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Abstract
Conclusion and Recommendations
This study has illustrated that MDR Gram-negative bacilli isolated from patients recruited from different departments at Sohag university hospitals is an important cause of health care-associated infections. 64.4% of isolated strains were found to be biofilm producers by tissue culture plate method and this character was reduced by exposure of these strains to EDTA, kojic acid and varying concentrations of copper oxide nanoparticles. Thus, these agents can be used for prevention of biofilm formation by MDR Gram-negative bacilli.Recommendations to decrease nosocomial infections by MDR Gram-negative bacilli: 1- Strict implementation of infection control measures particularly hand hygiene practice and reliable cleaning techniques to decrease spread of nosocomial infection.2- Proper cleaning, disinfection and sterilization of equipments and hospital environment to prevent cross transmission. 3- Active surveillance measures to identify patients infected with MDR Gram-negative bacilli especially biofilm producers. 4- Avoid the use of broad-spectrum antibiotics before doing antibiotic sensitivity to avoid treatment failure and development of resistance due to unnecessary use of this class of antibiotics.5- Search for alternatives to antibiotics.6- from our findings we recommend the use of EDTA, kojic acid and CuO NPs with the previously described concentrations for prevention of biofilm formation by MDR Gram-negative superbugs in different healthcare settings.7- Further studies are needed to test the effect of various concentrations of kojic acid as it is a natural compound with less expected side effects than EDTA and it is not widely tested yet.
Summary
Health care associated infections especially those caused by antimicrobial-resistant pathogens, is an area of public health concern throughout the world. The World Health Organization estimates that HCAI is the most frequently occurring adverse event in any healthcare system regardless of available resources. Out of every one hundred hospitalized patients, seven patients in developed countries and ten patients in developing countries acquire an HCAI.Multidrug-resistant bacteria remain the greatest challenge in public health care. The numbers of infections produced by such resistant strains are increasing globally. This acquired resistance of pathogens presents a key challenge for many antimicrobial drugs. Particularly problematic in this respect are the major human pathogens, e.g., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.Biofilm formation plays an important role in bacterial resistance protecting bacteria and allowing them to evade the action of antibiotics. Associated with the rise in antibiotic resistance is the lack of new antimicrobials. This has led to efforts being made all around the world to create new and more effective antimicrobial compounds as well as to develop novel delivery and targeting strategies, particularly for diseases involving biofilm formation.Recently various methods of biofilm control are being developed to overcome the complications of prolonged use of antibiotics such as antibiotic resistance which is widely spread among hospitalized patients, one of these methods is the use of metal chelators that renders the biofilm environment less convenient for the survival of microorganisms.Nanoparticles also provide an alternative strategy to target bacterial biofilms with potential to use both antibiotic free and antibiotic-coated approaches. Because most of these NPs-based platforms exert their action via distinct mechanisms of those used by traditional antibiotics, combined therapeutic regimens are promising strategies to tackle the surge of multidrug resistant bacteria by passing their defense mechanisms.Our study was carried out at Medical Microbiology and Immunology Department, Faculty of Medicine and Sohag University Hospitals in the period from January 2020 to January 2022. The study included 220 patients with different types of health care-associated infections, from which 118 (53.6%) patients were infected by MDR gram negative bacilli. The patient ages ranged from 6-85 years, the mean age ± SD was 39.14 ± 14.98. Males represented 55.08% and females represented 44.92% of cases.The highest percentage of isolation was from patients with surgical site infection (28.8%), while the lowest percentage was from asthmatic patients with chest infection (4.2%). The most frequent isolate was Pseudomonas aeruginosa 31.4%, followed by Klebsiella pneumoniae ssp pneumoniae 22%, Escherichia Coli 17.8%, Acinetobacter baumannii complex 11.9%, Enterobacter aerogenes 9.3%, Proteus mirabilis 6.8%, while Burkholderia cepacia group was isolated from one sample only (0.8%).The isolates were subjected to antibiotic sensitivity testing (disc diffusion method) according to CLSI 2020. Antibiotic susceptibility profile of the isolates was as follow:1- Escherichia coli: all E. coli isolates were resistant to piperacillin, amoxicillin- clavulanate, piperacillin-tazobactam and ceftazidime, while the highest sensitivity was to nitrofurantoin (95.2%).2- Enterobacter aerogenes: the highest resistance was to piperacillin, amoxicillin- clavulanate, and piperacillin- tazobactam (9.1% only were susceptible), while the highest sensitivity was to chloramphenicol (81.8%).
3- Klebsiella pneumonia ssp pneumoniae: all Klebsiella pneumonia isolates were resistant to amoxicillin-clavulanate, piperacillin-tazobactam and ceftazidime, while the highest sensitivity was to imipenem and meropenem (92.3% were susceptible).
4- Proteus mirabilis: all isolates were resistant to nitrofurantoin, while the highest sensitivity was to imipenem and meropenem (62.5% were susceptible).
5- Acinetobacter baumannii complex: the highest resistance rate was to piperacillin, cefotaxime, ceftriaxone and ceftazidime (14.3% isolates were susceptible), while the highest sensitivity was to trimethoprim- sulfamethoxazole (71.4% were susceptible).
6- Pseudomonas aeruginosa: the highest resistance rate was to piperacillin-tazobactam and ceftazidime (2.7% only were susceptible), while the highest sensitivity was to meropenem (89.2% were susceptible).
7- Burkholderia cepacia group: one isolate was detected and is found to be resistant to ceftazidime, trimethoprim-sulfamethoxazole and chloramphenicol, while sensitive to levofloxacin and meropenem.
The bacterial isolates were tested for their ability to form biofilm by tissue culture plate method. 76 (64.4%) of isolates were biofilm forming while non-biofilm forming isolates were 42 (35.6%). from the biofilm-forming isolates; 19 (25%) were strong biofilm-producers, 24 (31.6%) were moderate while most of the isolates 33 (43.4%) were weak biofilm-producers. The biofilm producing species were distributed as follows; 8 isolates (10.5%) belonged to Acinetobacter baumannii complex, one isolate (1.3%) was Burkholderia cepacia group, 13 (17.1%) were E. Coli, 6 (7.9%) were Enterobacter aerogenes, 19 (25%) were Klebsiella pneumoniae ssp pneumoniae, 3 (3.9%) were Proteus mirabilis and 26 (34.2%) were Pseudomonas aeruginosa.
In this study, we tested the effect of EDTA with concentration of 8 mg/ml and kojic acid with concentration of 6.25 mg/ml to verify their effectiveness as biofilm inhibitors. The results revealed that; the mean optical density reading OD± SD among biofilm forming isolates was 0.183± 0.11. EDTA was more powerful than kojic acid in inhibiting biofilm formation as it inhibited biofilm by 85.4 % with mean optical density reading 0.0267± 0.051, while kojic acid decreased biofilm formation by 65.5% with mean optical density 0.063± 0.108. This difference was of high statistical value (P-value < 0.0001).
We also tested the biofilm inhibition effect of different concentrations (125-2000 μg/ml) of copper oxide nanoparticles; a concentration dependent biofilm inhibition effect was observed with different concentrations of CuO NPs for all the tested isolates regardless the genus and the species of it. For Acinetobacter baumannii complex isolates, their mean OD reading was (0.227± 0.108) and the percentages of biofilm inhibition were (21.84%-93.57%) with mean OD reading (0.1665± 0.18- 0.0178± 0.043). For Burkholderia cepacia group isolate, the OD was (0.311) and percentages of biofilm inhibition were (7.39%- 97.1%) with OD of (0.288- 0.009). Regarding E. coli isolates, the mean OD reading was (0.1797± 0.125) and percentages of biofilm inhibition were (28.21%- 87.75%) with mean OD reading (0.129± 0.0875- 0.022± 0.068). For Pseudomonas aeruginosa isolates, the mean OD reading was (0.1665 ± 0.111) and the percentages of biofilm inhibition were (29.12%- 83.78%) with mean OD reading (0.118 ± 0.909- 0.027± 0.0308). For Klebsiella pneumoniae isolates, the mean OD reading was (0.198± 0.125) and the percentages of biofilm inhibition were (27.7%- 92.27%) with mean OD reading (0.143± 0.125- 0.0153± 0.057). Regarding Proteus mirabilis isolates, their mean OD reading was (0.208± 0.2016) and the percentages of biofilm inhibition were (25%- 89.42%) with mean OD reading (0.156± 0.133- 0.022± 0.036). Finally, the mean OD reading of biofilm forming Enterobacter aerogenes was (0.125± 0.047) and the percentages of biofilm inhibition were (24.8%- 83.2%) with mean OD reading (0.094± 0.047- 0.021± 0.044). The difference in all the readings of optical density before and after exposure to CuO NPs in all isolates was of a statistical significance (P-value < 0.05).