Background
During End Stage Renal Disease (ESRD), characterized by progressive kidney function loss, the glomerular filtration rate is below 15 mL/min/1.73 m
2. The kidney can no longer regulate the internal environment, and the patient requires support therapies like hemodialysis, peritoneal dialysis, and kidney transplantation to sustain life. Such support therapies are denominated Renal Replacement Therapies (RRT), being hemodialysis the most widely applied RRT [
1‐
3].
Pontoriero et al. [
4] have reported that hemodialysis patients are exposed to 400 L of water used to produce dialysis fluids every week. Despite interposition of a semi-permeable artificial membrane, this water comes into direct contact with the bloodstream. Therefore, knowing and monitoring the dialysis water chemical and microbiological purity is important.
Although dialysis fluid quality depends on a complex chain of devices and procedures and on the implemented quality control procedure, the best strategy to ensure patient safety is to prevent contamination in each dialysis process phase. Water constitutes 95% of the dialysate, and tubes, tanks, and taps represent potential reservoirs for microorganisms to form biofilms, which are extremely hard to eradicate by chemical or mechanical means [
5,
6].
Several procedures including physical, chemical, and physicochemical treatments are routinely used to disinfect hemodialysis monitors and the water treatment system [
7]. Sodium hypochlorite and peracetic acid disinfectants are commonly applied during disinfection.
Antiseptics and disinfectants play an important role in controlling infection because they act to minimize the spread of microorganisms. However, continued use of these products in hospitals and other health services can trigger bacterial resistance, contributing to antimicrobial resistance development.
Given that bacterial resistance and the risks associated with the use of disinfectants pose a constant challenge, the search for new compounds with antibacterial activity and the development of new products with disinfectant action are crucial.
Effective medicinal plant use has contributed to disseminating information about their therapeutic importance and medicinal effects, validating therapeutic knowledge that has been accumulated for centuries. Nevertheless, the chemical constituents of medicinal plants are not yet fully known [
8‐
12]. Identifying the active components of these plants should increase current knowledge about this inexhaustible natural source of medicinal compounds [
13].
The economic and ecological relevance of the species belonging to the genus
Copaifera has aroused researchers’ interest. According to Leandro et al. [
14] and Veiga Jr. and Pinto [
15],
Copaifera oleoresins contain mainly sesquiterpenes and diterpenes. The sesquiterpenes α-copaene, β-caryophyllene, β-bisabolene, α- and β-selinene, α-humulene, and δ- and γ-cadinene are worthy of note.
In vivo and in vitro evaluation has demonstrated that oils obtained from various
Copaifera species have anti-inflammatory, healing, antiedematogenic, antitumor, trypanocidal, and bactericidal activities [
16,
17].
Investigating natural products is clearly essential to the search for new molecules with antibacterial activity. In this sense, this work shall significantly contribute to research into the potential use of Copaifera species oleoresins against bacterial strains involved in hemodialysis.
Discussion
Given that
Copaifera oleoresins contain many easily deprotonable acid terpenes [
15,
18], the presence of this class of compounds in the oleoresins might contribute to the antibacterial activity observed in this study.
Plants are a source of great chemical and functional diversity, which has allowed investigations into an array of drugs for therapeutic use [
29].
Copaifera species oleoresins are produced by exudation of trunks from trees belonging to the genus
Copaifera. Studies have not evidenced that these oleoresins are cytotoxic to mammalian cells, induce behavioral changes, or cause lesions or hemorrhage in the stomach of rats treated with these extracts [
30,
31]. Therefore, determining the sensitivity of bacteria, especially Gram-positive organisms, to
Copaifera species oleoresins could prove a useful tool to combat these microorganisms [
32].
Gram-positive bacteria underlie community-acquired infections as well as hospital-acquired infections [
33]. Patients undergoing dialysis, mainly through venous access, are at 100 times higher risk of bacteremia than patients that do not require hemodialysis [
34]. Most of the times, the etiological agent of such infections is
S. aureus [
35]. In addition, the number of bacteria that are multiresistant to antimicrobial agents like beta-lactam antibiotics, fluoroquinolones, and macrolides has grown at an alarming rate [
36]. This has motivated the search for new antimicrobial agents.
Resistance of some of the tested isolates to
C. duckei oleoresin agrees with the report by Dos Santos et al. [
37], who also observed that
E. coli,
P. aeruginosa,
S. aureus, and
S. epidermidis are resistant to this oleoresin. Moreover, Pacheco et al. [
38] evaluated the antimicrobial activities of
Copaifera species oleoresins against Gram-positive and Gram-negative bacteria, to find that these oleoresins inhibit Gram-positive bacteria at different levels, but they are inactive against Gram-negative bacteria (
E. coli and
P. aeruginosa), in agreement with the present study.
Our results are not satisfactory for any of the other tested bacteria and partly agree with the data reported by Santos et al. [
39], who evaluated oleoresins obtained from three
Copaifera species (
C. martii,
C. officinalis, and
C. reticulata) against Gram-positive and Gram-negative bacteria as well as yeasts and dermatophytes. These authors found that
C. martii,
C. officinalis, and
C. reticulata oleoresin concentrations between 31.3 and 62.5 μg/mL inhibit Gram-positive bacteria, including
Bacillus subtilis,
Enterococcus faecalis,
S. aureus,
S. epidermidis, and methicillin-resistant
Staphylococcus aureus (MRSA).
Guissoni et al. [
40] reported that the oleoresins extracted from two
Copaifera species,
C. langsdorffii and
C. reticulata, can inhibit the growth of
E. coli,
K. pneumoniae,
S. marcescens,
S. aureus, including
S. aureus isolates (MRSA). According to these authors,
C. langsdorffii oleoresin presents MIC of 5000 μg/mL against all the tested bacteria, except for
E. coli, for which MIC is 620 μg/mL. In the present study, the MIC values determined for
C. reticulata oleoresin are not satisfactory against any of the assayed bacterial strains.
Papers on the antibacterial activity of
C. oblongifolia are rare, but
Copaifera species have been described to be efficient antibacterial agents. Masson et al. [
41] assessed the
C. langsdorffii oleoresin antibacterial activity against standard
S. aureus,
Streptococcus pyogenes,
E. faecalis,
P. aeruginosa, and
E. coli strains in vitro, to detect a broad inhibition spectrum for Gram-positive bacteria only. According to these authors, MIC values are 200, 400, and 1100 μg/mL against
S. aureus,
S. pyogenes, and
E. faecalis, respectively. Our MIC values against
S. aureus are more promising.
In contrast to Gram-negative bacteria, Gram-positive microorganisms are more sensitive to certain compounds. Antimicrobials act on the cell wall, and the different cell wall composition of these classes of bacteria may account for these results. In fact, Gram-positive bacteria present a thick cell wall consisting mainly of peptidoglycan, whereas Gram-negative bacteria display a stratified cell wall consisting of an outer membrane and a thin peptidoglycan layer [
42‐
44]. The unique structural cell wall properties of Gram-negative bacteria may have prevented
Copaifera species oleoresins from penetrating the cell wall; the external membrane contains lipopolysaccharides, which determine surface properties and alter cell permeability and susceptibility to the investigated oleoresins [
45].
Dialysis units have to follow a range of guidelines to disinfect the water distribution system. Among the various disinfecting agents employed in Brazil, sodium hypochlorite and peracetic acid are the most satisfactory [
46]. In this context, the present study aimed to evaluate the sodium hypochlorite and peracetic acid activities. Our results corroborate with data from a previous investigation [
47] reporting that 58% of the hemodialysis units that conduct disinfection use peracetic acid-based disinfectants. Here, disinfection occurs in 36% of the surveyed units every month, and these units follow the same guidelines followed in the unit where we collected the bacterial isolates for this work.
Compared to the usually recommended concentration of between 25,000 and 45,000 μg/mL [
48], we found that all the assayed strains are sensitive to sodium hypochlorite. This has been the standard disinfectant for water treatment and distribution systems in hemodialysis units. Nevertheless, events of bacteremia caused mainly by Gram-negative bacteria in dialysis patients have pointed out that this procedure is inadequate [
49‐
52].
Oliveira et al. [
53] tested disinfectants like quaternary ammonium salts; sodium hypochlorite at 0.5%, 1%, and 2%; glutaraldehyde at 2%; Lysoform®; aqueous ethanol solution at 70%; peracetic acid at 2%; and vinegar at 100% against 32
S. aureus isolates carried by insects within hospitals, to verify that the bacteria are only resistant to ethanol at 70% and vinegar.
The results we obtained for the assayed Copaifera species oleoresins and disinfectants against selected Staphylococcus strains show that Copaifera oleoresin 100 μg/mL, peracetic acid 7.81 μg/mL, and sodium hypochlorite 937.5 μg/mL inactivate all the bacteria. A comparative analysis reveals that C. duckei oleoresin is 12.8 times less efficient than peracetic acid but 9.37 times more efficient than sodium hypochlorite.
Interaction between compounds with antimicrobial activity has been used to reduce minimum inhibitory concentrations and to improve antimicrobial agent efficiency, once participating antimicrobials may act on different bacterial cell sites. Interactions are calculated on the basis of a mathematical equation and are defined as synergistic, additive, indifferent, or antagonist as compared to each isolated antimicrobial MIC [
54].
According to the criteria established by Rios and Recio [
55] and Gibbons [
56], MIC values of 100 μg/mL or lower are promising. Hence, just
C. duckei oleoresin presents antibacterial potential, and we only assayed this oleoresin in further tests.
The literature does not describe interaction between the
Copaifera oleoresins and the disinfectants tested herein, but there are some papers on the sysnergism between plant compounds and these same disinfectants against bacteria. One example is the report by Zago et al. [
57], who evaluated the synergistic potential of essential oils [(cinnamon (
Cinnamomum zeylanicum Blume Lauraceae), lemon grass (
Cymbopogon citratus (DC.) Stapf, Poaceae), peppermint (
Mentha piperita L. Lamiaceae), ginger (
Zingiber officinale Roscoe Zingiberaceae), clove (
Caryophillus aromaticus L. Myrtaceae), and rosemary (
Rosmarinus officinalis L. Lamiaceae)] combined with eight antimicrobial drugs (chloramphenicol, gentamicin, cefepime, tetracycline, sulfazotrim, cefalotin, ciprofloxacin, and rifampicin) against 12
S. aureus strains and 12
E. coli human isolates, to demonstrate that
S. aureus is the most susceptible to interaction between drugs and essential oils, and synergism occurs between lemon grass essential oil and eight of the tested drugs as well as between peppermint essential oil and seven of the tested drugs. In the case of
E. coli, synergism emerges only between rosemary essential oil and three of the tested drugs and between lemon grass essential oil and two of the tested drugs.
Moraes et al. [
20] assessed the synergistic antimicrobial action of
C. oblongifolia oleoresin with chlorhexidine dihydrochloride against bacteria that cause oral infections. Regarding chlorhexidine dihydrochloride combined with
C. oblongifolia oleoresin against
S. mutans (ATCC 25175),
L. casei (ATCC 11578),
P.gingivalis (ATCC 33277), and
P. micros (clinical isolate), the authors found that the effect is indifferent. As for
S. mitis (ATCC 49456) and
A. actinomycetemcomitans (ATCC 43717), the effect is additive.
Olmedo et al. [
58] examined the synergistic action between sodium hypochlorite and hydrogen peroxide, to observe that the combination of these compounds inactivates planktonic cells and inhibits biofilm formation by
E. coli,
Salmonella enterica subsp. enterica,
Klebsiella pneumonia, and
S. aureus standard strains as well as
S. Enterica,
K. oxytoca, and
E. coli clinical isolates.
Previous hospitalization, access type during dialysis, comorbidities, gender, time elapsed since the beginning of treatment with dialysis, and previous use of antibiotics contribute to
S. aureus colonization in dialysis patients [
59]. Hence, introducing new compounds with potential antibacterial action in disinfecting solutions is mandatory, especially to combat infection with
S. aureus.
Despite the development of some research into active combinations of conventional antibiotics, the scientific community has focused on identifying new antibacterial molecules, especially molecules of plant origin, to act as antibiofilm compounds and thus prevent biofilm formation [
60].
MICB
50 is defined as the lowest antibacterial agent concentration that can inhibit biofilm formation by approximately 50% [
25]. Moraes et al. [
20] investigated
C. oblongifolia oleoresin antibiofilm activity against bacteria that cause oral infections, to verify that this oleoresin inhibits 50% biofilm formation for the bacteria
Lactobacillus casei (ATCC 11578) and
Peptostreptococcus micros (clinical isolate) at 400 μ/mL,
Streptococcus mutans (ATCC 25175) and
Aggregatibacter actinomycetemcomitans (ATCC 43717) at 200 μ/mL, and
S. mitis (ATCC 49456) and
Porphyromonas gingivalis (ATCC 33277) at 100 μ/mL.
Leandro et al. [
61] evaluated the antibiofilm effect of the hydroalcoholic extract of
Copaifera trapezifolia rich in phenolic compounds against endodontic bacteria, to verify that the oleoresin at 200 μg/mL inhibits
P. gingivalis (ATCC 33277) and
P. micros (clinical isolate) biofilm formation by at least 50%.
Alencar et al. [
62] reported the
C. langsdorffii essential oil and oleoresin activities against
Staphylococcus,
Pseudomonas, and
Candida (resistant to azo compounds) and showed that a nanostructured suspension based on
C. langsdorffii essential oil or oleoresin presents efficient antibiofilm action.
Both sodium hypochlorite and peracetic acid have been tested against
S. aureus. However, comparison between studies is difficult due to lack of standardized methodologies and concentrations that act on biofilms. Many methodologies have been proposed to probe the microbiocidal action of various disinfectants. Das et al. [
63] were one of the first to pioneer the use of microplate methodology to report on the antimicrobial inhibitory effects of certain compounds on planktonic growth and adhered bacteria. These methodologies are based on visual alterations in color or on turbidimetric/colorimetric changes measured by spectrophotometric readings at a specific wavelength. A linear relationship is established between the inoculum size (10–10
7 CFU/mL) and the exposure time for individual wells, to obtain turbidity between 0.1 and 0.3 OD units within a certain time interval, generally between 1 and 24 h [
64]. Several dyes, such as crystal violet, have been proposed to verify microorganism growth after treatment [
65]. Another methodological possibility is plating in agar followed by incubation of bacterial inoculum aliquots before and after cell exposure [
66,
67].
Svidzinski et al. [
68] described that both peracetic acid and sodium hypochlorite at 0.1% (1000 μg/mL) act against MRSA staphylococcus, which agrees with our results demonstrating sodium hypochlorite activity at concentrations as low as 937.5 μg/mL. These authors also found that
S. aureus strain 15 is approximately 999 times more sensitive to peracetic acid.
Guimarães et al. [
69] studied how biocides (hydrogen peroxide at 7% combined with peracetic acid at 0.2%, sodium hypochlorite with 1% active chlorine, ethanol at 70% in aqueous solution and in gel, chlorhexidine digluconate at 0.5%, and povidone iodine at 10%) impact
S. aureus MRSA biofilm formation, to show that povidone iodine, sodium hypochlorite, and hydrogen peroxide combined with peracetic acid can reduce bacterial film formation by 90%.
Concerning sodium hypochlorite antibacterial activity, our results agree with the data reported by Silva et al. [
70], who developed
S. aureus and
P. aeruginosa biofilms on polyvinyl chloride (PVC) disks and treated them for 5 min with (a) aqueous chlorhexidine gluconate solution or (b) aqueous sodium hypochlorite solution at 3% and compared them with biofilm growth in buffer solution, to find that all the tested antimicrobials significantly reduce biofilm formation by both microorganisms. Cabeça et al. [
71] also evaluated the efficiency of disinfectants like iodine tincture (0.20%
w/
v), biguanide (0.50% w/v), quaternary ammonium compounds (0.50% w/v), peracetic acid (0.50%
v/v), and sodium hypochlorite (1.50% v/v) against planktonic cells (10
8 CFU/mL) and biofilms formed over sterile stainless steel disks of
Listeria monocytogenes,
S. aureus, and
E. coli reference strains, to verify that planktonic cells of all the organisms are sensitive to all the assayed antimicrobials. Biofim treatment with the disinfectants decreases the number of viable sessile cells. Sodium hypochlorite is the most effective agent, as corroborated by our results.
Toté et al. [
72] analyzed 12 disinfectants, including sodium hypochlorite at 1% (10,000 μg/mL) and peracetic acid at 0.3% (3000 μg/mL) diluted in water, against
S. aureus (ATCC 6538) and
P. aeruginosa (ATCC 700928) in the planktonic and biofilm growth modes. Samples were treated for 1, 5, 15, 30, and 60 min. The authors found that
P. aeruginosa planktonic cells are as sensitive as
S. aureus planktonic cells. Most biocides are effective after 1 min of contact with the microorganisms. Hydrogen peroxide and sodium hypochlorite are the most active biocides against sessile cells: they affect cell viability and diminish biofilm matrix, as also demonstrated herein.
Ueda and Kuwabara [
73] investigated
E. coli O:157,
Salmonella enteritidis, and
S. aureus biofilm sensitivities to various disinfectants and sanitizers, to verify that none of the disinfectants (acid, neutral, or alkaline) eliminate sessile cells effectively, all the sanitizers fail to inactivate the biofilm cells completely, and the most effective agent – sodium hypochlorite – still gives colony countings of 25 to 200 microorganisms/mL after treatment at concentrations recommended by the manufacturers. At the studied concentrations, the authors found that bezalkonium chloride, alkyl diaminoethylglycine hydrochloride, chlorhexidine digluconate, and polyhexamethylene biguanide inactivate most of the
E. coli and
S. enteritidis cells but not the
S. aureus cells. The authors concluded that
S. aureus is the most resistant microorganism evaluated in their study.
Królasik et al. [
74] assessed the efficiency of commercially available hydrogen peroxide- and peracetic acid-based disinfecting agents against
Listeria innocua,
Pseudomonas putida,
Micrococcus luteus, and
Staphylococcus hominis biofilms grown on stainless steel disks, to find that the disinfectants at 0.5% (5000 μg/mL) are ineffective against the bacterial biofilms after 10 min. However, the authors reported that after 30 min at 1% (10,000 μg/mL),
M. luteus counting reduces by 5 Log UFC/mL in the presence of the disinfectants. Given the results, the concentrations recommended by the manufacturers are ineffective against the assayed bacteria. In our assays, biofilm treatment with the recommended peracetic acid concentration of 0.1% (1000 μg/mL) for 24 or 48 h provides colony countings reduced by 11 Log UFC/mL in the case of the initial inocula of
S. aureus strains 2, 3, 9, 10, 13, and 15 and
S. epidermidis strains 57 and 68.
Gilbert et al. [
75] simultaneously evaluated five disinfectants diluted in culture medium containing peracetic acid (Proxitane 4002, Solvay Interox Ltd., Warrington, UK) against
E. coli and
S. epidermidis planktonic and biofilm cells. Disinfectant concentrations varied from 0 to 100 nanomoles (nmol)/L. Analyses considered the planktonic/biofilm cell ratio corresponding to 95% of dead cells within 30 min of exposure. The authors demonstrated that biofilm age affects results very little, but data heavily depend on the tested microorganism and disinfectant. They also showed that peracetic acid is the most effective agent against planktonic cells and significantly decreases biofilm cell activity at similar concentrations. One of the explanations for data dependence on concentration is that antimicrobial agents, especially agents that interfere in the membrane potential (for example, oxidizing agents such as peracetic acid), operate in many sites and through several mechanisms. Our results agree with the results of Gilbert et al. [
75] in the case of planktonic cells. However, we verified that sodium hypochlorite is more efficient against biofilm cells: IC
50 is 57.425% as compared to 33.060% achieved for peracetic acid.
In conclusion, MIC determination showed that C. duckei oleoresin is the most effective among the evaluated Copaifera species oleoresins: it inhibits the growth of S. aureus and S. epidermidis strains. Interaction between Copaifera oleoresins and disinfectants does not result in synergism. All the investigated strains form biofilms in the assayed conditions. Overall, the capacity of disinfectants to inhibit biofilm formation varies widely, and very low C. duckei oleoresin, vancomycin, peracetic acid, and sodium hypochlorite concentrations are necessary to achieve this effect. On the basis of the Minimum Biofilm Erradication Capacity, represented by IC50, sodium hypochlorite is the most effective antimicrobial tested herein.