The efficacy of N-acetyl cysteine, rifampicin, and ozone on the biofilm formed by the panresistant Klebsiella pneumoniae isolates

Aim and Introduction: In the present study, we searched for a solution for the infections of panresistant Klebsiella pneumoniae (PRKp) and the resistance which is difficult to handle and as a significant nosocomial infection factor in our hospital. We expect to administer the antibiofilm effective agents such as N-acetyl cysteine (NAC), rifampicin, and ozone by targeting the biofilm layer, and if they are found effective, to provide the effective antibiotics to be used in treatment, and to enable the cure of infection because no solution is yet known to be available with a new antibiotic. Material and Methods: K. pneumoniae isolates which created invasive infections in the samples collected from nine patients aged over 18 years who were hospitalized in Istanbul University Istanbul Faculty of Medicine Hospital between 15 June 2017- 15 July 2018 were identified using the classical biochemical methods. The zone diameters and minimum inhibitory concentration (MIC) values determined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used for disk diffusion method, gradient test, and broth microdilution methods in determining the sensitivity of these bacteria to antibiotics. The crystal violet staining method was used in demonstrating the biofilm forming capacity of the bacteria. The classification was performed in accordance with the biofilm forming capacities by measuring the optic density (OD) of the wells. Intracellular biofilm forming capability was identified in the single layer cell culture. Biofilm formation and the efficacy of NAC, rifampicin, and ozone on biofilm layer was demonstrated using the colony counting method, and laser screened confocal microscopy method. Results: Nine patients were included in the study. 5 (55.5%) patients were hospitalized in the Intensive Care Unit. Four patients were followed up with the diagnosis of bacteremia, three were diagnosed with ventilator-associated pneumonia (VAP), one patient had prosthetic infection, and the other one patient was diagnosed with complicated urinary tract infection. Six patients who developed PRKp infection were treated with meropenem, tigecycline, and colistin; two patients were administered imipenem, tigecycline, and colistin; and one patient was administered meropenem, ertapenem, and colistin combination. The mean hospital stay time was 50.4 (4-80 days) days. Although broad spectrum antibiotics were used, 7 (77.7%) patients died in a mean of 9 (4-21 days) days after the initiation of treatment. One of the surviving patients was received meropenem, ertapenem, and colistin treatment and whose catheter was replaced with the new one, the other surviving patient was administered meropenem, tigecycline, and colistin due to prosthetic infection, and the prosthesis was replaced with the new one. The isolates were shown to be resistant against all agents in all antimicrobial categories, and all were described as pan-drug resistant (PDR). Carbapenemase gene was detected in all (100%) strains resistant to carbapenem with the genotypic methods. The detected carbapenemase type was OXA-48 in all strains, and both OXA-48, and NDM-1 gene were detected in only one strain (no. 12). All (100%) the K. pneumoniae strains were detected to form biofilms. All (100%) isolates were found to form biofilms in high levels according to the classification in terms of the positive and negative control. NAC and rifampicin were administered to create a final concentration of 2 g/mL, and 0.1 g/mL to biofilm formed (24 hours) bacteria, and their effects at hour 2, hour 6, hour 24, and hour 72 were investigated using the colony counting method. No significant decrease was detected in the number of bacteria in all isolates compared with their controls (p value >0.05, >0.05, >0.05, >0.05 for NAC, and p value >0.05, >0.05, >0.05, >0.05 for rifampicin, respectively). Although there was a statistically significant decrease in the bacteria count at hour 6, and hour 24 compared with its control in the indicated time when ozone water was applied as to provide a final concentration of 0.6 g/mL (p value 0.05, 0.05, respectively), it was considered ineffective because no decrease was detected logaritmically as 2 log10 or over 2 log10. In addition, no significant decrease was detected in the number of bacteria in the biofilm in the investigation at hour 2 and hour 72 (p value >0.05 and >0.05, respectively). Although there was a statistically significant decrease in the bacterial count at hour 2, and hour 24 compared with its control in the indicated time when ozone water was applied as to provide a final concentration of 4.78 g/mL (p value <0.05, <0.05, respectively) by increasing the ozone amount in water, it was considered ineffective because no decrease was detected logaritmically as 2 log10 or over 2 log10. In addition, no statistically significant decrease was detected in the same concentration at hour 6. However, it was considered effective in detection of a decrease of 2.06-4.08 log10 interval in the bacterial count in the biofilm at hour 72 in all isolates. The effects of NAC, rifampicin, and ozone water on the living and death bacteria in the biofilm at hours 2, 6, and 24 were investigated as to provide the final concentration of 2 g/mL, 0.1 g/mL, and 4.78 g/mL in the biofilm forming (24 hours) bacteria using the laser screening confocal microscopy method. No significant decrease was detected in the living and death bacterial count in all isolates compared with their controls except the statistically significant decrease in the ozone water at hour 24 (p value for living bacteria was 0.05, and p value for death bacteria was 0.01). Ozone water was regarded ineffective at hour 24 as no decrease was determined logaritmically at 2 log10 or over 2 log10. Conclusion: Panresistant K. pneumoniae isolates are important pathogens that cause therapeutic problems in Turkey and worldwide. These bacteria that produce OXA-48 and NDM-1 type carbapenemase and are particularly colonised in the intensive care unit patients became endemic in our hospital. However, there is no data on the biofilm forming capabilities of these isolates. In addition, there is no study on the panresistant strains in the literature. There is need for different approaches to be used in the treatment of these isolates that form biofilms in addition to characterising the PRKp biofilm forming characteristics. The efficacy of the different concentrations of other agents such as NAC (2 mg/L), rifampicin (0.1 mg/L) and ozone (0.6 mg/L ve 4.78 mg/L) in treatment of such resistant strains was first tested in the present study. In accordance with the test results; NAC and rifampicin were found ineffective, however, ozone in a concentration of 4.78 mg/L enabled a decrease of ≥2 log10 in the number of bacteria. We suggest that more succesful results may be obtained in treatment with the use of higher ozone concentrations. There is a need for demonstrating the efficacy of these antibiofilm efficient agents with in vivo experiments with more strains and in vitro test. In addition, due to the lack of current treatment options owing to the biofilm forming capacities of bacteria and their being panresistant, emphasizes the prevention of the development of these infections and thus to the importance of the hospital control precautions. Key words: Panresistant Klebsiella pneumoniae, biofilm, N-acetyl cysteine, rifampicin, ozone