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Introduction
According to the Pan American Health Organization (PAHO) and World Health Organization (WHO), approximately 1.4 million people acquire a Health care Associated Infection (HAIs), affecting 1 of 20 hospitalized patients, affecting about 4.1 million patients, and approximately 37,000 patients die each year (1). Furthermore, the Acinetobacter baumannii complex is the most frequently isolated pathogen with 80% of all clinical isolates and has a potential resistance of 3.1% to carbapenems (2).
These cases have become the most frequent events in health care, affecting several patients annually in many countries of the world, generating an impact on the quality of life of patients. The economic expenses attributable to them estimated for the US a cost of care of 28 and 33 billion dollars a year (3).
In Colombia, the cost of care for HAIs is COP 1,190,879 per person, in antibiotic treatment (41% of the total value) and laboratory analysis (13.5%) (4). Specifically, there are records of the presence in the hospital facilities of bacteria resistant to bactericides in Valledupar. 50% of S. aureus strains showed resistance to methicillin, up to 14% were isolated from the emergency and surgery areas (5).
The reason why the Ministry of Health and Social Protection of Colombia in 2018 created the prevention, surveillance, and control Program of HAIs and antimicrobial resistance, was to provide elements to strengthen the prevention and control of HAIs. Considering among other measures, the appropriate cleaning and disinfection practices, including the disinfectants selection for hospital use (1).
Consequently, using inside the disinfectant’s composition natural ingredients with high biocidal power has been increasingly highlighted. Among those are extracts and essential oils of aromatic plants such as Cymbopogon citratus, Lippia alba (Mill), Eugenia caryophyllata (Thunb), Aloe vera (L.), Azadirachta indica (A. Juss), and others (6,7). The last one is a meliaceous family plant, currently used in the pharmacopeia, cosmetics, and phytosanitary products. This plant has active natural substances in leaves and seeds against multiple pathogens, including bacteria, fungi, and viruses (8). Its extract has been demonstrated to contain relevant properties at the health level (9,10), with antibacterial, anti-inflammatory, antiviral, hypoglycemic, and antiulcer activity, among others (11).
Furthermore, experimental studies indicate that the ethanol extract of leaves has antibacterial activity in vitro against Staphylococcus aureus (12). Specific structural components within the Neem extract, such as deacetylgedunin (DCG), presents a high level of coupling with the PLpro protein of SARS-CoV-2, which allows its inhibition (13).
Balakrishna et al. (7) developed a hand sanitizer gel containing Azadirachta indica, Ocimum sanctum and Citrus limon extracts, with an inhibitory effect on bacteria related to HAIs Escherichia coli and Staphylococcus aureus.
This research raised the evaluation of the bacteriostatic effect of the extract of the leaves and seeds of the Neem tree against microorganisms’ strains isolated from surfaces in procedure rooms, neonatal intermediate care unit, adult intermediate care unit, and surgery of a hospital institution in Valledupar.
Materials and methods
Location
The study was carried out in the laboratory of the University of Santander, in Valledupar, Colombia.
Microorganisms
The bacteria associated with IAAS, Acinetobacter baumanni, Bacillus subtilis, Enterobacter aerogenes, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus sp., and Streptomona malthophila, were isolated by the method of a swab from different surfaces such as the surgical procedure room and the intensive care unit (ICU) (adult and neonates ICUs) from a hospital in Valledupar township. The bacteria grown at 37 ° C amid nutrient agar and Muller Hilton cultures. The strains were identified by the API 20E method for enterobacteria and API 20NE for other gram-negative bacilli. 0.85% saline suspensions were prepared on the Macfarland 0.5 scale to conduct the tests.
Extract from A. indica (Neem)
The aqueous Neem extract was prepared according to the methodology described by Barrabí and Garcia (14), where the plant material was manually collected in sterile plastic bags with airtight closure. Leaves and seeds were obtained from 10-year-old Neem trees located in the Centro Biotecnológico del Caribe (CBC)-SENA in Valledupar, Colombia, (169 MAMSL and an average temperature of 28 °C). The material was washed with distilled water, allowed to dry at 28 °C. Afterward, 5 g was weighed on a precision analytical balance, and 200 ml of distilled water was added in a flask and boiled for 25 minutes. Finally, the mixture was filtered on filter paper in a Büchner funnel, and a final volume of 100 ml of 5% Neem extract was obtained. Three concentrations were prepared from the stock solution: 3, 4, and 5%.
Chemical products
This research evaluated the products frequently used to disinfect environments, instruments, and surfaces from the hospital. The first product corresponds to a pre-disinfectant enzymatic detergent (protease, lipase, and amylase; liquid presentation, pH 8.9) used for instrument cleaning. The second product is a microbial disinfectant based on formaldehyde, cetrimide, and glutaraldehyde (400, 600, and 1000 ppm).
In vitro Effectiveness Comparison
We prepared 9 ml of the enzymatic detergent, the disinfectant, and Neem extract at 3, 4, and 5% concentrations. Afterward, 1 ml of the microorganisms at 0.5 Macfarland scale was added, and let them mix during 15 minutes of contact. Finally, we inoculated the mix in-depth in plates containing 1 ml of the Muller Hinton agar suspension and incubated at 37 ºC. Each strain was assessed in triplicate. By calculating the percentage of dead cells: [1- (Mean CFU irrigant / Mean CFUinitial bacterial number)] × 100%. Where “mean CFU irrigant” refers to the measurement of the colony-forming units resulting from exposure with the antimicrobial or irrigant substances to be evaluated and “Mean CFUinitial bacterial number”, the measurement of the initial bacterial colony-forming units, before being in contact with antimicrobial substances (15). Likewise, the enzymatic detergent and disinfectant were used in concentrations of 3% and 100%, respectively, assuming a high organic matter concentration. The density of application solutions is assumed to be 1.
Results
In this research, when reviewing the results of the different treatments to which all the strains of bacteria evaluated were subjected, it was evidenced: 1) there was significant differences between extract of Neem concentrations and the bacterial strains evaluated 2) the efficiency of the disinfectant and growth inhibition bacterial, except in Bacillus subtilis strains (Table 1).
Table 1 In vitro inhibition of the aqueous extract of Neem leaves in the concentration 3, 4, and 5%
| Treatment | A. baumanii | B. subtilis | E. aerogenes | S. aureus | S. maltophilia | Micrococcus sp | S. epidermidis |
|---|---|---|---|---|---|---|---|
| Neem 3% | 99.84±0.04b | 0.00±0.00a | 99.77±0.07d | 99.87±0.05e | 0.00±±0.00a | 99.86±0.02a | 0.00±0.00a |
| Neem 4% | 99.95±0.01d | 0.00±0.00a | 99.75±0.02d | 99.85±0.02d | 0.00±0.00a | 99.89±0.01ab | 0.00±0.00a |
| Neem 5% | 100.00±0.00e | 0.00±0.00a | 99.85±0.02e | 99.96±0.01f | 0.00±0.00a | 99.97±0.01d | 0.00±0.00a |
| Enzymatic detergent 3% | 99.81±0.01a | 0.00±0.00a | 99.63±0.01a | 99.48±0.03a | 0.00±0.00a | 99.88±0.00a | 99.69±0.01b |
| Disinfectant 100%. | 100.00±0.00e | 0.00±0.00a | 100.00±0.00f | 100.00±0.00g | 99.91±0.01e | 100.00±0.00d | 100.00±0.00b |
* According to Tukey 0.05, the values in a column followed by the same letter do not differ significantly.
Additionally, it should be noted that the Neem extract was effective in inhibiting the growth of five out of seven bacteria under study, at all concentrations used, being greater in 5 %, at 15 minutes of contact, and without significant differences with the other treatments (P=0.00). In the case of bacteria Acinetobacter baumanii, Enterobacter aerogenes, Staphylococcus aureus, and Micrococcus sp., inhibitions of 99.48% were observed, while in the case of Bacillus subtilis, Stenotrophomonas maltophilia, and Staphylococcus epidermidis, no bactericidal effect was observed by the Neem extract.
The Bacillus subtilis and Stenotrophomonas maltophilia strains were the most resistants; Bacillus subtilis was also not susceptible to the enzymatic detergent nor the disinfectant. Stenotrophomonas maltophilia also presented resistance to enzymatic detergent.
Discussion
This research indicated that using a single type of sanitizing product, applied for 15 minutes, is not efficient enough for reducing all bacteria genus that cause infections associated with health care, which were isolated from hospital’s surfaces in the city of Valledupar. This finding highlights the importance of trials that allow the proper selection and suitable disinfectants use in hospital areas that facilitate the construction of a clear policy to prevent HAIs (16).
Furthermore, regarding the Bacillus subtilis resistance to disinfectants, it is associated with its characteristics as a sporulated microorganism, which forms a barrier that prevents the antimicrobial agents’ entrance since the complex membranes that surround the endospore act as an additional penetration factor (17). Besides, an interference with the culture medium could occur, and in the case of chemical products, a more prolonged action time is recommended.
The 99.96% effectiveness of the natural extract based on Neem leaves in concentrations of 3, 4, and 5% coincides with similar studies on Staphylococcus aureus isolated from cows milk with subclinical mastitis, where Giraldo (18) pointed out an 83% bacteriostatic activity of Neem extracts at a 5% concentration.
Valle (19) stated that the Neem leaf infusion is 100% effective against Oxyuris equi, at 15% concentration when used in oral administration of 60 ml. On the other hand, according to Dublin, Roque, and Estrada (20), several applications of Neem were required to achieve adequate effectiveness as an anthelmintic. Additionally, the treatments must be repeated to achieve cumulative effects, more significant bioactivity of the extract against bacteria Staphylococcus epidermidis and Stenotrophomonas maltophilia. In particular, those last emerging opportunistic pathogens with evidence of intrahospital transmission are resistant to multiple drugs and disinfectants, associated with their high mutagenic capacity (21,22).
Likewise, in the study carried out by Vásquez (23), it was observed that the seed has a 40% bacterial inhibition and the leaves a 70%. Regarding S. epidermidis, it was 60%, and there was no inhibition of S. aureus and E. coli. Unlike the current study, Neem leaf's aqueous extract inhibited S. aureus (99.96%) and did not inhibit S. epidermidis. Variability in fact, according to the part of the plant used, indicatesd the need to standardize the extraction process to regulate the biocidal effect of the oil.
Similarly, López et al. (24) evaluated the microbicidal activity of acetonic, ethanolic and methanolic extracts of Neem seeds, at concentrations of 1, 10, 25 and 50%, against Escherichia coli, Staphylococcus aureus, and the Bacteriophage P22, using two contact times (2.5 and 5 min), reporting that the 10% ethanolic extract of Neem, inhibited the growth of E. coli. In comparison, the methanolic and acetonic extracts did so from the concentration of 25% and 50%, respectively, while the Neem extracts did not achieve a total reduction of S. aureus. This contrasting result may be due to the reduced contact time since the S. aureus inhibition was 15 minutes in the current study.
Conclusions
In this research, it was evidenced that the aqueous Neem extract has biocidal properties against bacteria associated with IAAS, which indicates its potential as an active ingredient of products for hospital use. To improve the Neem extract's effectiveness, a phytochemical study and standardization of contact times for evaluation in a combined way on hospital surfaces are necessary.
Nevertheless, given the non-total effectiveness of any of the evaluated products, the combined use of them in a disinfection plan to control bacteria associated with IAAS is recommended.
