Since 1980, nosocomial infections, specially caused by Pseudomonas aeruginosa were classified as a big issue in Hospital, as a result of this problem, medical costs for health care systems have been high(20). Many studies has shown that efficacy of disinfectants and antibiotics are gradually reduced(21). There are several reasons of prevalence of resistance of disinfectants: inaccurate concentration, Inappropriate usage, and insufficient training to prepare and storage of Hospital disinfectants are among them(22). Compared to many antimicrobial resistance surveys about antibiotics, the number of global researches regard to resistance to biocides is yet very low. Due to clinical importance of Pseudomonas aeruginosa, the efficacy of five Hospital disinfectants was assessed against clinical isolates of Pseudomonas aeruginosa. Structural factors, bacterial efflux pumps, biofilm, toxins and enzymes are the main reasons of resistance to disinfectants and antibiotics in pseudomonas genus(23). Sodium hypochlorite 5%, showed the strongest bactericidal activity against isolated Pseudomonas aeruginosa followed by Dettol 4.8%, Sayasept- HP 2%, Chlorhexidine 2%, and Ethanol 70%.
In our study, the effectiveness of sodium hypochlorite in the concentration of active ingredient 0.078 was determined as 100% MBC of the studied isolates, which is more effective than all disinfectants in the present study. These results are consistent with another study performed on Pseudomonas aeruginosa and 0.5% sodium hypochlorite isolates. Sodium hypochlorite has also been shown to be more effective than ethanol and salvone (1.5 ٪ v / v chlorhexidine + 15 ٪ w / v cetrimide) in the study, and the results of our study show similar results(24). Also, the results of a study conducted in Brazil showed that sodium hypochlorite is more effective than ammonium tetravalent compounds against bacteria. In our study, sodium hypochlorite was more effective disinfectant than Sayaspet, which is a fifth generation of ammonium tetravalent compounds(25). The results of other studies on the active ingredient sodium hypochlorite confirm the results of our work (26–28).
Currently, EDTA has been approved as an antimicrobial agent to reduce the risk of bacterial biofilm formation and colonization. Another goal of the current study is to determine the synergistic effect of EDTA in combination with five other non-antibiotic antimicrobials. In the current survey the addition of EDTA increased the efficacy of selected disinfectants significantly. In a study reported that 4% tetra sodium EDTA is able to eradicate pre-formed biofilms of clinical isolates(29). some surveys revealed that combined antibiotics are more effective compared to single antibiotics(30). The efficacies of disinfectants currently are being investigated in order to decrease the rate of emerging resistance among clinically isolated bacteria. For decades, EDTA has been known as a potentiating and sensitizing agent. Several studies showed that EDTA biofilm disrupting is due to its ability to cations sequestering (Mg2+, Ca2 + and Fe3+), as a result increase the effect of other antimicrobial agents (31–33). Combination of antibiotics and other antimicrobial agents with disodium EDTA has been broadly studied (31, 34, 35). A study conducted on Candida and methicillin-resistant S. aureus (MRSA) on catheters, in this study a combination of ethanol (25 %), EDTA (30 mg/mL), and minocycline (3 mg/mL), eradicated pre-formed biofilms, synergistically(31). Another survey performed on commonly pathogens involved in canine otitis specially Pseudomonas, revealed that Combination of Tris-EDTA with Chlorhexidine 0.15% has excellent synergistic activity against all isolates (36). These trials propose that the combination of Chlorhexidine or ethanol with EDTA does not compromise the activity of one another. However, standard EDTA or disodium EDTA is not a potent and practical antimicrobial agent even when used at high level concentrations, and is not able to kill bacteria. On the other side, some studies showed that tetra-sodium EDTA has broad-spectrum antimicrobial activity on its own(29, 33). It was reported, tetra-sodium EDTA (40 mg/ mL) decreased biofilm colonization by Pseudomonas aeruginosa, S. epidermidis, Klebsiella pneumoniae, C. albicans and E. coli on catheter segments(33). In another survey killing ability of 4% tetra-sodium EDTA against clinically relevant pathogens was reported (29). Also There are many studies on Ethanol for its antimicrobial activity alone and combined with other antimicrobial agents, that indicated combination with other biocides increase the potent of Ethanol (32, 37, 38).
The role of biocides in bacterial antibiotics resistance is worrying since several changes in European law and claims of a risk of biocide use were promoted. All of the studies in this subject are significant to attain a better conception of the interaction between bacteria and biocides, and to make a proper risk assessment on the use of disinfectants and emerging resistance and cross-resistance in bacteria(39, 40). In this study, there was a significant difference between the results of antibiogram before and after exposure to sodium hypochlorite in most antibiotics. our results showed that 16 isolates were isolated from antibiotic-sensitive isolates, of these, 12 isolates became MDR isolates, and 4 isolates became XDR isolates. There are many studies that have emphasized the associations of biocide and antibiotic resistance in bacterial isolates. For instance, in 2016, Lloyd et al showed that in mercury contaminated place, the rate of resistance to antibiotics was higher in mercury-resistant bacteria in comparison to mercury-sensitive isolates(41). It is worth bearing in mind that, the ‘in-use’ concentration of disinfectants, in most times are 1,000 times greater than of their MIC, to gain a rapid killing of bacteria. Biocide at high level concentration usually interacts with several targets in the bacterial cell. For this reason, bacteria hardly become resistant via adaptation or other mechanisms. However, bacterial usually exposed to sub-inhibitory concentration of biocides. It has been shown that, bacteria exposing to sub-inhibitory concentrations of biocides result in increasing resistance to biocides and antibiotics in bacteria (42–45). In a study conducted on 2017, bacteria harboring biocide resistance genes were more probable to harbor an antibiotic resistance gene in comparison with bacteria lacking biocide resistance genes (46). In this study, we analyzed the correlation between biocides and antibiotics and positive connection were detected between sub-inhibitory biocides exposing and antibiotics resistance. Such relationships were extensively reported and often involved the up- regulation of efflux pumps (47).
Usage of disinfectant in Hospitals must be intently measured and re-evaluated due to the selection pressure effect of antimicrobials on the advent of resistant bacteria which could be spread to the Hospitalized patients. For this reason, resistance is inducible after exposure to sub-inhibitory level of disinfectant (sodium hypochlorite), which result in an increase in the isolates resistant to some antibiotics. It is noteworthy that evaluate some disinfectants and assess correlations with antibiotics resistant which should be taken into account for disinfection practices. It is not correct to consider bacteria that grow in low concentrations of disinfectants as resistant to biocides. This must be determined as ‘increasing MIC value’ or reducing susceptibility and as a result it is important to evaluate the bactericidal concentration instead of the inhibitory concentration of disinfectants (48). It should be noted that, results of different surveys and the methods employed must also be considered. However, opposed results show that there is not any correlation between antibiotic resistance and exposing to sub-lethal concentration of biocides (49), and it is not completely obvious that there is a correlation between biocide resistance and antibiotic resistance, and surveys still continue in this subject(50). In conclusion, it is clear that biocide concentration is a significant element in the bacterial resistance induction. if the disinfectant prepare at low concentrations, and if the diluted disinfectant is kept in a long time than manufacturer instruction, then bacteria are exposed to low concentration of biocide (48).
A vital key used by Pseudomonas aeruginosa to survive in harsh environment such as exposure to antibiotics agents is biofilm formation (51). The National Institute of Heart, Blood, and Lung reported that up to 80% of all infections caused by bacterial are related to biofilm formation (52). The results of our study showed that 117 (97.5%) isolates formed biofilm, which was similar to other studies (53). Antibiotic resistance has increased by biofilm formation, and resulted in using a higher concentrations of antibiotics in MDR Pseudomonas aeruginosa isolates infections (54). In a study, it was indicated that the rate of Pseudomonas aeruginosa isolates biofilm formation from Iranian patients varied from 48.5–99.5%. Generally, the biofilm formation ratio was reported as 87.6%. As well, 27.4%, 30.2%, and 47.7% of Pseudomonas aeruginosa isolates were weak, moderate, and strong biofilm producers, respectively (16). Accordingly, our data were in line with the results published in studies where 40–100% of Pseudomonas aeruginosa isolates produce biofilm (55, 56). Karami et al, reported 73% of both clinical and environmental isolates were biofilm producers (57). Also, other studies reveals the importance of biofilm formation by Pseudomonas aeruginosa (58, 59). In line with our study it was reported that 58.6% of MDR isolates produce strong biofilm. These results revealed a significant correlation between biofilm formation and MDR isolates (60). It should be noted that, In contrast to these findings, some studies from different parts of the world indicated a lower prevalence of biofilm formation, and as a result, there was no correlation between antibiotic resistance and biofilm producing (61–63). This issue possibly linked to other resistance mechanisms (efflux pumps, purines, chromosomal mutation, and plasmid acquisition) involved in antibacterial resistance (64).
Another aim of the present study was to investigate the prevalence of qacE∆1, qacE and sug-E1 genes, and their relationship with resistance to antibiotics and biocides in Pseudomonas aeruginosa. In the current study, the prevalence of efflux pump genes was very high, and due to the high prevalence of qacE and sug-E1 genes, no association was found between these genes and resistance to disinfectants and antibiotics. Of the 21 isolates carrying the qacE∆1 gene, 16 isolates were MDR / XDR, indicating an association between this gene and antibiotic resistance. In a study conducted in Egypt, the qacE∆1 gene was identified in 57.8% of multidrug-resistant isolates, and 21.4% in susceptible strains, which confirms the results of our study (65). In our study, 95% prevalence of sug-E1 gene was reported, which is similar to the results of a study conducted in Australia, While the prevalence of qacE∆1 gene was 46.2%, it was higher than the prevalence reported in our study (66).