibility of floating bacteria (as determined by MIC) and of adherent bacteria (as determined by MRC) was determined and compared. The biofilm cells were found to be markedly more resistant than planktonic cells. The highest increase in resistance was found with tobramycin (1024-8192 folds), cefoperazone (512-8192 folds) and imipenem (128-2048 folds), while ciprofloxacin and amikacin were the least affected by biofilm formation; their MRC/MIC ratios were 8-512 and 64-256, respectively.

 

The aminoglycosides tested in this study; namely amikacin and tobramycin showed variable activity against biofilms with amikacin being less affected by biofilm resistance. Several mechanisms account for resistance of biofilms to aminoglycosides. They include the delayed penetration of the biofilm matrix barrier, production of aminoglycosides inactivating enzymes, sequestering of aminoglycosides by glucans, overexpression of efflux pumps in addition to low oxygen and slow growth.26-30

 

The susceptibilities of biofilm bacteria to the β-lactams imipenem and cefoperazone were different. Cefoperazone was less active than imipenem against established biofilms. Slow growth in addition to the coupling between delayed diffusion through the biofilm matrix barrier and inhibiting activity of β-lactamase in the matrix may be responsible for the biofilm resistance to β-lactams .31, 32 Stability to β-lactamases accounts for the low resistance of biofilm bacteria to imipenem.33-34

 

Ciprofloxacin showed higher activity against P.aeruginosa established biofilms. The penetration of fluoroquinolones through the biofilm matrix is not delayed.35 On the other hand, Low oxygen concentration, low metabolism and slow growth rates increase the resistance of biofilms to fluoroquinolones.29, 36 Overexpression of efflux pumps and persister cell formation are other factors that contribute to the biofilm resistance to ciprofloxacin.28, 37 The high antibiotic resistance of biofilm cells as compared to free-

 

 

living floating cells was reported in previous studies.24, 38, 39, 40, 41

 

To combat the high level of antimicrobial resistance associated with biofilm formation, there is an urgent need to test for antibiofilm agents and to use them in combination with antibiotics to enhance their activity against biofilms. 

 

In this study, complete or partial synergy between antibiotics and tested antibiofilm agents was observed with 87% of isolates. The strongest effect was found for the combinations of piroxicam with each of tobramycin and amikacin (synergy rate of 100%). High rates of synergy (80%) were also found with the combinations of piroxicam with ciprofloxacin and sodium ascorbate with cefoperazone. Potentiation rates of 60% were obtained with ketoprofen combined with each of tobramycin and amikacin. Low augmentation rates (20%) were shown by the combinations of ketoprofen with imipenem, diclofenac with each of tobramycin and amikacin, sodium ascorbate with each of tobramycin, ciprofloxacin and amikacin and piroxicam with cefoperazone. No complete synergy was found with the combinations of imipenem with each of piroxicam, diclofenac and sodium ascorbate (with partial synergy rate of 100%), cefoperazone with each of diclofenac sodium and ketoprofen (with partial synergy rate of 80%). No complete or partial synergy was observed with ketoprofen when combined with ciprofloxacin. Piroxicam was more synergistic than other tested agents, followed by sodium ascorbate, ketoprofen and diclofenac sodium.

 

In the presence of ¼ MRC of the tested agents, piroxicam showed more potentiating action than other agents in combination with ciprofloxacin, amikacin and tobramycin in 80% of isolates, with imipenem and cefoperazone in 20% of isolates, diclofenac sodium exerted higher augmenting activity with ciprofloxacin in 20% isolates, while ketoprofen in combination with imipenem was more potentiating in 20% of isolates. Sodium ascorbate was observed to have more augmenting effect than the tested NSAIDs when combined with cefoperazone in all isolates. Comparing the synergistic activities of ketoprofen, diclofenac sodium and sodium ascorbate, sodium ascorbate was more synergistic when combined with amikacin and ciprofloxacin in 20% of isolates. The synergistic effect of diclofenac was higher with tobramycin in 20 % of isolates. Ketoprofen was more synergistic when combined with tobramycin and amikacin in 40% of isolates.

 

Tobramycin and amikacin were found to be more affected than ciprofloxacin, cefoperazone and imipenem by combination with NSAIDs and sodium ascorbate. Comparing the synergy of antibiotics when combined with piroxicam, amikacin and tobramycin were the most augmented, followed by ciprofloxacin, cefoperazone and imipenem. In the presence of sodium ascorbate, cefoperazone was more potentiated than amikacin, tobramycin, imipenem and ciprofloxacin. The synergy observed by combining diclofenac sodium with antibiotics was higher with tobramycin and amikacin cefoperazone than with imipenem, cefoperazone and ciprofloxacin. Similarily, The synergistic effect of ketoprofen was higher with tobramycin and amikacin, while lower effect was found with imipenem, cefoperazone and ciprofloxacin.

 

The antibacterial and antibiofilm activities of NSAIDs were reported in previous studies. Diclofenac showed a broad spectrum antibacterial activity, and showed synergy when combined with antibiotics.  Diclofenac could potentiate aminoglycosides such as streptomycin, against Salmonella typhimurium and Mycobacterium tuberculosis, and gentamicin against Listeria monocytogenes. Moreover, diclofenac augmented cephalosporins, such as ceftriaxone against E. coli.42-45 Riordan et al.46 reported that diclofenac increased the susceptibility of S. aureus to ciprofloxacin, norfloxacin and ofloxacin by decreasing the expression of efflux systems. Furthermore, Perilli et al.47 found that diclofenac sodium could disorganize biofilm formed by Staphylococcus epidermidis on contact lens and significantly reduce the number of adherent bacteria, and Sankaridurg et al.48 reoprted that diclofenac sodium at 5 mg/ml inhibited the colonization of contact lens with Pseudomonas aeruginosa.

 

Ascorbic acid used in combination with erythromycin for a period of 4 weeks for the treatment of children with cystic fibrosis, a biofilm-based infection could eliminate the slimy mucous and viable cells of P. aeruginosa on the 4th week of treatment.49, 50

 

In conclusion, piroxicam, diclofenac sodium, ketoprofen and sodium ascorbate augmented the activity of antibiotics against biofilms formed by Pseudomonas aeruginosa and piroxicam showed the highest synergistic effect.

 

REFERENCES:

1.       Hancock REW and Speert DP. Antibiotics for Pseudomonas and related infections. In Cystic fibrosis – current topics, Edited by Dodge JA et al. John Wiley and Sons Ltd, 1996; vol. 3: pp. 245–266.

2.       Harmsen M et al. An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal. FEMS Immunology and Medical Microbiology. 59; 2010: 253–268.

3.       Lopez D, Vlamakis H and Kolter R. Biofilms. Cold Spring Harbor Perspectives in Biology. 2; 2010: a000398.

4.       Davies JC and Bilton D. Bugs, biofilms, and resistance in cystic fibrosis. Respiratory Care. 54; 2009: 628–640.

5.       Hoiby N et al. Antibiotic resistance of bacterial biofilms. International Journal of Antimicrobial Agents. 35; 2010: 322–332.

6.       Costerton JW, Stewart PS and Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 284; 1999: 1318–22.

7.       Drenkard E. Antimicrobial resistance of Pseudomonas aeruginosa biofilms. Microbes and Infection. 5; 2003: 1213–1219.

8.       Werner E et al. Stratified growth in Pseudomonas aeruginosa biofilms. Applied and Environmental Microbiology. 70; 2004: 6188–6196.

9.       Borriello G et al. Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrobial Agents and Chemotherapy. 48; 2004: 2659–2664.

10.     Lewis K. Multidrug tolerance of biofilms and persister cells. Current Topics in Microbiology and Immunology. 322; 2008: 107–131.

11.     Donlan RM and Costerton JW. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews. 15; 2002: 167–193.

12.     Bjarnsholt T et al. 2005. Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. Microbiology. 151; 2005: 373–383.

13.     Widmer AF et al. Killing of nongrowing and adherent Escherichia coli determines drug efficacy in device-related infections. Antimicrobial Agents and Chemotherapy. 35; 1991: 741–746.

14.     Annadurai S et al. Antibacterial activity of the anti-inflammatory agent diclofenac sodium. Indian Journal of Experimental Biology. 36; 1998: 86–90.

15.     Umaru T et al. Antimicrobial activity of non-steroidal anti-inflammatory drugs with respect to immunological response: Diclofenac sodium as a case study. African Journal of Biotechnology. 8; 2009: 7332–7339.

16.     Dastidar SG et al. 2000. The antibacterial action of diclofenac shown by inhibition of DNA synthesis. International Journal of Antimicrobial Agents. 14; 2000: 249–251.

17.     Abbas HA, Serry FM and EL-Masry EM. Combating Pseudomonas aeruginosa biofilms by potential biofilm inhibitors. Asian Journal of Research in Pharmaceutical Sciences. 2; 2012: 66-72.

18.     Alem MAS and Douglas LJ. Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candida albicans. Antimicrobial Agents and Chemotherapy. 48; 2004: 41–47.

19.     Novak JS and Fratamico PM. Evaluation of ascorbic acid as a quorum-sensing analogue to control growth, sporulation, and enterotoxin production in Clostridium perfringens. Journal of Food Science. 69; 2004: 72–78.

20.     Serry FME et al. The role of haemolysin transport system in antimicrobial resistance of haemolytic strains of Escherichia coli and the effect of potential efflux inhibitors. Journal of Pure and Applied Microbiology. 2; 2008: 307–318.

21.     Stepanovic S et al. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods. 40; 2000: 175–179.

22.     Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. 7th ed. CLSI Document M7-A7. Wayne, PA. 2006.

23.     Černohorská L and Votava M. Determination of minimal regrowth concentration (MRC) in clinical isolates of various biofilm-forming bacteria. Folia Microbiologica. 49; 2004: 75–78.

24.     Černohorská L and Votava M. Antibiotic synergy against biofilm-forming Pseudomonas aeruginosa. Folia Microbiologica. 53; 2008: 57–60.

25.     Costerton JW, Montanaro L and Arciola CR. Biofilm in implant infections: its production and regulation. International Journal of Artificial Organs 28; 2005:1062–1068.

26.     Shigeta M et al. Permeation of antimicrobial agents through Pseudomonas aeruginosa biofilms: a simple method. Chemotherapy 43; 1997: 340–345.

27.     Hoiby N et al.  Pseudomonas aeruginosa and the biofilm mode of growth. Microbes and Infection. 3; 2001: 1–13.

28.     Mah TF et al. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature. 426; 2003: 306–310.

29.     Walters MC et al. Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrobial Agents and Chemotherapy. 47; 2003: 317–323.

30.     Duguid, IG et al. Effect of biofilm culture upon the susceptibility of Staphylococcus epidermidis to tobramycin. Journal of Antimicrobial Chemotherapy. 30; 1992: 803–810.

31.     Tanaka G et al. Effect of the growth rate of Pseudomonas aeruginosa biofilms on the susceptibility to antimicrobial agents: beta-lactams and fluoroquinolones. Chemotherapy. 45; 1999: 28–36.

32.     Giwercman B et al. Induction of β-lactamase production in Pseudomonas aeruginosa biofilm. Antimicrobial Agents and Chemotherapy. 35; 1991: 1008–1010.

33.     Hill D et al. Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions. Journal of Clinical Microbiology 43; 2005: 5085–5090.

34.     El Gamal MI and Oh CH. Current status of carbapenem antibiotics. Current Topics in Medicinal Chemistry. 10; 2010:1882–1897.

35.     Suci PA et al. Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms. Antimicrobial Agents and Chemotherapy. 38; 1994: 2125–2133.

36.     Evans DJ et al. Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate. Journal of Antimicrobial Chemotherapy. 27; 1991: 177–184

37.     Brooun A, Liu S and Lewis K.A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms.  Antimicrobial Agents and Chemotherapy. 44; 2000: 640–646.

38.     Ceri H et al. The Calgary Biofilm Device: a new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. Journal of Clinical Microbiology. 37; 1999: 1771–1776.

39.     Cirioni O et al.  Effect of the combination of clarithromycin and amikacin on Pseudomonas aeruginosa biofilm in an animal model of ureteral stent infection. Journal of Antimicrobial Chemotherapy. 66; 2011: 1318–1323.

40.     Goto T et al. In vitro bactericidal activities of beta lactamases, amikacin, and fluoroquinolones against Pseudomonas aeruginosa biofilm in artificial urine. Urology. 53; 1999: 1058–1062.

41.     Abd El-Aziz et al. Evaluation of the combination of N-acetylcysteine and or sodium salicylate with ciprofloxacin bacterial adhesion and biofilm formation on urinary catheters. The International Arabic Journal of Antimicrobial Agents. 2; 2012:1–12.

42.     Joly V et al.  Enhancement of the therapeutic effect of cephalosporins in experimental endocarditis by altering their pharmacokinetics with diclofenac. The Journal of pharmacology and experimental therapeutics. 246; 1988: 695–700.

43.     Annadurai S et al.  Experimental studies on synergism between aminoglycosides and the antimicrobial anti-inflammatory agent diclofenac sodium. Journal of Chemotherapy. 14; 2002: 47–53.

44.     Dutta NK et al. Activity of diclofenac used alone and in combination with streptomycin against Mycobacterium tuberculosis in mice. International Journal of Antimicrobial Agents. 30; 2007: 336–340.

45.     Mazumdar K et al. The anti-inflammatory non-antibiotic helper compound diclofenac: an antibacterial drug target. European Journal of Clinical Microbiology& Infectious Diseases 28; 2009: 881–891.

46.     Riordan JT et al. Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac. Annals of Clinical Microbiology and Antimicrobials 10; 2011: 30.

47.     Perilli R et al. Alteration of organized structure of biofilm formed by Staphylococcus epidermidis on soft contact lenses.  Journal of Biomedical Materials Research. 49; 2000: 53–57.

48.     Sankaridurg PR et al. Non Steroidal Anti Inflammatory Drugs Inhibit Bacterial Colonization on Soft Contact Lenses. Investigative Ophthalmolpgy& Visual Science. 44; 2003: 1434.

49.     Rawal BD. 1974. Inhibition of Pseudomonas aeruginosa by ascorbic acid acting alone and in combination with erythromycin: role of magnesium ions. IRCS Medical Science 2; 1974: 1670.

50.     Rawal BD, Mckay G and Blackhall MI. Inhibition of Pseudomonas aeruginosa by ascorbic acid acting singly and in combination with antimicrobials: in-vitro and in-vivo studies. The Medical Journal of Australia. 1; 1974: 169–174.

 

 

 

 

Received on 31.07.2012       Modified on 15.08.2012

Accepted on 25.08.2012      © RJPT All right reserved

Research J. Pharm. and Tech. 5(8): August 2012; Page 1124-1129