Essential oils against bacterial isolates from CF patients

January 4 2021

Sources: Essential oils against bacterial isolates from cystic fibrosis patients by means of antimicrobial and unsupervised machine learning approaches. Sci Rep 10, 2653 (2020). https://doi.org/10.1038/s41598-020-59553-8

Full article and illustrations available in SVB 2020

Recurrent and chronic respiratory tract infections in patients with cystic fibrosis (CF) cause progressive lung damage and represent the main cause of morbidity and mortality. Staphylococcus aureus (S. aureus) is one of the bacteria that causes the earliest infections in infants and children with CF. It appears at the beginning of adolescence. The patients are therefore chronically infected.
by non-fermentable gram-negative bacteria. Pseudomonas aeruginosa (P. aeruginosa) is the most important and the most recurrent. The intensive use of antimicrobial drugs to fight lung infections inevitably leads to the appearance of bacterial strains that are resistant to antibiotics. New antimicrobial compounds should be identified to overcome antibiotic resistance in these patients.

Interesting data have recently been reported in the literature on the use of compounds of natural origin that have inhibited the bacterial growth of S. aureus and P. aeruginosa in vitro. Of these, essential oils seemed to be the most promising. In this work, an in-depth study on 61 essential oils (EO) is reported against a panel of 40 isolated clinical strains from CF patients.
To reduce the in vitro procedure and make the study as convergent as possible, machine learning clustering algorithms were first applied to select a reduced number of representative strains from the panel of 40. This approach allowed us to easily identify three EOs capable of strongly inhibiting bacterial growth in all bacterial strains. It is interesting to note that the antibacterial activity of EOs is absolutely not linked to the antibiotic resistance profile of each strain. Given the results obtained, a clinical use of EHs could be suggested.

Cystic Fibrosis (CF), one of the most common fatal genetic diseases in the Caucasian population, is an autosomal recessive condition that affects 70,000 people worldwide (Cystic Fibrosis Canada). The defective gene, identified in 1989, is the cystic fibrosis transmembrane regulator (CFTR), which is carried by 4% of people (among Caucasians). As the CFTR encodes a chloride channel from the surface of epithelial cells, patients with CF manifest various multi-organ problems caused by the alteration of sodium and chloride secretion across cell membranes and by the resulting luminal dehydration. Impaired mucociliary clearance, which should eliminate all microbes entering the respiratory tract, leads to the production of thick, dehydrated mucus in the lungs of people with CF, which promotes chronic bacterial colonization of the respiratory tract.

The microbiology of the respiratory tract in CF is particular. In the early stages of life, it is characterized by the presence of the gram-positive bacterium Staphylococcus aureus (S. aureus). Overall, in 2017, more than half of those affected had at least one positive culture for methicillin-sensitive S. aureus (MSSA). The highest prevalence of methicillin-resistant S. aureus (MRSA) occurs in individuals between 10 and 30 years of age, while MRSA peaks in patients under 10 years of age (Cystic Fibrosis Foundation, 2017). Register
of the annual Data Report patients. (https://www.cff.org/Research/Researcher-Resources/Patient-Registry/2017 - Patient-Registry-Annual-Data-Report.pdf).

During early adolescence, the lungs of CF patients are chronically infected by unfermented gram-negative bacteria. Of these, Pseudomonas aeruginosa (P. aeruginosa) is the most important and the most recurrent, so that 30% of children with CF and up to 80% of adults with CF (25 years and older) have lungs chronically colonized by this pathogen. P. aeruginosa isolated from respiratory secretions has a high phenotypic diversity and develops genetic mutations over time to adapt and survive in the complex environment of the respiratory tract of the person with CF. The mucoid phenotype of P. aeruginosa, defined by the overproduction of exopolysaccharide alginate in the lungs of CF patients, is a characteristic of chronic infection and predictive of a poor prognosis. Indeed, P. aeruginosa mucoid has also been associated with eradication failure and, compared to its non-mucoid counterpart, it has increased resistance to multiple antibiotics and to host immune effectors.

Thanks to current treatments, the life expectancy of CF patients has continued to increase, reaching an average lifespan of 40 years. Assuming a positive trend of improved clinical care that follows its current pace, CF patients born in 2010 are expected to live to be 50 years old.
The intensive use of antimicrobial drugs to fight lung infections inevitably leads to the emergence of bacterial strains that are resistant to antibiotics. Novel antimicrobial compounds need to be identified to overcome antibiotic resistance during the treatment of lung infections caused by CF.

Recent research has revealed that a few small molecules, such as peptides or mannosides, have shown promising effectiveness in the prevention and treatment of infections in vivo by bacterial and fungal biofilms. However, because of their mechanism of action based on a specific binding to a main target, it is known that the use of small molecules makes it possible to select strains that are more and more resistant. It is interesting to note that, in recent literature, some reports on the use of naturally derived compounds have shown in vitro the possibility of inhibiting the development of infections associated with CF. Essential oils, in particular, seemed to be the most promising agents among the natural compounds tested.
This analysis reports on an extensive study involving 61 essential oils (EOs) against a panel of 40 bacterial strains isolated from CF patients.
In order to reduce the in vitro procedure and to make the investigation as convergent as possible, the following workflow was adopted. Unsupervised machine learning algorithms and techniques, as implemented in Python language, were first applied to sample a smaller number of representative strains (RS) from among the 40 members of the panel. To this end, a number of categorical descriptors were collected and used to group isolated CF strains together. The cluster centroids indicated the RS to be studied for their sensitivity to a list of commercial EOs at fixed doses. Three EOs showed great effectiveness in reducing the growth of microorganisms and were therefore rapidly tested against all available clinical isolates. The three EOs confirmed the initial hypothesis that demonstrated their ability to inhibit bacterial growth. Gas chromatography coupled with mass spectrometry (GC-MS) was then performed on the three EOs in order to study the chemical components most likely to be the main responsible for antibacterial activity.

Discussion
Long-term administration of antibiotics to prevent and treat respiratory tract infections in patients with CF has been shown to be associated with the emergence of microorganisms resistant to multi-drug antimicrobials (MDR).
In particular, the acquisition of Meca/mecc genes in S. aureus and the accumulation of resistance mechanisms after exposure to antibiotics in P. aeruginosa, two key lung pathogens in people with CF, which are of concern in this context.

Multidrug resistance significantly limits effective treatment options, affecting patient clinical outcomes and prognosis. This is why the identification and development of new antibacterial agents is fundamental to improving the survival and quality of life of people with CF. It is therefore desirable to develop antimicrobial agents with new molecular mechanisms that could make it possible to fight bacterial infectious diseases without spreading antibacterial resistance.

Unsupervised machine learning algorithms applied to a panel of 40 S. aureus and P. aeruginosa strains isolated from CF patients made it possible to select a smaller number of representative strains using phenotypic and genotypic characteristics as categorical descriptors. Therefore, the antibacterial activity of all the EOs tested was evaluated initially on nine bacterial strains
selected: six representative strains of P. aeruginosa and three representative strains of S. aureus.
The activity of the 61 EOs was also evaluated on reference strains. Antimicrobial tests identified 3 HEs (CEO, BEO and CCPEO) out of the 61 tested, which showed greater antibacterial activity on the bacterial strains selected previously and on the reference strains. The antibacterial activity of the 3 selected EOs was then extended to all the strains of the two species. It is interesting to note that the three EOs showed maximum antimicrobial power on all the strains studied. Nothing can yet be ruled out about the role of chemical compounds. Future studies involving the application of machine learning will be devoted to the analysis of the importance of the chemical component either in the modulation of the biofilm or in its antibacterial powers.
Several documents have been written in order to elucidate the mechanism of antimicrobial action of EOs. For example, Cinna aldehyde, the main component of cinnamon, is capable of disrupting the transmembrane potential of P. aeruginosa.

In addition, EOs of various origins (lavender, lemongrass, marjoram, marjoram, peppermint, tea tree, and rosewood) show antimicrobial activity against the Burkholderia cepacia complex by inducing changes in the fatty acid composition of the membrane, followed by membrane rupture. In addition, ethylene oxide from Alluaudia procera was active against S. aureus ATCC25923, a multidrug-resistant strain.
The reported data confirmed the possibility of using EOs as therapeutic strategies in multi-drug resistant strains, probably due to the heterogeneous composition of the oils themselves.

In particular, during this work, we found an antibacterial activity of EOs that was unrelated to the antibiotic resistance profile of each strain. This observation is particularly relevant, as it suggests the potential use of EOs by topical administration taking into account the complexity of the microbiota's drug resistance profile in each patient.

In conclusion, the approach applied here made it possible to reduce the experimental steps and it was possible to identify the most promising EOs based on probabilistic evaluations that confirmed their broad spectrum of antibacterial potency with a reduced set of experiments.
According to a bibliographical survey (www.scopus.com), accessed on December 13, 2019, keywords: oil
essential, antibacterial activity and resistance), no evidence of resistance to the antibacterial activity of EOs has yet been reported. This is a particularly important characteristic for antibacterial candidates to be administered in the case of a chronic condition such as CF.

Indeed, some articles report an increase in sensitivity to antibiotics after treatment.
with essential oils.
Although a plethora of publications did not show the development of HE resistance, a very recent publication suggested the induction of efflux pumps and multidrug resistance in P. aeruginosa by Cinna aldehyde, the main component of cinnamon. Therefore, in the light of recent reports, much remains to be clarified about the effect of essential oils on the multidrug resistance of bacteria.

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