The characteristics of the essential oils.
Carbapenem-resistant Gram-negative organisms are increasingly isolated from lower respiratory tract infections. Limited treatment options are the main problems for physicians and clinical microbiologists who have to face such clinical cases. Bacteriological diagnosis, starting with accurate Gram smear performed from properly collected specimens and ending with antibiotic susceptibility testing, is essential. Morphological characters of bacterial cells provide important clues about the nature of infection, prior to bacterial isolation and identification. Attempts to find complementary options for the respiratory contamination and treatment of carbapenem-resistant Gram-negative bacillary pneumonia led us to test the susceptibility of 21 essential oils. Among them, Thymus vulgaris, Eugenia caryophyllata, Origanum vulgare, Melaleuca alternifolia and Aniba rosaeodora essential oils proved to be efficient against Acinetobacter baumannii carbapenem-resistant strain and Escherichia coli ATCC 25922. In an attempt to evaluate the magnitude of environmental spreading of the carbapenemase genes, 40 carbapenemase sequences of different organisms were compared. Carbapenemases show striking similarities inside each beta-lactamase class (A, D, and B), no matter their origin—environmental organisms or clinical isolates. Class B carbapenemases are most widely distributed, metallo-beta-lactamases being present in bacteria as well in Archaea.
- carbapenemase producer
- nosocomial pneumonia
- essential oils
- environmental pollution
Antibiotic-resistance genes are nowadays a constant presence not only in the hospital environment but are also more and more demonstrated in various ecosystems [7–9]. Antibiotic-resistance genes naturally already exist in organisms living in most diverse environments. Surely, antibiotic-resistance genes have an essential role in maintaining of inter-species equilibrium on specific ecosystems . All living things from prokaryotes to eukaryotes are constantly exposed to a huge mixture of organic and inorganic compounds. Even if, nowadays, accurate methods exist to isolate and to characterize antibiotic-resistant microorganisms, it is not possible to calculate the influence of a myriad factors that interfere in every environment. An interesting study demonstrated the utility of transmission electron microscopy for observing of aquatic microorganism structural abnormalities in different environmental conditions . The terrestrial ecosystem is also prone to be reshaped by human activities . Intensive farming implies antibiotics, so the spread of intestinal bacteria which harbor antibiotic-resistance genes is an immediate consequence. This does not imply that once a certain bacteria species is present in a certain geographic area its antibiotic-resistance pattern remains unchanged. Atmospheric conditions, notably rainfalls, could contribute to spreading of contaminants from the soil to groundwater and greatly alter the count of microorganisms. Different bacterial species do not behave the same, antibiotic-resistance patterns differently changed, but certain beta-lactamines could be used as indicators of antibiotic resistance at least for
The physicians require bacteriological diagnosis on admission of the patients in ICUs and for surveillance of any nosocomial infection. For respiratory infections, Gram smears from sputum, endotracheal aspirate, or bronchoalveolar lavage are mandatory. Very often in ICUs, respiratory infections are due to carbapenem-resistant Gram-negative bacilli. In our opinion, testing volatile EOs, as complementary substances, for prevention of respiratory infections is not a futile idea. Finally, last but not least, wide use of antibiotics alters not only the hospital environment but also disturbs other ecological niches (water and soil). These topics will be our concern in the next sections.
2. Carbapenem resistance
The carbapenems are sometimes the last-resort antibiotics for treating of extended spectrum beta-lactamase (ESBL) producing Gram-negative bacteria. But carbapenem resistance is increasingly reported in
3. Microbiological investigations
Microbiological evaluation is mandatory for an adequate therapeutic regimen; accurate identification of the bacterial species is essential to avoid administration of broad-spectrum antibiotics. The optimal recovery of the pathogens ultimately depends on the accuracy of sample collection (sputum, endotracheal aspirate, or bronchoscopically obtained specimens by bronchoalveolar lavage). Bacteriological assessment of lower respiratory tract infections begins with care evaluation of Gram-stained smear performed from respiratory tract specimen. The low-power scanning provides a first sight of the quality of the sample—for sputum more than 10 squamous epithelial cells show oropharyngeal contamination. The examination with the oil immersion provides more details regarding bacterial morphology. The importance of this step in the management of bacterial pneumonia is well recognized. As illustrated in Figures 1 and 2, it is possible to anticipate the diagnosis toward a nonfermenter or an
Since this chapter is not intended to be a highly elaborate description of bacterial diagnosis of Gram-negative bacillary pneumonia, further details about isolation and bacterial identification were not be reviewed. However, the microscopic examination of clinical sample offers essential clues about the nature of bacterial infection. For busy clinicians—in ICUs the physicians have always needed a microbiologic response as quickly as possible—these details, provided in advance, could make the difference.
4. Therapeutic issues
4.1. Prevention of respiratory contamination
A particular issue of ventilator associated pneumonia (VAPs) is the risk of infection with multidrug-resistant strains and carbapenem-resistant bacilli too. Not surprisingly, severely injured patients—VAPs and burn patients—are most prone to infection with carbapenem-resistant species. The chief question for carbapenem-resistant Gram-negative bacillary pneumonia is how to efficiently prevent them. First of all, could these infections be stopped? In the hospital environment, hand hygiene and alcohol-based disinfection remain, undeniably, the sanitation gold standard. Rigorous monitoring of patients at admission and an accurate history are early stages in identification of patients with documented multidrug-resistant strains for further isolation or, at least clustering separately to prevent the risk of cross-contamination [25, 26]. Respiratory contamination depends on so many circumstances, almost impossible to eliminate, that the specific strategies are designed in order to reduce VAP, rather than to eliminate such infections . What else could be taken in consideration apart from already established strategies? As it was underlined in introduction, from the ancient times, people are aware of the so-called air purification performed intuitively by burning scented substances or widespread use of all sorts of perfumes, plant extracts, or spices.
4.2. Antibiotic therapy
Antibiotic regimens of carbapenem-resistant bacillary pneumonia often rely only on few antibiotics. Although there is not an ideal therapeutic regimen for the treatment of pneumonia due to carbapenem-resistant species, Polymixin B, Tigecycline, and Amikacine remain the most valid options . A prerequisite for adequate treatment of VAPs is intravenous administration of the suitable antibiotic. Aerosolized antibiotics delivery has been experimentally studied in order to reduce the side effects of systemic administration of antibiotics. Efficiency of these methods relies on the antibiotics’ ability on crossing the alveolar-capillary membrane . An abundant literature is devoted to the issue of carbapenem-resistant strains. Because antibiotic resistance continuously evolved, clinical guidelines rapidly changed, therefore, a unique treatment scheme is almost impossible to establish. Clearly, we must look at the information provided by extensive epidemiological studies to up-date infection control and treatment options .
4.3. Inhibitory activity of essential oils
In spite of specific protocols implemented in ICUs, it is worthwhile to consider additional methods to prevent respiratory contamination. We should be considering the inhibitory effect of some essential oils (EOs), underlining the efficiency of volatile substances. EOs are more and more regarded as complementary to antibiotic therapy [31–35]. In our previous work, we demonstrated the high activity of EOs against
4.3.1. Materials and methods
We are interested in evaluating of the efficiency of some EOs against carbapenem-resistant
|1||Thyme||Lamiaceae||Fares||Internal use, aromatherapy, massage|
|2||Clove||Myrtaceae||Fares||Internal use, aromatherapy, massage|
|3||Eucalyptus||Myrtaceae||Fares||Internal use, aromatherapy, massage|
|4||Juniper||Cupressaceae||Fares||Internal use, aromatherapy, massage|
|5||Lavander||Lamiaceae||Fares||Internal use, aromatherapy, massage|
|6||Mint||Labiatae||Fares||Internal use, aromatherapy, massage|
|7||Pine||Pinaceae||Fares||Internal use, aromatherapy, massage|
|8||Rosemary||Lamiaceae||Fares||Internal use, aromatherapy, massage|
|9||Tea tree||Myrtaceae||Fares||Internal use, aromatherapy, massage|
|10||Oregano||Lamiaceae||Steaua Divina||Internal use, aromatherapy, massage|
|11||Negril||Ranunculaceae||Steaua Divina||Internal use, aromatherapy, massage|
|12||Lemon||Rutaceae||Steaua Divina||Internal use, aromatherapy, massage|
|16||Seeds of apricots||Rosaceae||Herbavit||Internal use|
22.214.171.124. Diffusimetric method
Figures 4–7 showing EOs inhibitory activity, demonstrate antibacterial activity for the two Gram-negative species tested. Before we comment on the implication of these results, it is worth considering the obvious antibacterial effect of some EOs. In particular, it is worth mentioning that we obtain more precise results—the smallest SDs—when 5 μl EOs are tested.
Aromatogram method (Figures 8 and 9) reveals more interesting evidence of usefulness of volatile effects of at least seven essential oils:
Synthetic results are listed in Table 2. At first glance, we noticed a remarkable antibacterial activity for
|2 μl||5 μl||10 μl||2 μl||5 μl||10 μl|
|Mean (SD)||Mean (SD)||Mean (SD)||Mean (SD)||Mean (SD)||Mean (SD)|
|1||Thyme||38.33 mm (10.41)||66.67 mm (5.77)||70.00 mm (5.00)||28.00 mm (3.61)||65.00 mm (8.66)|
|2||Clove||21.67 mm (6.51)||32.33 mm (2.52)||46.67 mm (2.89)||19.00 mm (5.29)||29.33 mm (2.31)|
|3||Eucalyptus||9.67 mm (2.52)||16.00 mm (3.61)||NI||8.00 mm (1.73)||16.00 mm (3.46)||NI|
|4||Juniper||16.33 mm (2.52)||23.00 mm (1.73)||NI||14.67 mm (4.62)||27.33 mm (2.52)||NI|
|5||Lavander||10.33 mm (0.58)||14.67 mm (2.52)||19.67 mm (5.51)||11.67 mm (0.58)||20.33 mm (3.51)||NI|
|6||Mint||13.67 mm (2.31)||20.67 mm (6.51)||39.00 mm (6.56)||7.00 mm (0.00)||15.00 mm (1.00)||NI|
|8||Rosemary||12.33 mm (±4.62)||16.33 mm (±1.53)||NI||10.33 mm (±3.51)||18.67 mm (±1.53)||NI|
|9||Tea tree||15.67 mm|
|24.67 mm (3.51)||26.33 mm (4.16)||18.33 mm (7.64)||23.33 mm (2.89)||11.00 mm (6.56)|
|10||Oregano||46.00 mm (±2.00)||70.00 mm (0.00)||77.67 mm (2.52)||29.67 mm (0.58)||40.33 mm (1.53)||76.00 mm (1.73)|
|13||Fennel||6.00 mm (0.00)||6.33 mm (0.58)||NI||6.00 mm (0.00)||NI||NI|
|16||Seeds of apricots||NI||NI||NI||NI||NI||NI|
|21||Rosewood||16.00 mm (1.00)||32.33 mm (11.59)||80.00 mm (0.00)||14.67 mm (0.58)||30.33 mm (9.50)||50.00 mm (2.00)|
In this study, commercial undiluted EOs were tested. There are some differences on viscosity, dispersion, vaporization, and other physical properties that, no doubt, influence the antibacterial activity. Although no proof can be given, the presence of antibiotic-resistance genes does not influence the efficiency of EOs. Therefore, an intuitive feeling turned our attention to the utility of these products in prevention or, why not, treatment of antibiotic-resistant Gram-negative bacillary respiratory infections. Although there are precise methods for identifying the chemicals with antibacterial activity, these are not important for our purpose. Note that here we are speaking of a screening of some commercial EOs that anyone can buy without medical prescription. Some authors demonstrate discrepancy of antimicrobial activity of EOs from different herbal varieties [37–40]. Of course, as we observed in our study, accurate description of physicochemical properties are needed , but for clinical purpose, the overall activity of EOs is relevant. In our opinion, a plant product should be considered as a distinct entity, and we can be certain that each component is synergistic to each other in a manner that exceeds the individual action of separated molecules. As we are speaking of living things, plant properties greatly depends on geographic area of collection, weather influence, manufacturing protocols, preservation conditions, species variety, and so on. Because natural plant products are considered safe, diverse possible applications were investigated: in agriculture for preventing crop diseases, monitoring soil characteristics [42, 43], or as industrial preservation solutions .
5. Environmental source of carbapenem-resistant strains
5.1. General considerations
Extensive studies are devoted to the ecotoxicity of industrial compounds or of pharmaceutical wastes . Even though existing tools permit measurement of the concentration of any chemical in a certain geographic area, it is almost impossible to accurately estimate the influence of external factors that, no doubt, interfere with the spread or chemical transformation of any substance—like antibiotics. When biology occurs, the problems become more complicated. Nowadays, a different approach in follow-up to the intricate relationships of abundant microorganisms from a specific environment is needed. Antibiotics, like any other chemicals, do not differ in the way of spreading, accumulation, and changing certain environmental characteristics. It is not an exaggeration to state that multidrug-resistant microorganisms from hospital facilities are the nightmare of health practitioners. Analyzing bacterial species one by one provides certain information, but the big picture contains much more. Before starting to accumulate new data, there are huge unexplored resources, like public databases. Our concern was related to the magnitude of change on environmental microbiome by medical activities, especially the use of carbapenems. We extensively searched carbapenemases, or similar beta-lactamases, in protein databases available, and we compared their similitudes depending on their isolation source. The goal of searching similitudes between carbapenemase of clinical samples and their counterpart of environmental origin was to assess the influence of medical activities in changing soil and water microbiota.
5.2. Data collection and methods
Herein, we have focused on comparison of the carbapenemases, from different sources, deposited in public databases—NCBI/National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/protein). The preliminary results were briefly presented in the First Conference of the Romanian Association of Laboratory Medicine . Carbapenemase sampling was carried out to cover all beta-lactamase classes. In summary, 40 FASTA carbapenemase sequences were collected, and then a pairwise sequence alignment (EMBOSS Needle Program) and multiple sequence alignment (Clustal Omega tool) were performed [47–49]. The default settings were used. Table 3 lists the characteristics of the sequences used for further comparisons. Sequences for Classes A, B, and D belonging to Archaea and bacteria were selected. Alignment sequence based on protein crystal structure was not possible due to lack of crystal structures available for beta-lactamases isolated from environmental samples.
|Class||Source||Organism (accession number) isolation source||Beta-lactamase||Residues|
|MBL-fold||ES||MBL fold metallo-hydrolase||305|
|MBL fold metallo-hydrolase||395|
|MBL fold metallo-hydrolase||293|
|MBL fold metallo-hydrolase||306|
|uncultured bacterium (ALJ52278)/river sediment, Haithe River, China||IMI-3||280|
|SFC-1 E166A mutant||283|
|CphA complex with Biapenem||227|
|Uncultured bacterium (AJF40233)/river water, Portugal||OXA-48-like||248|
|E||Oxa-48 complex with Fpi-1602||243|
|Oxa-48 complex with Avibactam||242|
5.3. Results and discussion
The major limit of the present study is the scarcity of carbapenemases of environmental source deposited in the public databases. Most of environmental beta-lactamases found belong to Classes B and D. The archaeal beta-lactamases found are metallo-hydrolase, and metal ions probably contribute to enzymes stability in extreme environmental conditions. Searching was extended to Eukaryota domain of life, but any sequences were included in the present study. Only two beta-lactamases-like of eukaryotic origin were retrieved—
On the other hand, environmental Classes A, B, and D carbapenem-hydrolyzing enzymes are very similar with carbapenemases of clinical sources. Some carbapenemases, such as IMI-2, IMI-3, NDM-1, or OXA-48, are the same regardless their origin as observed by sequences pairwise comparison [50, 51]. For further statistical analysis, identity percent was recovered. Student’s
Mean (CI 95)
Mean (CI 95)
|51.07 (±70.37)||40.30 (±42.34)||0.65|
|51.07 (±70.37)||40.30 (±42.34)||0.65|
|51.07 (±70.37)||40.30 (±42.34)||0.65|
|15.47 (±0.75)||15.56 (±3.04)||0.93|
|14.93 (±1.11)||14.48 (±2.88)||0.69|
|11.00 (±5.81)||13.18 (±2.57)||0.25|
|15.27 (±2.67)||15.38 (±1.59)||0.89|
|16.50 (±1.51)||16.60 (±2.21)||0.91|
|54.87 (±17.00)||31.40 (±18.06)||0.02|
|Uncultured (ALJ52278)||73.03 (±26.33)||40.20 (±26.23)||0.03|
|69.85 (±29.32)||28.54 (±2.37)||0.02|
|67.78 (±25.43)||27.94 (±2.63)||0.01|
|60.93 (±21.63)||25.58 (±2.28)||0.01|
|Uncultured (AJF40233)||80.35 (±34.61)||32.26 (±3.02)||0.02|
Multiple sequence alignment of Classes A and D carbapenemases demonstrate that the active-site residues are very well conserved. Class B carbapenems, which mediate resistance to all beta-lactamases except aztreonam, are particularly interesting; they are found in bacteria, Archaea, and similar proteins, even in eukaryotes. Further, multiple sequence alignment (Figure 10) highlights the notable differences between Archaea and bacteria. Residues involved in Zn2+ ions are very well conserved with the exception of Cys221, which is replaced by Asp in Archaea. However, nearby Cys residues were noticed. Since their crystal structures are not solved, we can just assume their involvement in metal ion binding.
The observation that Archaea contain different beta-lactamases demonstrates that human behavior has not profoundly altered natural environment. Or some microorganism communities have regulatory mechanisms so flexible that rapidly adapt at new environmental factors. For example,
6. Final remarks
Carbapenems remain a valid option for the treatment of ESBL
Essential oils have, no doubt, beneficial effects, but some providers excessively claim many possible effects—from antibacterial activity to neurological benefits—not always consistent with reality. Moreover, nowadays, there exists all sort of mixtures, and we can only hope that the ingredients are chosen following logical connections between their activities. Herein, these problems were not debated, but it is not a trivial question about the validity of all plant extracts. Just notice that the only mixture tested (an inhalant) proved no antibacterial activity, at least against
One important question arises from here—what else, apart from widely using of antibiotics, could influence the persistence of antibiotic genes in hospital facilities? Industrial wastes could remain for a long period of time in the environment, notably in groundwater . Carbapenemases from hospital sources are, no doubt, the major factor in the evolution of these enzymes, hospital residues being, definitely, a source of wastewater pollution. Further, the carbapenemases produced by natural environmental bacteria and Archaea significantly contribute to selection of new mutations. Soil bacterial species greatly influences our life; therefore, a genome-scale metabolic network  has proved to be a valid approach to evaluate the complex dynamics of soil bacterial species, mostly in geographic areas near huge hospitals with many departments. Also, innate resistance to antibiotics has raised over time a growing interest for a rational design of new antibacterial compounds .
Carbapenem-resistant Gram-negative bacilli are one of the leading causes of nosocomial pneumonia. They are particularly involved in the outbreaks in ICUs. These strains are very often multidrug resistant, putting additional pressure on physicians and clinical microbiologists. Bacteriological diagnosis provides essential evidence for carbapenem-resistant Gram-negative bacillary pneumonia. From the very beginning, the Gram smear from respiratory specimens shows to be indispensable for an accurate diagnosis. Preventing respiratory infections in ICUs is a challenging issue; antibiotic prescription for any kind of acute respiratory tract infection does not benefit the patients. Besides, carbapenem-resistant bacilli already exist not only in clinical units but also alter environmental microbiota. It is time for a different approach in dealing with the antibiotic-resistance issues. An endless struggle with microorganisms does not work; these tiny creatures have incredible resources to deal with any new chemotherapeutic agent. We do not have the slightest idea of the long-term impact of widespread antibiotic use on environmental microorganisms. Careful analysis of existing data, like the evidence deposited in public databases, and reconsidering the antibacterial efficiency of natural products, such as EOs, could help at dealing with multidrug-resistant organisms.
I gratefully acknowledge the financial support received from the Fares Bio Vital Laboratories, Orăştie, România for covering part of the cost to publish in open access.
Conflicts of Interest
The author declare no conflict of interest. The funder had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.