Abstract
Objective
The aim of the study was to study the efficacy of 2 different lasers in vitro, in disrupting biofilm and killing planktonic pathogenic bacteria.
Materials and methods
Biofilms of a stable bioluminescent of Staphylococcus aureus Xen 31 were grown in a 96-well microtiter plate for 3 days. The study included 7 arms: ( a ) control; ( b ) ciprofloxacin (3 mg/L, the established minimum inhibitory concentration [MIC]) alone; ( c ) shock wave (SW) laser alone; ( d ) near-infrared (NIR) laser alone; ( e ) SW laser and ciprofloxacin; ( f ) SW and NIR lasers; ( g ) SW, NIR lasers, and ciprofloxacin. The results were evaluated with an in vivo imaging system (IVIS) biophotonic system (for live bacteria) and optical density (OD) for total bacteria.
Results
Without antibiotics, there was a 43% reduction in OD ( P < .05) caused by the combination of SW and NIR suggesting that biofilm had been disrupted. There was an 88% reduction ( P < .05) in live biofilm. Ciprofloxacin alone resulted in a decrease of 28% of total live cells (biofilm remaining attached) and 58% of biofilm cells (both P > .05). Ciprofloxacin in combination with SW and SW + NIR lasers caused a decrease of more than 60% in total live biomass and more than 80% of biofilm cells, which was significantly greater than ciprofloxacin alone ( P < .05).
Conclusions
We have demonstrated an effective nonpharmacologic treatment method for methicillin-resistant Staphylococcus aureus (MRSA) biofilm disruption and killing using 2 different lasers. The preferred treatment sequence is a SW laser disruption of biofilm followed by NIR laser illumination. Treatment optimization of biofilm is possible with the addition of ciprofloxacin in concentrations consistent with planktonic MIC.
1
Introduction
Chronic rhinosinusitis (CRS) is the most commonly treated upper respiratory tract infection and the fifth most commonly treated chronic disease in the United States. Many studies have shown that Staphylococcus aureus as one of the most common organisms causing CRS (18.6–36.6%) . More worryingly, methicillin-resistant (or multidrug-resistant) S aureus (MRSA) infection rate of MRSA-causing CRS is 9.22% incidence. Liberal antibiotic use in ear, nose, and throat infections plays a possible role in the increasing emergence of MRSA and MRSA biofilm-causing CRS. Furthermore, a comparison of the rates of recovery of MRSA between the periods 2001 and 2003 and 2004 and 2006 in acute and chronic maxillary sinusitis illustrated a significant increase in the rate of recovery of this organism in patients with acute and chronic maxillary sinusitis from 27% to 61% of all Staphylococcus CRS infections .
Taken the 2% to 24% rate of primary functional endoscopic sinus surgery (FESS) failure , new causes rather than anatomical causes should be considered. Indeed, the rising role of biofilms in CRS is overwhelming. Wormald found that biofilms may play an active role in perpetuating inflammation in CRS patients and may explain the recurrent and resistant nature of this disease. Therefore, removing biofilms may be important in the management of recalcitrant CRS .
The current antibacterial treatment modalities often require a hundred- or thousand-fold the planktonic minimum inhibitory concentration or minimum bactericidal concentration antibiotic dosage to be effective against the same bacteria when they are in biofilms, which is not systemically feasible because of toxicity .
Experiments have shown that antibiotics can eradicate planktonic and possibly surface biofilm bacteria thus sparing the inner protected biofilm bacteria, which may explain biofilms being potential cause for chronic infections with acute exacerbations .
Taken all those considerations of MRSA as an emerging pathogen in CRS, increasing FESS failures and increasing biofilm role in CRS, we set to look for a new frontier in the treatment of CRS when antibiotic fail. The study by Desrosiers et al showed promise with biofilm disruption by using citric acid/zwitterionic surfactant (CAZS). As other studies focused solely on killing biofilms, our study tries to combine these approaches. The biofilms are initially disrupted then destroyed. We used nontoxic, host tissue-friendly mechanical and optical energies, using the shock wave (SW) to disrupt the biofilm, then the near-infrared (NIR) to kill planktonic bacteria.
2
Materials and methods
2.1
Bacterial cultures
We grew biofilms from S aureus Xen 31 (Caliper LifeSciences, Hopkinton, MA, USA), a stable bioluminescent clinical MRSA construct derived from S aureus ATCC 33591. Bioluminescent bacteria only produce light when they are alive and metabolically active. Thus, we used light emission as detected with the in vivo imaging system (IVIS) Lumina II system (Caliper LifeSciences), as an indicator of biofilm activity. We calibrated the light emission by the light-emitting MRSA strains with extent of biofilm formation by determining the correlation between geometric means from the culture data (colony-forming unit per square centimeter [CFU/cm 2 ]) and the light data photons/s/cm2/sr) by linear regression of the log-log data. The relationship was log light emission = 0.17 log CFU + 3.75. There was a reasonable agreement between the 2 methods with an R 2 value of 0.77. The biofilm concentration ranged from 2.4×10 3 to 4.3×10 8 CFU/cm 2 . The 96-well plates were inoculated with a stationary phase (18 hours) culture grown in brain-heart infusion broth. The culture was diluted 1:1000 to achieve approximately 1 × 10 7 CFU/mL. After 24 hours, the medium was replaced with fresh medium to produce a 48-hour biofilm. After the laser treatments, ciprofloxacin (0.3 μ /mL) was added and the cultures were incubated for a further 24 hours for total of 72-hour incubation.
2.2
Lasers
We used 2 lasers. The first was a Q-switched Nd-YAG SW laser (ARCLaser, Nuremberg, Germany, and Valam, New York, NY). The probe was coupled to an optical fiber of 300 nm. The second was a NIR diode laser (ARCLaser, Nuremberg, Germany, and Valam, New York, NY), with a wavelength of 940 nm coupled to a 300-nm optical fiber. The Q-switched Nd-YAG laser was set for a frequency of 1 pulse per second for this experiment, whereas the output energy for the experiment’s laser system was between 8 and 12 mJ. The biofilm was exposed to 10 pulses of SW placed in each of the tested wells. The NIR laser was applied with an energy level of 3 W with a distance between the well and the probe set constantly to cover the entire well diameter of 0.7 cm diameter for 180 seconds, delivering a total energy density of 1400 J/cm 2 .
2.3
Study arms
The study included 7 arms: ( a ) 48-hour control; ( b ) SW laser alone; ( c ) NIR lasers alone; ( d ) ciprofloxacin (0.3 mg/L) alone; ( e ) SW and NIR lasers; ( f ) SW laser and ciprofloxacin; and ( g ) SW, NIR lasers, and ciprofloxacin. The effect of lasers on biofilm was measured immediately in the nonantibiotic arms and the antibiotic arms before ciprofloxacin addition; thus, these arms had 12 wells each. The “before rinsing” measurements included biofilm bacteria and planktonic bacteria. The “after rinsing” measurements indicated biofilm bacteria only. The rinsing step involved rinsing 3 times with sterile phosphate buffered saline. In addition to the IVIS system, we measured the optical density (OD) using a plate reader (Beckman DU 650 spectrophotometer, Beckman Coulter Inc, Fullerton, CA) at 595 nm. The IVIS analysis provided a measure of active bacteria, and the OD provided a measure of the total biomass (live and dead bacteria). For the arms with ciprofloxacin, we allowed additional 24-hour incubation before measuring the light emission and OD before and after rinsing. As minimum inhibitory concentration or minimum bactericidal concentration is not established for biofilms, only reduction of the biomass was assessed.
2.4
Statistics
Statistical analysis using Microsoft EXCEL for Windows was used. All parametric data, including OD analysis and IVIS analysis readings, were characterized by a mean and SD or SE from 12 replicate wells. In addition to the 2 sets of 12 controls, we also had control wells. A Student t test was used to determine whether differences in the various treatments were significant ( P < .05).
2
Materials and methods
2.1
Bacterial cultures
We grew biofilms from S aureus Xen 31 (Caliper LifeSciences, Hopkinton, MA, USA), a stable bioluminescent clinical MRSA construct derived from S aureus ATCC 33591. Bioluminescent bacteria only produce light when they are alive and metabolically active. Thus, we used light emission as detected with the in vivo imaging system (IVIS) Lumina II system (Caliper LifeSciences), as an indicator of biofilm activity. We calibrated the light emission by the light-emitting MRSA strains with extent of biofilm formation by determining the correlation between geometric means from the culture data (colony-forming unit per square centimeter [CFU/cm 2 ]) and the light data photons/s/cm2/sr) by linear regression of the log-log data. The relationship was log light emission = 0.17 log CFU + 3.75. There was a reasonable agreement between the 2 methods with an R 2 value of 0.77. The biofilm concentration ranged from 2.4×10 3 to 4.3×10 8 CFU/cm 2 . The 96-well plates were inoculated with a stationary phase (18 hours) culture grown in brain-heart infusion broth. The culture was diluted 1:1000 to achieve approximately 1 × 10 7 CFU/mL. After 24 hours, the medium was replaced with fresh medium to produce a 48-hour biofilm. After the laser treatments, ciprofloxacin (0.3 μ /mL) was added and the cultures were incubated for a further 24 hours for total of 72-hour incubation.
2.2
Lasers
We used 2 lasers. The first was a Q-switched Nd-YAG SW laser (ARCLaser, Nuremberg, Germany, and Valam, New York, NY). The probe was coupled to an optical fiber of 300 nm. The second was a NIR diode laser (ARCLaser, Nuremberg, Germany, and Valam, New York, NY), with a wavelength of 940 nm coupled to a 300-nm optical fiber. The Q-switched Nd-YAG laser was set for a frequency of 1 pulse per second for this experiment, whereas the output energy for the experiment’s laser system was between 8 and 12 mJ. The biofilm was exposed to 10 pulses of SW placed in each of the tested wells. The NIR laser was applied with an energy level of 3 W with a distance between the well and the probe set constantly to cover the entire well diameter of 0.7 cm diameter for 180 seconds, delivering a total energy density of 1400 J/cm 2 .
2.3
Study arms
The study included 7 arms: ( a ) 48-hour control; ( b ) SW laser alone; ( c ) NIR lasers alone; ( d ) ciprofloxacin (0.3 mg/L) alone; ( e ) SW and NIR lasers; ( f ) SW laser and ciprofloxacin; and ( g ) SW, NIR lasers, and ciprofloxacin. The effect of lasers on biofilm was measured immediately in the nonantibiotic arms and the antibiotic arms before ciprofloxacin addition; thus, these arms had 12 wells each. The “before rinsing” measurements included biofilm bacteria and planktonic bacteria. The “after rinsing” measurements indicated biofilm bacteria only. The rinsing step involved rinsing 3 times with sterile phosphate buffered saline. In addition to the IVIS system, we measured the optical density (OD) using a plate reader (Beckman DU 650 spectrophotometer, Beckman Coulter Inc, Fullerton, CA) at 595 nm. The IVIS analysis provided a measure of active bacteria, and the OD provided a measure of the total biomass (live and dead bacteria). For the arms with ciprofloxacin, we allowed additional 24-hour incubation before measuring the light emission and OD before and after rinsing. As minimum inhibitory concentration or minimum bactericidal concentration is not established for biofilms, only reduction of the biomass was assessed.
2.4
Statistics
Statistical analysis using Microsoft EXCEL for Windows was used. All parametric data, including OD analysis and IVIS analysis readings, were characterized by a mean and SD or SE from 12 replicate wells. In addition to the 2 sets of 12 controls, we also had control wells. A Student t test was used to determine whether differences in the various treatments were significant ( P < .05).
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