INTRODUCTION:

Pseudomonas aeruginosa is one of the most important opportunistic human pathogens and is a leading cause of nosocomial infections and it is the most common cause of Intensive Care Unit-associated pneumonia.^1-4 Pseudomonas aeruginosa can form biofilms.^5,6 Biofilms are multicellular aggregates of microbial cells that are encased in a self-produced extracellular matrix of polysaccharides, proteins and nucleic acids.^{7- 11}

Biofilm formation presents a great problem in treatment of microbial infections due to protection of microbes against the host immumity and the high increase in resistance of biofilm bacteria to antimicrobial agents.^12,13 Resistance mechanisms include lowered diffusion of antibiotics through the biofilm matrix, low oxygen, nutrients and metabolic activity. Moreover, formation of persisters, high bacterial cell density and activation of general stress response contribute to the biofilm antimicrobial resistance.^{14, 15}

N-acetylcysteine (NAC) is a mucolytic agent used in medical treatment of chronic bronchitis^{16, 17} and it has antibacterial properties. The molecule is a thiol containing antioxidant that disrupts disulfide bonds in mucus^{18, 19} and competitively inhibits cysteine utilization^20,21. NAC decreases biofilm formation by a variety of bacteria^22-24and reduces the production of an extracellular polysaccharide matrix, while promoting the disruption of mature biofilms^{23, 25} Ambroxol is used as an expectorant in the treatment of bronchial asthma and chronic bronchitis²⁶. Furthermore, it has antioxidant and anti-inflammatory properties²⁷. Ambroxol can inhibit biofilm formation by a combination of its antiadhesive effect, quorum sensing inhibiting effect in addition to its ability to reduce the production of the biofilm matrix in P. aeruginosa.^28-30 This study aimed to study the potentiation of antibiotics by NAC and ambroxol against pre-formed P. aeruginosa biofilms.

MATERIALS AND METHODS:

Bacterial strains

Pseudomonas aeruginosa (5 isolates) isolated from intensive care unit patients in Zagazig university Hospitals by endotracheal aspiration.

Assay of biofilm

Quantification of biofilm was performed according to Stepanovic et al. ³¹ The test isolates, from 24-hour tryptone soya agar plates, were inoculated into 5 mL quantities of tryptone soya broth supplemented with 1% glucose (TSB_glu) in Falcon tubes to achieve a turbidity equivalent to that of a 0.5 McFarland standard. The negative control tubes contained TSB_glu only. After incubation of the tubes for 24 h at 37 ºC; the content of each tube was aspirated, and the tubes were washed thrice with sterile saline. The tubes were vigorously shaken to remove any non-adherent bacteria and 99% methanol was added for 15 minutes for fixation of adherent cells. The tubes were then decanted, left to dry, stained with 2% Hucker crystal violet for 5 minutes and the excess stain was washed under running tap water. After the tubes were air dried, the dye bound to the adherent cells was resolubilized with 33% (v/v) glacial acetic acid and the Optical density (OD) was measured at 570 nm using Spectrophotometer (UV-1800 Shimadzu, Japan). The cut-off OD (ODc) was defined as three times standard deviations above the mean OD of the negative control. According to the measured ODs of the bacterial biofilms, the test isolates were categorized into four groups, non-biofilm forming (OD ≤ ODc), weak biofilm forming (OD > ODc, but ≤ 2x ODc), moderate biofilm forming (OD >2x ODc, but ≤ 4x ODc), and 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 mucolytics were determined by the agar dilution method according to Clinical Laboratory and Standards Institutes Guidelines (CLSI).³² Bacterial inocula were standardized against the 0.5 McFarland standard and diluted with sterile saline to achieve an approximate cell density of 10⁷ CFU/mL. A standardized inoculum was deliveded to the surface of Mueller-Hinton agar containing antibiotic dilutions and the control plates, so that the final inoculum on the agar contains approximately 10⁴ CFU per spot. Antimicrobial-free plates were used as growth control. The inoculated agar plates were allowed to stand at room temperature until the liquid was absorbed into the agar. 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 completely inhibited growth.

Determination of minimum regrowth concentration

The minimum regrowth concentrations (MRCS) of anbtibiotics, NAC and ambroxol were determined based on the method described by Černohorská and Votava.³³ For each isolate, an inoculum was prepared in TSB to have a turbidity that matches 0.5 McFarland standard. Aliquots of 75 μL of the inoculated medium were added to the U wells of the polystyrene microtiter plates, and the plates were incubated for 24 h at 37°C. For removal of the non-adherent cells, the wells were washed thrice with PBS under aseptic conditions. The plates were dried in an inverted position and volumes of 100 μL of appropriate two-fold dilutions of the tested compunds in Mueller–Hinton broth were transferred into the dried wells with pre-formed biofilms. The microtiter plates were incubated for 18–20 h at 37 ºC and the wells were washed thrice with PBS under aseptic conditions, filled with 100 µL TSB. After incubation for 24 h at 37 ºC, the minimum regrowth concentration (MRC) was determined. MRC is the minimum concentration of the antimicrobial agent which inhibits regrowth of the cells. Each experiment was repeated three times with positive and negative controls in all experiments.

The effect of combinations on NAC and ambroxol with antibiotics on the pre-formed biofilms

The synergistic antibiofilm effects against were determined according to Černohorská and Votava.³⁴ The antibiotics tested were ciprofloxacin, amikacin, tobramycin, cefoperazone and imipenem. Volumes of 100 μL of the individual dilutions of the respective antibiotics and NAC or ambroxol in Mueller Hinton broth (50 μL of antibiotic and 50 μL of sub-MRCs of NAC or ambroxol) were transferred into the microtiter plate wells with pre-formed biofilms. The microtiter plates were incubated for 18–20 h at 37 ºC and the wells were washed thrice with PBS under aseptic conditions, filled with 100 µL TSB. After incubation for 24 h at 37 ºC, the MRCs of antibiotics were determined. Each experiment was repeated three times with positive and negative controls in all experiments.

RESULTS:

Assay of biofilm. The five clinical Pseudomonas aeruginosa isolates showed strong biofilm forming ability.

Antibiotic susceptibility of planktonic and biofilm cells of Pseudomonas aeruginosa isolates. For the 5 clinical P. aeruginosa strains 8-fold to 8192-fold higher MRC values than MIC were obtained (Table 1).

Table 1. Ratio of minimum biofilm regrowth inhibiting concentrations to minimum inhibitory concentrations of antibiotics against Pseudomonas aeruginosa isolates.

Isolate

MRC/MIC ratio

Ciprofloxacin

Cefoperazone

Tobramycin

Amikacin

Imipenem

PA1

PA2

PA3

PA4

PA5

512

128

4096

8192

4098

1024

512

1024

4096

2048

8192

256

128

256

1024

2048

512

Antibiofilm activity of NAC and ambroxol

NAC and ambroxol showed direct antibiofilm activities. NAC Could inhibit regrowth of biofilm bacteria at concentrations ranging between 5 and 80 mg/mL and ambroxol produced similar effect at lower concentrations (15 mg/mL) except for one isolate (30mg/mL) (Table 2).

Table 2. Minimum regrowth concentrations of NAC and ambroxol.

Isolate

MRC (mg/mL)

NAC

Ambroxol

PA1

PA2

PA3

PA4

PA5

Synergy between antibiotics and antibiofilm agents

Sub-MRCs of NAC and ambroxol potentiated the activity of antibiotics against pre-formed biofilms. Ambroxol reduced the MRCs of the tested antibiotics by up to 16-512 folds (Tables 3), while NAC decreased the MRCs by up to 4-256 folds. The synergistic effect of ambroxol was demonstrated in 60% of the isolates for tobramycin, 80% for amikacin and imipenem and 100% for ciprofloxacin and cefoperazone. On the other hand, NAC augmented the antibiotic action on biofilms in 20% of the isolates for ciprofloxacin, 40% for amikacin, 60% for tobramycin and 100% for cefoperazone, while the augmenting effect of NAC when combined with imipenem was shown in 20% of isolates.

DISCUSSION:

Biofilm-related infections account for about 60% of bacterial infections.¹² Biofilm infections are difficult to eradicate due to alteration of the gene expression of bacteria within biofilms.³⁵ This alteration leads to enhanced protection against macrophages and antibiotics, compared to free living cells.³⁶ In order to determine the role of biofilm in resistance to antibiotics, the differences in antibiotic susceptibility between planktonic populations (erxpressed as MIC) and biofilm populations (expressed as MRC) of the clinical isolates of Pseudomonas aeruginosa tested in this study were determined. It was found that the susceptibility of biofilm cells to different antibiotics decreased as demonstrated by the ratio of MRC to MIC.

The highest MRC/MIC ratios (highest biofilm resistance) were found with cefoperazone (512-8192), tobramycin (1024-8192) and imipenem (128-2048). Whereas, lower ratios were obtained with amikacin (64-256) and ciprofloxacin (8-512).

Different biofilm susceptibilities to aminoglycosides were found in this study. Amikacin showed higher antibiofilm activity than that of tobramycin. The biofilm resistance could be attributed to several factors. The coupling between retarded diffusion by the biofilm matrix and inactivation by aminoglycosides inactivating enzymes decreases biofilm susceptibility to aminoglycosides.³⁷ The ndvB gene, which is responsible for synthesis of glucans, is linked to biofilm-specific resistance to aminoglycosides. Glucans bind and sequester aminoglycosides like tobramycin, preventing them from reaching their targets.³⁸ The ATP binding cassette (ABC) family efflux system that is more highly expressed in biofilm than in planktonic cells, PA1874-PA1877, contributes to P. aeruginosa biofilm-specific resistance tobramycin.³⁹ Oxygen limitation and low metabolic rate were found to increase P. aeruginosa biofilms resistance to tobramycin.⁴⁰

Similarly, the β-lactamas imipenem and cefoperazone showed different activities against biofilm bacteria. Cefoperazone demonstrated low biofilm regrowth inhibiting action. The mechanisms of resistance of biofilm to β-lactam antibiotics include slow growth rates ⁴¹ and retarded biofilm matrix diffusion combined with degradation by β-lactamase accumulated in the biofilm matrix.⁴²Imipenem, on the other hand was more effective against biofilms. One factor that may explain the low biofilm resistance to the carbapenem imipenem found in the present study is their stability to β-lactamases.^{43, 44}

Lower biofilm resistance rates were found with the fluoroquinolone ciprofloxacin against biofilms formed by P.aeruginosa. Fluoroquinolones, unlike aminoglycosides, can penetrate readily the biofilm matrix without delay.^45,46,47 Resistance of biofilms to fluoroquinolones is mediated by decreased oxygen concentration, slow growth and lowered metabolic rates.^12,40 The efflux pump PA1874-PA1877 was involved in P. aeruginosa biofilm-specific resistance to ciprofloxacin. Persister cell formation was found to increase resistance of P. aeruginosa biofilms to ciprofloxacin.⁴⁸

Table 3. The effect of sub-MRCs of ambroxol hydrochloride and N-acetyl cysteine on the minimum regrowth inhibiting concentrations of antibiotics.

Antibiotic	Sub-MRC of antibiofilm agent	Folds decrease in MRC of antibiotic for isolate
		PA1		PA2		PA3		PA4		PA5
		Amb*	NAC	Amb	NAC	Amb	NAC	Amb	NAC	Amb	NAC
Ciprofloxacin Cefoperazone Tobramycin Amikacin Imipenem	½ ¼ ½ ¼ ½ ¼ ½ ¼ ½ ¼	8 8 64 32 16 16 8 4 16 8	2 ND** 8 8 4 2 2 ND 4 4	32 32 16 16 4 4 8 2 32 32	2 2 32 16 8 4 2 ND 128 2	8 4 256 32 2 2 8 8 64 16	2 2 16 8 4 2 4 4 32 2	16 16 16 16 8 8 4 4 4 2	4 4 8 4 4 4 2 2 2 2	8 8 32 16 8 2 32 8 512 128	2 ND 8 8 256 128 64 16 32 2

Amb*, ambroxol; ND**, No decrease in MRC of antibiotic

This clear difference in susceptibility between bacteria in biofilm and in planktonic form was demonstrated by many studies. In agreement with this study, Agarwal et al.⁴⁹ found that Pseudomonas aeruginosa in biofilm showed a 4-fold greater resistance against ciprofloxacin and gentamicin compared with free-living forms. Černohorská and Votava³⁴ reported a high resistance of 20 clinical isolates of Pseudomonas aeruginosa biofilm populations to individual antibiotics. Higher resistance was found for cefoperazone, while resistance to amikacin and ciprofloxacin was lower. These results are in agreement with this study. In accordance with the present study, Ceri et al.⁵⁰ demonstrated decreased susceptibility of Pseudomonas aeruginosa biofilm by 4 folds for tobramycin, 8 folds for amikacin, 16 folds for ciprofloxacin, > 1024 folds for imipenem. Cirioni et al.⁵¹ reported a 4-fold increase in MIC of amikacin against biofilm cells of Pseudomonas aeruginosa compared to planktonic cells.The results of this study were different from those of other studies. Kádár et al.⁵² investigated the effect of imipenem, ciprofloxacin, amikacin and tobramycin on biofilm-formation in 14 P. aeruginosa clinical isolates and found that imipenem was more potent than other antibiotics tested against biofilms.

Several strategies have been employed to inhibit biofilm formation and decrease the biofilm-associated antimicrobial resistance. Preventing the initial adhesion of bacteria to a surface is an effective method to prevent the formation of biofilm.⁵³ Moreover, intereference with quorum sensing and cleavage of essential components of the biofilm matrix such as polysaccharides or extracellular DNA, are two other examples of these strategies.⁵⁴

NAC was reoprted to have an antibiofilm activity. NAC can disperse the biofilms formed by Pseudomonas aeruginosa.⁵⁵ NAC decreases extracellular polysaccharides production and promotes disruption of mature biofilms.^{22, 56} NAC was found to reduce adhesion of Staphylococci and some Gram-negative bacilli to different abiotic materials.^25,
57 The mode of action of biofilm inhibition of NAC may be via chelation of calcium, which is a component essential for the maintenance of the extracellular biofilm matrix by controlling the degree of cross-linking.^55,58 Addition of dispersants like NAC to antibiotics can enhance the activity of antibiotics against the adherent bacteria by decreasing the number of adherent bacteria and detachment of adherent bacteria.^{59, 60} Moreover, the disruption of biofilms by NAC increases the antimicrobial susceptibility of biofilm bacteria.⁶¹ As a result, the combination of NAC to antibiotics would be synergistic.²⁵ Another mode of action may be the prevention of bacterial adhesion to abiotic surfaces. The initial adhesion of bacteria to surfaces is dependent on the wettability of the substratum. Olofsson et al.²⁵ reported that NAC binds to solid steel surface, increasing the wettability of the surface and thus, decreased bacterial adhesion and even detached bacteria that were adhering to solid surfaces.

In this study, NAC showed biofilm regrowth inhibiting activity at concentrations of 5 to 80 mg/mL. Complete or partial synergy was observed with all tested antibiotics. Tobramycin was the most augmented antibiotic (MRCs were decreased by up to 4-256 folds), followed by imipenem (up to 2-128 folds), amikacin (up to 2-64 folds) and cefoperazone (up to 8-32 folds), while ciprofloxacin was the least potentiated (with up to 2-4 folds decrease in MRCs).

In accordance with the synergistic effect of NAC agents with antibiotics on biofilm, Zhao and Liu⁶² showed that NAC (2.5 mg/mL) and ciprofloxacin (> 2 MIC) showed significant antibiofilm activity against biofilms formed by P. aeruginosa. NAC-ciprofloxacin combinations decreased the viable biofilm bacteria. This combination was synergistic at NAC of 0.5 mg/mL and CIP at 1/2MIC. Moreover, El-Feky et al.⁶³ demonstrated that ciprofloxacin-NAC combination showed higher inhibitory effect on biofilm formation by Pseudomonas aeruginosa and higher disruptive effect than ciprofloxacin alone on the pre-formed biofilms by two strains of Pseudomonas aeruginosa. This combination reduced biofilm synthesis by 97%-98.4% at MIC of ciprofloxacin and 2 mg/mL of NAC and by 99.8%-99.98% at 2 MIC of ciprofloxacin and 4 mg/mL of NAC, compared to reduction by 68.2%-74% and 90%-92.5% at MIC and 2MIC of ciprofloxacin alone. Similar effect was observed on the disruption of mature bioflims. Ciprofloxacin reduced pre-formed biofilms of Pseudomonas aeruginosa by 81.4%-87.4% and 90.5%-94% at concentrations equal to MIC and 2MIC, while ciprofloxacin combined with NAC at the same ratios disrupted mature biofilms by 96%-97.2% and 99.8%-99.9%.

Marchese et al.²³ found that NAC at 2 mg/mL in combination with the cell wall acting antibiotic fosfomycin 2 mg/mL against four slime-producing uropathogenic E. coli biofilms, reduced pre-formed mature biofilms by 60-73%, and produced 99-99.9% decrease in the viability of sessile cells, compared to 41-49% biofilm reduction and 36-85.7% viable counts reduction produced by fosfomycin (2 mg/mL) alone. Also, Aslam et al.⁵⁶ studied the effect of NAC combined with the protein synthesis inhibiting antibiotic tigecycline at 80 mg/ml and 1 mg/ml, respectively, on biofilm embeded bacteria on vascular catheters, and found that NAC-tigecycline combination was synergistic against Staphylococcus aureus and Staphylococcus epidermidis.

Our results showed that the MRCs of ambroxol were 15-30 mg/mL. Like NAC, ambroxol showed complete or partial synergy with the tested antibiotics. According to the maximum decrease in MRCs of antibiotics in the presence of sub-MRCs of ambroxol, imipenem was the most potentiated (with up to 4-512 folds decrease in MRCs), followed by cefoperazone (8-256 folds), ciprofloxacin and amikacin (up to 4-32 folds), while the lowest decrease in MRCs was found for the combination of tobramycin with ambroxol (2-16 folds). Ambroxol showed higher synergism with tested antibiotics than NAC.

Comparing the magnitude of antibiotic potentiation by ambroxol and NAC, it was found that ambroxol was more effective. In the presence of ¼ MRC of the tested mucolytics, ambroxol demonstrated more augmenting action than NAC in combination with ciprofloxacin in 100% of isolates, cefoperazone, amikacin and imipenem in 80% of isolates and tobramycin in 40% of isolates. On the other hand, NAC showed more potentiating effect when combined with cefoperazone, tobramycin and imipenem in only 20% of isolates, while equivalent effects were reoprted for both ambroxol and NAC with cefoperazone and imipenem in 20% of isolates and tobramycin in 40% of isolates. Furthermore, in the presence of ½ MRC of the tested mucolytics, ambroxol was more augmenting than NAC to the antibiotics on the biofilm cells. Ambroxol demonstrated higher augmenting effect with ciprofloxacin in 100% of isolates, with cefoperazone, amikacin and imipenem in 80% of isolates and with tobramycin in 40% of isolates. On the contrary, NAC showed more potentiating action with tobramycin in 60% of isolates, with cefoperazone, amikacin and imipenem in 20% of isolates.

In agreement with the current study, Li et al.²⁸ reported that ambroxol can promote the permeability of ciprofloxacin through P. aeruginosa biofilms and reduces the contents of extracellular polymeric substances. Moreover, at concentrations of 1.875 mg/mL and 3.75 mg/mL, ambroxol showed anti quorum sensing activity in P. aeruginosa, and by this effect it could reduce the viable count of biofilm bacteria and interfere with adhesion to abiotic surfaces³⁰. Furthermore, Li et al.²⁸ reported the ability of ambroxol to disrupt the biofilms at 3.75 mg/mL. In addition, Li et al.⁶⁴ demonstrated the P. aeruginosa biofilm-disrupting capacity of ambroxol on the endotracheal tube in a rat model with decrease in the viable count of the biofilm bacteria. The anti-QS properties of ambroxol can extend its potential uses against organisms associated with chronic infections. Ambroxol can be used in the treatment of patients with cystic fibrosis or in the prevention of biofilms on indwelling devices.⁶⁵ In conclusion, ambroxol and NAC enhance the activity of antibiotics against biofilms formed by Pseudomonas aeruginosa and ambroxol produced more potentiating effect than NAC.

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